Engineered glucose dehydrogenases and methods for the reductive amination of ketone and amine compounds

Abstract

The present application provides engineered glucose dehydrogenase polypeptides having imine reductase activity, polynucleotides encoding the engineered polypeptides, host cells capable of expressing the engineered polypeptides, and methods of using the engineered polypeptides and a series of ketone and amine substrate compounds to prepare secondary amine and tertiary amine product compounds.

Description

[0001] This application claims priority to U.S. Patent Application Serial No. 62 / 841,666, filed on May 1, 2019, which is hereby incorporated by reference in its entirety for all purposes. Technical Field

[0002] The present invention provides engineered glucose dehydrogenase polypeptides having imine reductase activity that can be used to produce secondary amines, as well as compositions and methods utilizing these engineered polypeptides.

[0003] Reference to a sequence listing, table or computer program

[0004] An official copy of the sequence listing is submitted with the specification via EFS-Web in an ASCII formatted text file named "CX2-188WO1_ST25.txt", created on April 22, 2020, and 2.26 megabytes in size. The sequence listing submitted via EFS-Web is part of the specification and is incorporated herein by reference in its entirety.

[0005] background

[0006] Chiral secondary amines are important building blocks for the pharmaceutical industry. However, there are only a few known biocatalytic pathways to produce such chiral amine compounds. Existing chemical methods use reduction methods based on chiral boron reagents, transition metals, or protecting group strategies, which require multi-step syntheses for the overall reduction of imines.

[0007] There are several reports in the literature on the biocatalytic synthesis of stable cyclic amines. Imine reductases or "IREDs" were purified and characterized from Streptomyces sp. GF3587 and GF3546 and were shown to stereoselectively reduce 2-methyl-1-pyrroline (see, Mitsukura et al., Biosci. Biotech. Biochem., 75: 1778-1782 [2011)]; Huber et al. Chem. Cat. Chem., 6: 2248-2252

[2014] ). Reduction to acyclic amines and amino acids with alkyl groups using natural imine reductases has also been shown (Gand et al., J. Mol. Catal. B: Enzym., 110: 126-132

[2014] ). Recently, other authors reported IREDs, also known as reduced amines (RedAm), which catalyze synthetically interesting reductive aminations (Grogan, Curr. Opin. Chem. Biol., 2018, 43, 15-22; France et al., ChemCatChem., 2018, 10(3), 510-514; Aleku et al., Nat. Chem., 2017, 9, 961-969 and ChemCatChem., 2018, 10(3), 515-519; Sharma et al., ACS Catalysis 2018, 8(12), 11534-11541 and Adv. Synth. Catal., 2017, 359(12), 2011-2015; Roiban et al., ChemCatChem., 2017, 9(24), 4475-4479).

[0008] Glucose dehydrogenase (GDH), the original member of the short-chain dehydrogenase reductase family, is well known for its NAD(P)-dependent oxidation of D-glucose to gluconolactone. It has been widely used for the regeneration of nicotinamide cofactor. Recently, the multifunctional imine reductase activity of GDH was reported, with several GDH enzymes from different organisms asymmetric reduction of cyclic imine salts to cyclic chiral amines (Roth et al., ChemBioChem 2017, 18, 1703–1706). This preliminary report raised the question of whether GDH, similar to IRED, can be used to generate a range of chiral secondary amines, especially including acyclic amines, amino acid esters, and amino acids with alkyl chains.

[0009] Overview

[0010] The present invention provides novel biocatalysts and related methods for synthesizing chiral secondary amines by direct reduction of imine substrates. The biocatalysts of the present disclosure are engineered polypeptide variants of wild-type genes from Bacillus subtilis, which encode glucose dehydrogenase with the amino acid sequence of SEQ ID NO: 2. A variant of the wild-type glucose dehydrogenase (SEQ ID NO: 4) contains the following residue differences compared to SEQ ID NO: 2: E170K, Q252L, I165M, P194T, which are used as the starting point for protein engineering. These engineered polypeptides are capable of catalyzing the conversion of imines into secondary amines.

[0011] The present invention provides an engineered polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the reference sequence SEQ ID NO: 4, comprising at least one substitution or a set of substitutions at one or more positions selected from the group consisting of: 96, 96 / 118, 147, 155, 155 / 253, 195, 200, and 256, wherein the positions are numbered with reference to SEQ ID NO: 4. In some additional embodiments, the engineered polypeptide comprises at least one substitution or a set of substitutions selected from the group consisting of: 96A, 96C / 118M, 147A, 147I, 147S, 155A / 253H, 155N, 195G, 200W, 256A, 256S, 256T, and 256V, wherein the positions are numbered with reference to SEQ ID NO: 4. In some embodiments, the engineered polypeptide comprises at least one substitution or set of substitutions selected from the group consisting of E96A, E96C / T118M, H147A, H147I, H147S, F155A / Y253H, F155N, I195G, F200W, F256A, F256S, F256T, and F256V, wherein the positions are numbered with reference to SEQ ID NO: 4. In some additional embodiments, the engineered polypeptide comprises an amino acid sequence having at least 80% sequence identity to any even-numbered sequence listed in SEQ ID NO: 4 to SEQ ID NO: 30.

[0012] The present invention also provides an engineered polypeptide, wherein the engineered polypeptide comprises a polypeptide having a sequence similar to the reference sequence SEQ ID NO:14 has an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity comprising at least one substitution or a set of substitutions selected from the group consisting of: 17, 95 / 96, 95 / 96 / 118, 95 / 96 / 118 / 156 / 200 / 253, 95 / 96 / 147, 95 / 96 / 147 / 200, 95 / 96 / 147 / 200 / 253, 95 / 96 / 155 / 156, 95 / 96 / 155 / 195 / 200, 95 / 96 / 159 / 195 / 253, 95 / 155 / 156, 95 / 155 / 159 / 200, 95 / 155 / 200, 96, 96 / 147 / 195 / 200 / 253, 96 / 155, 96 / 155 / 159, 96 / 155 / 159 / 195, 96 / 155 / 159 / 200, 96 / 156 / 159 / 195, 96 / 156 / 159 / 195 / 200, 96 / 195, 147, 147 / 155, 147 / 155 / 156, 147 / 155 / 156 / 200, 147 / 155 / 159, 147 / 195 / 200 / 253, 155, 155 / 156, 155 / 156 / 195 / 200 / 253, 155 / 159 / 200 and 253, wherein the positions refer to SEQ ID NO:14.In some embodiments, the engineered polypeptide comprises at least one substitution or a set of substitutions selected from the group consisting of: 17T, 95V / 96A / 118M / 156T / 200W / 253H, 95V / 96A / 147Q / 200W / 253H, 95V / 96C, 95V / 96C / 118M, 95V / 96C / 147Q, 95V / 96C / 147Q / 200W, 95V / 96C / 155A / 195G / 200W, 95V / 96C / 155N / 156Q, 95V / 96C / 159T / 195G / 253H, 95V / 155N / 15 6A, 95V / 155N / 159C / 200W, 95V / 155N / 200W, 96A / 147Q / 195G / 200W / 253H, 96C / 155A, 96C / 155N / 159C、96C / 155N / 159C / 195G、96C / 155N / 159C / 200W、96C / 155N / 159T、96C / 156A / 159T / 195G / 200W, 96C / 156L / 159T / 195G, 96C / 195G, 96S, 147Q / 155N, 147Q / 155N / 156A / 200W, 147Q / 155N / 156T, 147Q / 155N / 159C, 147Q / 195G / 200W / 253H, 147T, 155N, 155N / 156A, 155N / 156T / 195G / 200W / 253H, 155N / 159C / 200W, 155Q, 155R, 253G and 253S, wherein the positions are numbered with reference to SEQ ID NO:14.In some additional embodiments, the engineered polypeptide comprises at least one substitution or a set of substitutions selected from the group consisting of S17T, L95V / E96A / T118M / V156T / F200W / Y253H, L95V / E96A / I147Q / F200W / Y253H, L95V / E96C, L95V / E96C / T118M, L95V / E96C / I147Q, L95V / E96C / I147Q / F200W, L95V / E96C / F155A / I195G / F200W, L95V / E96C / F155N / V156Q, L95V / E96C / A159T / I195G / Y253H, L95V / F155N / V1 56A, L95V / F155N / A159C / F200W, L95V / F155N / F200W, E96A / I147Q / I195G / F200W / Y253H, E96C / F155A, E96 C / F155N / A159C, E96C / F155N / A159C / I195G, E96C / F155N / A159C / F200W, E96C / F155N / A159T, E96C / V156 A / A159T / I195G / F200W, E96C / V156L / A159T / I195G, E96C / I195G, E96S, I147Q / F155N, I147Q / F155N / V156 In some additional embodiments, the engineered polypeptide comprises an amino acid sequence having at least 80% sequence identity to any even-numbered sequence listed in SEQ ID NO: 32 to SEQ ID NO: 104.

[0013] The present invention also provides an engineered polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the reference sequence SEQ ID NO:34, comprising at least one substitution or a set of substitutions selected from the group consisting of: 17, 17 / 21, 17 / 21 / 42, 17 / 21 / 42 / 155 / 260, 17 / 21 / 49, 17 / 21 / 49 / 51, 17 / 21 / 49 / 51 / 96, 17 / 21 / 49 / 51 / 260, 17 / 21 / 49 / 51 / 96, 17 / 21 / 51 / 155 / 260. 6、17 / 21 / 96 / 155、17 / 21 / 96 / 195 / 260、17 / 21 / 96 / 253、17 / 21 / 147 / 155 / 253、17 / 21 / 155 / 253、17 / 21 / 195、17 / 21 / 253、17 / 42、17 / 42 / 49 / 195 / 260、17 / 42 / 96、17 / 44、17 / 44 / 47 / 51 / 96、17 / 44 / 47 / 51 / 96 / 114 / 147 / 195 / 199 / 253、17 / 44 / 47 / 51 / 96 / 114 / 147 / 199、17 / 44 / 47 / 51 / 96 / 114 / 195 / 199、1 7 / 44 / 47 / 51 / 96 / 147 / 253、17 / 44 / 47 / 51 / 178 / 195、17 / 44 / 47 / 195 / 199、17 / 44 / 51 / 114 / 195 / 253、17 / 46 / 47 / 51 / 96 / 147、17 / 46 / 47 / 51 / 96 / 195 / 253、17 / 46 / 47 / 51 / 155、17 / 46 / 47 / 51 / 155 / 195 / 199、17 / 46 / 47 / 96 / 195、17 / 46 / 47 / 178、17 / 46 / 96 / 253、17 / 47、17 / 47 / 51、17 / 47 / 51 / 96 / 114 / 147 / 1 95、17 / 47 / 51 / 96 / 114 / 155 / 178 / 253、17 / 47 / 51 / 96 / 195、17 / 47 / 51 / 96 / 195 / 199、17 / 47 / 96 / 114 / 155 / 253、17 / 47 / 96 / 147 / 155 / 195 / 199、17 / 47 / 96 / 147 / 195 / 199 / 253、17 / 47 / 96 / 155、17 / 47 / 96 / 195 / 199、17 / 47 / 114、17 / 47 / 114 / 155 / 195 / 199 / 253、17 / 47 / 147 / 195 / 199 / 253、17 / 49、17 / 49 / 51、17 / 49 / 51 / 96 / 195 / 253、17 / 49 / 51 / 155 / 253、17 / 49 / 51 / 155 / 253 / 260、17 / 49 / 51 / 155 / 260、17 / 49 / 51 / 253、17 / 49 / 96、17 / 49 / 96 / 155 / 253、17 / 49 / 96 / 195、17 / 49 / 260、17 / 51、17 / 51 / 96 / 114、17 / 51 / 96 / 155 / 178、17 / 51 / 96 / 155 / 195 / 199 / 253、17 / 51 / 96 / 155 / 199、17 / 51 / 96 / 155 / 253、17 / 51 / 96 / 178 / 195 / 199、17 / 51 / 96 / 195、17 / 51 / 96 / 260、17 / 51 / 114、17 / 51 / 114 / 253、17 / 51 / 147、17 / 51 / 155 / 195、17 / 51 / 178 / 195 / 253、17 / 51 / 253、17 / 96、17 / 96 / 114 / 147 / 155、17 / 96 / 114 / 147 / 155 / 178 / 195 / 199、17 / 96 / 147、17 / 96 / 147 / 155、17 / 96 / 155 / 178、17 / 96 / 155 / 178 / 253、17 / 96 / 155 / 195、17 / 96 / 155 / 195 / 199、17 / 96 / 178、17 / 96 / 178 / 195、17 / 96 / 195、17 / 96 / 195 / 199、17 / 96 / 195 / 199 / 253、17 / 96 / 253、17 / 114、17 / 114 / 253、17 / 147 / 155 / 253、17 / 147 / 178、17 / 147 / 199、17 / 155、17 / 155 / 178、17 / 155 / 178 / 195、17 / 155 / 195、17 / 155 / 195 / 253、17 / 155 / 199、17 / 155 / 253、17 / 178、17 / 178 / 195 / 253、17 / 195、17 / 195 / 199、17 / 195 / 199 / 253、17 / 195 / 253、17 / 199、17 / 253、17 / 260、21 / 49 / 51、21 / 49 / 96 / 195、21 / 49 / 96 / 253、21 / 49 / 195、21 / 51 / 96 / 155、21 / 96、21 / 96 / 195、21 / 96 / 253、44 / 46 / 47 / 51 / 114 / 178 / 253、44 / 46 / 47 / 51 / 155 / 178 / 253、44 / 46 / 47 / 96 / 155 / 195、44 / 46 / 51 / 96 / 147 / 178 / 195 / 199、44 / 46 / 51 / 96 / 155 / 195 / 199 / 253、44 / 46 / 51 / 96 / 195、44 / 46 / 51 / 147 / 155 / 178 / 195 / 253、44 / 47 / 51 / 96 / 114、44 / 47 / 51 / 96 / 177 / 178 / 199 / 253、44 / 47 / 51 / 114 / 178 / 253、44 / 47 / 96 / 114、44 / 47 / 96 / 195 / 253、44 / 47 / 147 / 155、44 / 47 / 147 / 155 / 199、44 / 96 / 155 / 178、46 / 47 / 51 / 96 / 114 / 178、46 / 47 / 51 / 114 / 147 / 195 / 199、46 / 47 / 51 / 155 / 195 / 199、46 / 47 / 96 / 114 / 195 / 199、46 / 47 / 96 / 155 / 178 / 195 / 253、46 / 47 / 155 / 195 / 199、46 / 114 / 147 / 155 / 178 / 195 / 199、46 / 114 / 195 / 199 / 253、47 / 51 / 96、47 / 51 / 96 / 114、47 / 51 / 96 / 147 / 155 / 195 / 199、47 / 51 / 96 / 147 / 195 / 199、47 / 51 / 96 / 178 / 195 / 199 / 253、47 / 51 / 96 / 195、47 / 51 / 96 / 195 / 199 / 253、47 / 51 / 114 / 253、47 / 51 / 155 / 178 / 195、47 / 51 / 155 / 195 / 253、47 / 96 / 114、47 / 96 / 114 / 253、47 / 96 / 155 / 178 / 195、47 / 96 / 155 / 195、47 / 96 / 155 / 195 / 199、47 / 96 / 178 / 195 / 253、47 / 96 / 178 / 253、47 / 96 / 195 / 199 / 253、47 / 96 / 195 / 253、47 / 114、47 / 114 / 155 / 195 / 199、47 / 114 / 178、47 / 147 / 195、47 / 155 / 253、47 / 178 / 195、47 / 178 / 195 / 199、47 / 195 / 199、49 / 51 / 96、49 / 96、49 / 96 / 253、51 / 96、51 / 96 / 114 / 155 / 195 / 199 / 253、51 / 96 / 155 / 178、51 / 96 / 155 / 178 / 195 / 253、51 / 96 / 155 / 195、51 / 96 / 155 / 195 / 253、51 / 96 / 195 / 253、51 / 96 / 199、51 / 96 / 253、51 / 114 / 155 / 195 / 199、51 / 114 / 195 / 199、51 / 147 / 253、51 / 155 / 195 / 199、51 / 195、51 / 253、96、96 / 114 / 147 / 155 / 195 / 199、96 / 114 / 155 / 178 / 195 / 199 / 253、96 / 114 / 155 / 195 / 253、96 / 114 / 199、96 / 147、96 / 147 / 155 / 178 / 1 95 / 253, 96 / 147 / 178 / 195 / 199, 96 / 147 / 195, 96 / 147 / 195 / 199, 96 / 147 / 253, 96 / 155 / 178 / 199, 96 / 155 / 195, 96 / 155 / 199, 96 / 155 / 253, 96 / 178 / 195, 96 / 178 / 195 / 199, 96 / 195, 96 / 195, 96 / 195 / 253 195 / 199、96 / 195 / 253、96 / 199、96 / 199 / 253、96 / 253、114、114 / 147 / 155 / 253、114 / 178 / 195 / 199 / 253、114 / 195、114 / 195 / 199 / 253、132、147 / 155、149、154、155 / 178、155 / 178 / 19 5 / 199, 155 / 195, 155 / 195 / 199, 155 / 195 / 199 / 253, 155 / 195 / 253, 155 / 253, 157, 167, 170, 178, 178 / 195, 178 / 199, 195, 196, 198, 214, 218, 251, 252, 256, 257, 259 and 260, wherein the positions are numbered with reference to SEQ ID NO:34. In some additional embodiments, the engineered polypeptide comprises at least one substitution or a set of substitutions selected from the group consisting of: 17T, 17T / 21R, 17T / 21R / 42T, 17T / 21R / 42T / 155R / 260G, 17T / 21R / 49A, 17T / 21R / 49A / 51L, 17T / 21R / 49A / 51L / 96S, 17T / 21R / 49A / 51L / 260G, 17T / 21R / 49A / 96S / 155R / 260G, 17T / 21R / 49A / 260G, 1 7T / 21R / 51L / 96S, 17T / 21R / 51L / 195N, 17T / 21R / 96S, 17T / 21R / 96S / 155R, 17T / 21R / 96S / 195N / 260G, 17T / 21R / 96S / 253 L, 17T / 21R / 147T / 155R / 253L, 17T / 21R / 155R / 253L, 17T / 21R / 195N, 17T / 21R / 253L, 17T / 42T, 17T / 42T / 49A / 195N / 260G,17T / 42T / 96S、17T / 44N、17T / 44N / 47A / 51A / 96A、17T / 44N / 47A / 195N / 199R、17T / 44N / 47L / 51A / 96A / 114A / 147T / 195G / 199R / 253G、17T / 44N / 47L / 51A / 96S / 114A / 147T / 199R、17T / 44N / 47L / 51A / 96T / 114A / 195G / 199R、17T / 44N / 47S / 51A / 96T / 147T / 253G、17T / 44N / 47S / 51A / 178S / 195N、17T / 44N / 47S / 195G / 199R、17T / 44N / 51A / 114A / 195N / 253G、17T / 46W / 47A / 96C / 195G、17T / 46W / 47A / 178S、17T / 46W / 47L / 51A / 96C / 195G / 253R、17T / 46W / 47L / 51A / 96T / 147T、17T / 46W / 47L / 51A / 155Q、17T / 46W / 47L / 51A / 155R / 195G / 199R、17T / 46W / 96C / 253G、17T / 47A / 51A、17T / 47A / 51A / 96T / 114A / 147T / 195G、17T / 47A / 51A / 96T / 114A / 155R / 178S / 253R、17T / 47A / 96A / 155Q、17T / 47A / 96T / 147T / 195G / 199R / 253G、17T / 47A / 147T / 195G / 199R / 253S、17T / 47L / 51A / 96T / 195G、17T / 47L / 51A / 96T / 195G / 199R、17T / 47L / 96A / 114A / 155Q / 253G、17T / 47L / 114A、17T / 47L / 114A / 155R / 195G / 199R / 253S、17T / 47S、17T / 47S / 96C / 147T / 155Q / 195G / 199R、17T / 47S / 96C / 195N / 199R、17T / 49A、17T / 49A / 51L、17T / 49A / 51L / 96S / 195N / 253L、17T / 49A / 51L / 155R / 253L、17T / 49A / 51L / 155R / 253L / 260G、17T / 49A / 51L / 155R / 260G、17T / 49A / 51L / 253L、17T / 49A / 96S、17T / 49A / 96S / 155R / 253L、17T / 49A / 96S / 195N、17T / 49A / 260G、17T / 51A / 96C / 155Q / 195G / 199R / 253G、17T / 51A / 96C / 155R / 178S、17T / 51A / 96C / 195N、17T / 51A / 96S / 114A、17T / 51A / 96S / 155Q / 199R、17T / 51A / 96T / 155R / 195G / 199R / 253S、17T / 51A / 96T / 178S / 195G / 199R、17T / 51A / 114A、17T / 51A / 114A / 253R、17T / 51A / 147T、17T / 51A / 155R / 195G、17T / 51A / 178S / 195G / 253G、17T / 51L、17T / 51L / 96S / 155R / 253L、17T / 51L / 96S / 260G、17T / 51L / 253L、17T / 96A / 147T / 155R、17T / 96A / 155Q / 195G / 199R、17T / 96A / 155R / 178S / 253G、17T / 96A / 195N / 199R、17T / 96C、17T / 96C / 114A / 147T / 155Q、17T / 96C / 178S / 195G、17T / 96C / 195G / 199R、17T / 96S / 114A / 147T / 155Q / 178S / 195G / 199R、17T / 96S / 155R / 178S、17T / 96S / 155R / 195N、17T / 96S / 178S、17T / 96S / 195N、17T / 96S / 253L、17T / 96T、17T / 96T / 147T、17T / 96T / 195G / 199R、17T / 96T / 195G / 199R / 253R、17T / 96T / 253R、17T / 114A、17T / 114A / 253R、17T / 147T / 155Q / 253G、17T / 147T / 178S、17T / 147T / 199R、17T / 155Q、17T / 155Q / 178S、17T / 155Q / 195G / 253S、17T / 155Q / 199R、17T / 155R、17T / 155R / 178S、17T / 155R / 178S / 195G、17T / 155R / 195G、17T / 155R / 253G、17T / 155R / 253L、17T / 178S、17T / 178S / 195N / 253S、17T / 195G、17T / 195G / 199R、17T / 195G / 199R / 253S、17T / 195N、17T / 195N / 199R、17T / 195N / 253L、17T / 199R、17T / 253L、17T / 260G、21R / 49A / 51L、21R / 49A / 96S / 195N、21R / 49A / 96S / 253L、21R / 49A / 195N、21R / 51L / 96S / 155R、21R / 96S、21R / 96S / 195N、21R / 96S / 253L、44N / 46W / 47A / 51A / 155R / 178S / 253G、44N / 46W / 47L / 51A / 114A / 178S / 253G、44N / 46W / 47L / 96C / 155R / 195G、44N / 46W / 51A / 96A / 147T / 178S / 195G / 199R、44N / 46W / 51A / 96T / 155R / 195G / 199R / 253G、44N / 46W / 51A / 96T / 195G、44N / 46W / 51A / 147T / 155R / 178S / 195G / 253G、44N / 47A / 51A / 96T / 177T / 178S / 199R / 253S、44N / 47L / 96T / 114A、44N / 47S / 51A / 96S / 114A、44N / 47S / 51A / 114A / 178S / 253S、44N / 47S / 96T / 195N / 253G、44N / 47S / 147T / 155Q、44N / 47S / 147T / 155Q / 199R、44N / 96C / 155R / 178S、46W / 47A / 51A / 114A / 147T / 195G / 199R、46W / 47A / 51A / 155R / 195G / 199R、46W / 47L / 51A / 96T / 114A / 178S、46W / 47L / 96C / 155R / 178S / 195G / 253S、46W / 47L / 96T / 114A / 195N / 199R、46W / 47L / 155R / 195N / 199R、46W / 114A / 147T / 155Q / 178S / 195G / 199R、46W / 114A / 195G / 199R / 253S、47A / 51A / 96C / 178S / 195G / 199R / 253G、47A / 51A / 96T / 195G、47A / 51A / 155R / 195G / 253G、47A / 96S / 114A、47A / 96S / 195N / 253S、47A / 96T / 155Q / 178S / 195G、47A / 96T / 155Q / 195N / 199R、47A / 96T / 178S / 195N / 253R、47A / 114A、47A / 147T / 195G、47A / 178S / 195N / 199R、47L / 51A / 96C / 147T / 195G / 199R、47L / 51A / 96C / 195G / 199R / 253G、47L / 51A / 96S / 147T / 155Q / 195G / 199R、47L / 51A / 155Q / 195G / 253S、47L / 96A / 195G / 199R / 253S、47L / 96T / 114A / 253R、47 L / 96T / 178S / 253G、47L / 114A / 155R / 195G / 199R、47L / 114A / 178S、47L / 195N / 199R、47S / 51A / 96A、47S / 51A / 96C / 114A、47S / 51A / 96C / 195G、47S / 51A / 114 A / 253S、47S / 51A / 155R / 178S / 195N、47S / 96S / 155R / 195G、47S / 96T / 195G / 2 53S、47S / 155R / 253G、47S / 178S / 195G、49A / 51L / 96S、49A / 96S、49A / 96S / 253 L、51A / 96C / 114A / 155Q / 195G / 199R / 253G、51A / 96C / 155Q / 195G / 253G、51A / 96C / 155R / 178S、51A / 96C / 155R / 178S / 195G / 253R、51A / 96S / 155Q / 195G、51A / 96S / 199R、51A / 96T / 195G / 253G、51A / 96T / 199R、51A / 96T / 253R、51A / 114A / 155Q / 195G / 199R、51A / 114A / 195G / 199R、51A / 147T / 253S、51A / 155Q / 195N / 199R、51A / 195G、51A / 195N、51A / 253G、51L / 96S、51L / 195N、96A、96A / 114A / 155R / 178S / 195G / 199R / 253G、96A / 147T、96A / 147T / 155R / 178S / 195G / 253G、 96A / 147T / 195G、96A / 147T / 195G / 199R、96A / 147T / 253R、96A / 195G、96A / 19 5G / 199R、96C、96C / 114A / 147T / 155R / 195G / 199R、96C / 147T / 195G / 199R、96C / 155R / 195G、96C / 178S / 195G / 199R、96C / 195G / 199R、96C / 199R、96C / 253S、96G、96K、96N、96S、96S / 114A / 155Q / 195N / 253S、96S / 147T、96S / 155R / 253L、96S / 195N, 96S / 195N / 253L, 96S / 253G, 96S / 253S, 96T, 96T / 114A / 199R, 96T / 147T / 178S / 195N / 199R , 96T / 155Q / 178S / 199R, 96T / 155Q / 199R, 96T / 178S / 195G, 96T / 178S / 195N, 96T / 195G, 96T / 195G / 25 3G, 96T / 195G / 253R, 96T / 195N / 199R, 96T / 199R, 96T / 199R / 253S, 96T / 253G, 114A, 114A / 147T / 155Q / 253S, 114A / 178S / 195G / 199R / 253S, 114A / 195G, 114A / 195G / 199R / 253G, 132L, 147T / 155R, 149D, 1 49E, 154E, 155Q / 178S, 155Q / 178S / 195G / 199R, 155Q / 178S / 195N / 199R, 155Q / 195G / 253G, 155Q / 195 N, 155Q / 195N / 199R, 155R / 195G / 199R / 253G, 155R / 195N / 199R / 253R, 155R / 195N / 253L, 155R / 253S, 157N, 167G, 167Q, 170Q, 178A, 178G, 178K, 178S, 178S / 195N, 178S / 199R, 195G, 195N, 195R, 195S, 196G, 196H, 198H, 214G, 218P, 251A, 251L, 252A, 252T, 256I, 257E, 257W, 259D, 259E and 260E, wherein the positions are numbered with reference to SEQ ID NO:34. In some additional embodiments, the engineered polypeptide comprises at least one substitution or a set of substitutions selected from the group consisting of S17T, S17T / K21R, S17T / K21R / K42T, S17T / K21R / K42T / N155R / R260G, S17T / K21R / K49A, S17T / K21R / K49A / E51L, S17T / K21R / K49A / E51L / E96S, S 17T / K21R / K49A / E51L / R260G, S17T / K21R / K49A / E96S / N155R / R260G, S17T / K21R / K49A / R260G , S17T / K21R / E51L / E96S, S17T / K21R / E51L / I195N, S17T / K21R / E96S, S17T / K21R / E96S / N155R,S17T / K21R / E96S / I195N / R260G、S17T / K21R / E96S / Y253L、S17T / K21R / I147T / N155R / Y253L、S17T / K21R / N155R / Y253L、S17T / K21R / I195N、S17T / K21R / Y253L、S17T / K42T、S17T / K42T / K49A / I195N / R260G、S17T / K42T / E96S、S17T / D44N、S17T / D44N / E47A / E51A / E96A、S17T / D44N / E47A / I195N / K199R、S17T / D44N / E47L / E51A / E96A / G114A / I147T / I195G / K199R / Y253G、S17T / D44N / E47L / E51A / E96S / G114A / I147T / K199R、S17T / D44N / E47L / E51A / E96T / G114A / I195G / K199R、S17T / D44N / E47S / E51A / E96T / I147T / Y253G、S17T / D44N / E47S / E51A / P178S / I195N、S17T / D44N / E47S / I195G / K199R、S17T / D44N / E51A / G114A / I195N / Y253G、S17T / N46W / E47A / E96C / I195G、S17T / N46W / E47A / P178S、S17T / N46W / E47L / E51A / E96C / I195G / Y253R、S17T / N46W / E47L / E51A / E96T / I147T、S17T / N46W / E47L / E51A / N155Q、S17T / N46W / E47L / E51A / N155R / I195G / K199R、S17T / N46W / E96C / Y253G、S17T / E47A / E51A、S17T / E47A / E51A / E96T / G114A / I147T / I195G、S17T / E47A / E51A / E96T / G114A / N155R / P178S / Y253R、S17T / E47A / E96A / N155Q、S17T / E47A / E96T / I147T / I195G / K199R / Y253G、S17T / E47A / I147T / I195G / K199R / Y253S、S17T / E47L / E51A / E96T / I195G、S17T / E47L / E51A / E96T / I195G / K199R、S17T / E47L / E96A / G114A / N155Q / Y253G、<h2 style=";text-align:left;direction:ltr">S17T / E47L / G114A、S17T / E47L / G114A / N155R / I195G / K199R / Y253S、S17T / E47S、S17T / E47S / E96C / I147T / N155Q / I195G / K199R、S17T / E47S / E96C / I19 5N / K199R、S17T / K49A、S17T / K49A / E51L、S17T / K49A / E51L / E96S / I195N / Y253L、S17T / K49A / E51L / N155R / Y253L、S17T / K49A / E51L / N155R / Y253L / R260 G、S17T / K49A / E51L / N155R / R260G、S17T / K49A / E51L / Y253L、S17T / K49A / E96S、S17T / K49A / E96S / N155R / Y253L、S17T / K49A / E96S / I195N、S17T / K49A / R260G、S17T / E51A / E96C / N155Q / I195G / K199R / Y253G、S17T / E51A / E96C / N1 55R / P178S、S17T / E51A / E96C / I195N、S17T / E51A / E96S / G114A、S17T / E51A / E96S / N155Q / K199R、S17T / E51A / E96T / N155R / I195G / K199R / Y253S、S17T / E51A / E96T / P178S / I195G / K199R、S17T / E51A / G114A、S17T / E51A / G114A / Y2 53R、S17T / E51A / I147T、S17T / E51A / N155R / I195G、S17T / E51A / P178S / I195 G / Y253G、S17T / E51L、S17T / E51L / E96S / N155R / Y253L、S17T / E51L / E96S / R2 60G、S17T / E51L / Y253L、S17T / E96A / I147T / N155R、S17T / E96A / N155Q / I195 G / K199R、S17T / E96A / N155R / P178S / Y253G、S17T / E96A / I195N / K199R、S17T / E96C、S17T / E96C / G114A / I147T / N155Q、S17T / E96C / P178S / I195G、S17T / E 96C / I195G / K199R、S17T / E96S / G114A / I147T / N155Q / P178S / I195G / K199R、S17T / E96S / N155R / P178S、S17T / E96S / N155R / I195N、S17T / E96S / P178S、S17T / E96S / I195N、S17T / E96S / Y253L、S17T / E96T、S17T / E96T / I147T、S17T / E96T / I195G / K199R、S17T / E96T / I195G / K199R / Y253R、S17T / E96T / Y253R、S17T / G114A、S17T / G114A / Y253R、S17T / I147T / N155Q / Y253G、S17T / I147T / P178S、S17T / I147T / K199R、S17T / N155Q、S17T / N155Q / P178S、S17T / N155Q / I195G / Y253S、S17T / N155Q / K199R、S17T / N155R、S17T / N155R / P178S、S17T / N155R / P178S / I195G、S17T / N155R / I195G、S17T / N155R / Y253G、S17T / N155R / Y253L、S17T / P178S、S17T / P178S / I195N / Y253S、S17T / I195G、S17T / I195G / K199R、S17T / I195G / K199R / Y253S、S17T / I195N、S17T / I195N / K199R、S17T / I195N / Y253L、S17T / K199R、S17T / Y253L、S17T / R260G、K21R / K49A / E51L、K21R / K49A / E96S / I195N、K21R / K49A / E96S / Y253L、K21R / K49A / I195N、K21R / E51L / E96S / N155R、K21R / E96S、K21R / E96S / I195N、K21R / E96S / Y253L、D44N / N46W / E47A / E51A / N155R / P178S / Y253G、D44N / N46W / E47L / E51A / G114A / P178S / Y253G、D44N / N46W / E47L / E96C / N155R / I195G、D44N / N46W / E51A / E96A / I147T / P178S / I195G / K199R、D44N / N46W / E51A / E96T / N155R / I195G / K199R / Y253G、D44N / N46W / E51A / E96T / I195G、D44N / N46W / E51A / I147T / N155R / P178S / I195G / Y253G、D44N / E47A / E51A / E96T / A177T / P178S / K199R / Y253S、D44N / E47L / E96T / G114A、D44N / E47S / E51A / E96S / G114A、D44N / E47S / E51A / G114A / P178S / Y253S、D44N / E47S / E96T / I195N / Y253G、D44N / E47S / I147T / N155Q、D44N / E47S / I147T / N155Q / K199R、D44N / E96C / N155R / P178S、N46W / E47A / E51A / G114A / I147T / I195G / K199R、N46W / E47A / E51A / N155R / I195G / K199R、N46W / E47L / E51A / E96T / G114A / P178S、N46W / E47L / E96C / N155R / P178S / I195G / Y253S、N46W / E47L / E96T / G114A / I195N / K199R、N46W / E47L / N155R / I195N / K199R、N46W / G114A / I147T / N155Q / P178S / I195G / K199R、N46W / G114A / I195G / K199R / Y253S、E47A / E51A / E96C / P178S / I195G / K199R / Y253G、E47A / E51A / E96T / I195G、E47A / E51A / N155R / I195G / Y253G、E47A / E96S / G114A、E47A / E96S / I195N / Y253S、E47A / E96T / N155Q / P178S / I195G、E47A / E96T / N155Q / I195N / K199R、E47A / E96T / P178S / I195N / Y253R、E47A / G114A、E47A / I147T / I195G、E47A / P178S / I195N / K199R、E47L / E51A / E96C / I147T / I195G / K199R、E47L / E51A / E96C / I195G / K199R / Y253G、E47L / E51A / E96S / I147T / N155Q / I195G / K199R、E47L / E51A / N155Q / I195G / Y253S、E47L / E96A / I195G / K199R / Y253S、E47L / E96T / G114A / Y253R、E47L / E96T / P178S / Y253G、E47L / G114A / N155R / I195G / K199R、E47L / G114A / P178S、E47L / I195N / K199R、E47S / E51A / E96A、E47S / E51A / E96C / G114A、E47S / E51A / E96C / I195G、E47S / E51A / G114A / Y253S、E47S / E51A / N155R / P178S / I195N、E47S / E96S / N155R / I195G、E47S / E96T / I195G / Y253S、E47S / N155R / Y253G、E47S / P178S / I195G、K49A / E51L / E96S、K49A / E96S、K49A / E96S / Y253L、E51A / E96C / G114A / N155Q / I195G / K199R / Y253G、E51A / E96C / N155Q / I195G / Y253G、E51A / E96C / N155R / P178S、E51A / E96C / N155R / P178S / I195G / Y253R、E51A / E96S / N155Q / I195G、E51A / E96S / K199R、E51A / E96T / I195G / Y253G、E51A / E96T / K199R、E51A / E96T / Y253R、E51A / G114A / N155Q / I195G / K199R、E51A / G114A / I195G / K199R、E51A / I147T / Y253S、E51A / N155Q / I195N / K199R、E51A / I195G、E51A / I195N、E51A / Y253G、E51L / E96S、E51L / I195N、E96A、E96A / G114A / N155R / P178S / I195G / K199R / Y253G、E96A / I147T、E96A / I147T / N155R / P178S / I195G / Y253G、E96A / I147T / I195G、E96A / I147T / I195G / K199R、E96A / I147T / Y253R、E96A / I195G、E96A / I195G / K199R、E96C、E96C / G114A / I147T / N155R / I195G / K199R、E96C / I147T / I195G / K199R、E96C / N155R / I195G、E96C / P178S / I195G / K199R、E96C / I195G / K199R、E96C / K199R、E96C / Y253S、E96G、E96K, E96N, E96S, E96S / G114A / N155Q / I195N / Y253S, E96S / I147T, E96S / N155R / Y253L, E96S / I195N, E96S / I195N / Y253L, E96S / Y2 53G, E96S / Y253S, E96T, E96T / G114A / K199R, E96T / I147T / P178S / I195N / K199R, E96T / N155Q / P178S / K199R, E96T / N155Q / K199R, E9 6T / P178S / I195G, E96T / P178S / I195N, E96T / I195G, E96T / I195G / Y253G, E96T / I195G / Y253R, E96T / I195N / K199R, E96T / K199R, E9 6T / K199R / Y253S, E96T / Y253G, G114A, G114A / I147T / N155Q / Y253S, G114A / P178S / I195G / K199R / Y253S, G114A / I195G, G114A / I195 G / K199R / Y253G, V132L, I147T / N155R, V149D, V149E, L154E, N155Q / P178S, N155Q / P178S / I195G / K199R, N155Q / P178S / I195N / K19 9R、N155Q / I195G / Y253G、N155Q / I195N、N155Q / I195N / K199R、N155R / I195G / K199R / Y253G、N155R / I195N / K199R / Y253R、N155R / I19 5N / Y253L, N155R / Y253S, H157N, L167G, L167Q, K170Q, P178A, P178G, P178K, P178S, P178S / I195N, P178S / K199R, I195G, I195N, I195R, I195S, N196G, N196H, E198H, P214G, I218P, T251A, T251L, L252A, L252T, F256I, Q257E, Q257W, G259D, G259E and R260E, wherein the positions are numbered with reference to SEQ ID NO:34. In some additional embodiments, the engineered polypeptide comprises an amino acid sequence having at least 80% sequence identity to any even-numbered sequence set forth in SEQ ID NO: 106 to SEQ ID NO: 702.

[0014] The present invention provides an engineered polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the reference sequence SEQ ID NO: 112, comprising at least one substitution or a set of substitutions at one or more positions selected from the group consisting of: 21 / 42 / 96 / 178 / 195 / 253, 21 / 42 / 96 / 195, 21 / 96, 21 / 96 / 147 / 195, 21 / 96 / 149, 21 / 96 / 155 / 195 / 253, 21 / 96 / 167, 21 / 96 / 167 / 195, 21 / 96 / 178, 21 / 96 / 179 / 195 / 251 / 253, 21 / 42 / 96 / 195 96 / 195, 21 / 147 / 149, 21 / 147 / 253, 21 / 149 / 178 / 195, 21 / 155 / 178 / 253 / 256, 21 / 178 / 195 / 256, 42 / 96 / 149 / 253 / 256, 94, 96, 96 / 114 / 155 / 198 / 218 / 253 / 256 / 257, 96 / 114 / 155 / 252 / 253 / 257, 96 / 114 / 155 / 253, 96 / 114 / 196 / 214 / 253 / 256, 96 / 114 / 214 / 253 / 256, 96 / 147, 96 / 147 / 149, 96 / 147 / 1 49 / 155、96 / 147 / 167 / 195、96 / 149 / 155 / 167 / 178 / 253、96 / 149 / 155 / 195、96 / 149 / 178 / 179 / 251 / 256、96 / 149 / 178 / 253、96 / 149 / 198 / 252 / 253 / 257、96 / 155、96 / 155 / 167、96 / 155 / 167 / 178、96 / 155 / 195、96 / 155 / 196 / 218 / 253 / 256 / 257、96 / 155 / 196 / 252 / 253、96 / 155 / 198、96 / 155 / 252 / 253、96 / 167, 96 / 178, 96 / 178 / 179 / 195, 96 / 195, 96 / 196, 96 / 196 / 198, 96 / 196 / 201 / 256, 96 / 251 / 253 / 256, 96 / 253 / 257, 96 / 256 / 257, 98, 103, 114 / 253 / 256 / 257, 147, 147 / 149, 149 / 167, 149 / 195, 149 / 218, 149 / 253, 149 / 257, 155 / 167, 155 / 178 / 195, 155 / 178 / 195 / 251 / 253, 155 / 195 / 251 / 253 / 256,155 / 195 / 253 / 256, 155 / 196 / 198 / 253, 167, 167 / 195, 167 / 195 / 251 / 253, 196, 196 / 253 / 256 / 257, 198 / 253 / 256, 198 / 256, 205, 214 / 253 / 256, 251 and 257, wherein the positions are numbered with reference to SEQ ID NO:112. In some additional embodiments, the engineered polypeptide comprises at least one substitution or a set of substitutions selected from the group consisting of 21R / 42T / 96C / 178S / 195N / 253G, 21R / 42T / 96S / 195R, 21R / 96C / 147T / 195R, 21R / 96C / 149E, 21R / 96C / 178S, 21R / 96C / 195R, 21R / 96G / 155N / 195R / 253G, 21R / 96G / 167Q / 195N, 21R / 96K / 167Q, 21R / 96S, 21R / 96C / 147T / 195R, 21R / 96C / 149E, 78S, 21R / 96S / 179N / 195N / 251A / 253G, 21R / 147T / 149E, 21R / 147T / 253G, 21R / 149E / 178S / 195N, 21R / 155N / 178S / 253G / 256I, 2 1R / 178S / 195R / 256I, 42T / 96C / 149E / 253G / 256I, 94A, 96A / 149E / 155N / 167Q / 178S / 253G, 96A / 155N / 167Q, 96C, 96C / 114G / 155N / 252A / 253G / 257W, 96C / 114G / 155N / 253R, 96C / 114G / 196G / 214G / 253G / 256I, 96C / 147T, 96C / 147T / 149E / 155C, 96C / 147T / 167 Q / 195N、96C / 149D / 198H / 252A / 253R / 257E、96C / 149E / 155N / 195N、96C / 149E / 155N / 195R、96C / 155N / 196G / 218P / 253G / 256I / 25 7E、96C / 155N / 196G / 252T / 253G、96C / 155N / 198H、96C / 155N / 252T / 253R、96C / 195N、96C / 196G / 198H、96C / 196G / 201P / 256I、96G / 149E / 178S / 253G、96G / 155N / 167Q、96G / 155N / 195R、96K / 155N、96K / 178S、96S、96S / 114G / 155N / 198H / 218P / 253R / 256I / 257E、96S / 114G / 214G / 253G / 256I, 96S / 147T / 149E, 96S / 149E / 178S / 179N / 251A / 256I, 96S / 155N, 96S / 155N / 167Q / 178S, 96S / 167Q, 96S / 178S, 96S / 178S / 179N / 195N, 96S / 195N, 96S / 196G, 96S / 251A / 253G / 256I, 96S / 253G / 257E, 96S / 256I / 257E, 98A, 1 03L, 114G / 253G / 256I / 257E, 147A, 147R, 147T, 147T / 149E, 149D / 257E, 149E / 167Q , 149E / 195N, 149E / 218P, 149E / 253G, 155N / 167Q, 155N / 178S / 195R, 155N / 178S / 1 95R / 251A / 253G, 155N / 195N / 251A / 253G / 256I, 155N / 195N / 253G / 256I, 155N / 196G / 198H / 253G, 167Q, 167Q / 195N, 167Q / 195N / 251A / 253G, 196A, 196G / 253G / 256I / 257W, 198H / 253G / 256I, 198H / 256I, 205E, 214G / 253G / 256I, 251L and 257E, where the positions are numbered with reference to SEQ ID NO:112. In some embodiments, the engineered polypeptide comprises at least one substitution or a set of substitutions selected from the group consisting of K21R / K42T / E96C / P178S / G195N / S253G, K21R / K42T / E96S / G195R, K21R / E96C / I147T / G195R, K21R / E96C / V149E, K21R / E96C / P178S, K21R / E96C / G195R, K21R / E96G / R155N / G195R / S253G, K21R / E96G / L167Q / G195N, K21R / E96K / L167Q, K21R / E96S, K21R / E96S / P178S, K21R / E96S / K179N / G195N / T251A / S253G, K21R / I147T / V149E, K21R / I147T / S253G, K 21R / V149E / P178S / G195N, K21R / R155N / P178S / S253G / F256I, K21R / P178S / G195R / F256I, K42T / E96C / V149E / S253G / F256I, G94A,E96A / V149E / R155N / L167Q / P178S / S253G、E96A / R155N / L167Q、E96C、E96C / A114G / R155N / L252A / S253G / Q257W、E96C / A114G / R155N / S253R、E96C / A114G / N196G / P214G / S253G / F256I、E96C / I147T、E96C / I147T / V149E / R155C、E96C / I147T / L167Q / G195N、E96C / V149D / E198H / L252A / S253R / Q257E、E96C / V149E / R155N / G195N、E96C / V149E / R155N / G195R、E96C / R155N / N196G / I218P / S253G / F256I / Q257E、E96C / R155N / N196G / L252T / S253G、E96C / R155N / E198H、E96C / R155N / L252T / S253R、E96C / G195N、E96C / N196G / E198H、E96C / N196G / A201P / F256I、E96G / V149E / P178S / S253G、E96G / R155N / L167Q、E96G / R155N / G195R、E96K / R155N、E96K / P178S、E96S、E96S / A114G / R155N / E198H / I218P / S253R / F256I / Q257E、E96S / A114G / P214G / S253G / F256I、E96S / I147T / V149E、E96S / V149E / P178S / K179N / T251A / F256I、E96S / R155N、E96S / R155N / L167Q / P178S、E96S / L167Q、E96S / P178S、E96S / P178S / K179N / G195N、E96S / G195N、E96S / N196G、E96S / T251A / S253G / F256I、E96S / S253G / Q257E、E96S / F256I / Q257E、P98A、E103L、A114G / S253G / F256I / Q257E、I147A、I147R、I147T、I147T / V149E、V149D / Q257E、V149E / L167Q、V149E / G195N、V149E / I218P、V149E / S253G、R155N / L167Q、R155N / P178S / G195R、R155N / P178S / G195R / T251A / S253G, R155N / G195N / T251A / S253G / F256I, R15 5N / G195N / S253G / F256I, R155N / N196G / E198H / S253G, L167Q, L167Q / G195N, L 167Q / G195N / T251A / S253G, N196A, N196G / S253G / F256I / Q257W, E198H / S253G / F256I, E198H / F256I, Q205E, P214G / S253G / F256I, T251L and Q257E, wherein the positions are numbered with reference to SEQ ID NO: 112. In some additional embodiments, the engineered polypeptide comprises an amino acid sequence having at least 80% sequence identity to any even-numbered sequence listed in SEQ ID NO: 704 to SEQ ID NO: 876.

[0015] The present invention provides an engineered polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the reference sequence SEQ ID NO: 736, comprising at least one substitution or a set of substitutions at one or more positions selected from the group consisting of: 21 / 47 / 96 / 114 / 155 / 251, 21 / 47 / 149 / 257, 21 / 47 / 155 / 178 / 257, 21 / 47 / 178, 21 / 47 / 178 / 251 / 256 / 257, 21 / 96 / 114 / 155 / 178, 21 / 114 / 155 / 251 / 257, 21 / 114 / 178 / 179, 21 / 155 / 178 / 209 / 256, 25 / 155, 25 / 178 / 256, 42 / 47 / 155 / 178, 47 / 114 / 149 / 178 / 251, 47 / 114 / 178 / 257, 47 / 149, 47 / 178, 47 / 178 / 179 / 251 / 257, 47 / 178 / 251 / 257, 96 / 114 / 155 / 198, 96 / 114 / 155 / 253 / 257, 96 / 253, 114 / 149 / 155 / 251 / 256, 114 / 149 / 178 / 256 / 257, 114 / 155 / 195 / 198 / 256, 114 / 209 / 256 / 257, 149, 149 / 256 / 257, 155, 155 / 178 / 179, 155 / 178 / 198 / 252 / 253, 155 / 195 / 198, 155 / 195 / 198 / 253 / 256 / 257, 155 / 256 / 257, 178 / 253, 178 / 256, 179 / 256 / 257, 251 and 253, wherein the positions are numbered with reference to SEQ ID NO:736.In some additional embodiments, the engineered polypeptide comprises at least one substitution or a set of substitutions selected from the group consisting of 21R / 47A / 96A / 114G / 155N / 251A, 21R / 47A / 149E / 257E, 21R / 47A / 155N / 178S / 257E, 21R / 47A / 178S, 21R / 47A / 178S / 251A / 256I / 257W, 21R / 96A / 114G / 155N / 178S, 21R / 114G / 155N / 251A / 257E, 21R / 114G / 178S / 179N, 21R / 155N / 178S / 209L / 256I, 25L / 155N, 25L / 178S / 256I, 42T / 47A / 155N / 178S, 47A / 114G / 149E / 178S / 251A, 47A / 114G / 178S / 257E, 47A / 149E, 47A / 178S, 47A / 178S / 179N / 251A / 257E、47A / 178S / 251A / 257E、96T / 114G / 155N / 198L、96T / 114G / 155N / 253G / 257W、96T / 253G、114G / 149E / 15 5N / 251A / 256I, 114G / 149E / 178S / 256I / 257E, 114G / 155N / 195N / 198L / 256I, 114G / 209L / 256I / 257E, 149E, 1 49E / 256I / 257E, 155N, 155N / 178S / 179N, 155N / 178S / 198H / 252T / 253G, 155N / 195N / 198H / 253G / 256I / 257W, 155N / 195N / 198L, 155N / 256I / 257E, 178S / 253G, 178S / 256I, 179N / 256I / 257E, 251I, 251L and 253G, wherein the positions are numbered with reference to SEQ ID NO:736.In some embodiments, the engineered polypeptide comprises at least one substitution or a set of substitutions selected from the group consisting of K21R / L47A / E96A / A114G / R155N / T251A, K21R / L47A / V149E / Q257E, K21R / L47A / R155N / P178S / Q257E, K21R / L47A / P178S, K21R / L47A / P178S / T251A / F256I / Q257W, K21R / E96A / A114G / R155N / T251A / Q257E, K21R / A114G / P178S / K179N, K21R / R155N / P178S / V209L / F256I, I25L / R155N, I25L / P178S / F256I, K42T / L47A / R155 N / P178S, L47A / A114G / V149E / P178S / T251A, L47A / A114G / P178S / Q257E, L47A / V149E, L47A / P178S, L47A / P178S / K179N / T251A / Q 257E, L47A / P178S / T251A / Q257E, E96T / A114G / R155N / E198L, E96T / A114G / R155N / S253G / Q257W, E96T / S253G, A114G / V149E / R15 5N / T251A / F256I, A114G / V149E / P178S / F256I / Q257E, A114G / R155N / G195N / E198L / F256I, A114G / V209L / F256I / Q257E, V149E, V 149E / F256I / Q257E, R155N, R155N / P178S / K179N, R155N / P178S / E198H / L252T / S253G, R155N / G195N / E198H / S253G / F256I / Q257W, R155N / G195N / E198L, R155N / F256I / Q257E, P178S / S253G, P178S / F256I, K179N / F256I / Q257E, T251I, T251L and S253G, wherein the positions are numbered with reference to SEQ ID NO:736. In some additional embodiments, the engineered polypeptide comprises an amino acid sequence having at least 80% sequence identity to any even-numbered sequence set forth in SEQ ID NO:878 to SEQ ID NO:954.

[0016] The present invention provides an engineered polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the reference sequence SEQ ID NO: 908, comprising at least one substitution or a set of substitutions at one or more positions selected from the group consisting of: 21 / 42 / 149, 21 / 42 / 149 / 214 / 253, 21 / 42 / 179 / 214 / 244 / 253, 21 / 42 / 253 / 260 / 261, 21 / 149, 21 / 149 / 179, 21 / 149 / 179 / 253, 21 / 149 / 179 / 253 / 260, 21 / 149 / 179 / 253 / 260 / 261, 21 / 149 / 179 / 261, 21 / 149 / 260 / 261, 21 / 149 / 261, 21 / 179 / 214 / 253, 21 / 179 / 253, 21 / 179 / 261, 21 / 21 4 / 253、21 / 253、40、42 / 149 / 179 / 253 / 260、42 / 149 / 179 / 260 / 261、42 / 149 / 214 / 253、42 / 179 / 253 / 261、42 / 253、104、111、149、149 / 179、149 / 179 / 253、149 / 179 / 253 / 260 / 261、149 / 179 / 260、149 / 179 / 260 / 261, 149 / 179 / 261, 149 / 253, 149 / 261, 160, 178, 179 / 214 / 253, 179 / 253, 179 / 253 / 260, 179 / 253 / 260 / 261, 179 / 253 / 261, 179 / 260 / 261, 179 / 261, 253, 253 / 260 / 261, 253 / 261 and 255, wherein the positions are numbered with reference to SEQ ID NO:908.In some additional embodiments, the engineered polypeptide comprises at least one substitution or a set of substitutions selected from the group consisting of 21K / 42T / 149E, 21K / 42T / 149E / 214A / 253G, 21K / 42T / 179R / 214A / 244F / 253G, 21K / 42T / 253G / 260Q / 261E, 21K / 149E, 21K / 149E / 179N, 21K / 149E / 179N / 253G, 21K / 149E / 179N / 261E, 21K / 149E / 179R, 21 K / 149E / 179R / 253G / 260Q / 261E, 21K / 149E / 179R / 253R / 260Q, 21K / 149E / 260Q / 261E, 21K / 149E / 261E, 21K / 179N / 253G, 21K / 179R / 214A / 253G, 21K / 179R / 253G, 21K / 179R / 261E, 21K / 214A / 253G, 21K / 253G, 40T, 42T / 149E / 179N / 260Q / 261E, 42T / 14 9E / 179R / 253G / 260Q, 42T / 149E / 214A / 253G, 42T / 179N / 253G / 261E, 42T / 253G, 104L, 111L, 149E, 149E / 179N / 253G, 149E / 1 79N / 253G / 260Q / 261E, 149E / 179R, 149E / 179R / 253G, 149E / 179R / 260Q, 149E / 179R / 260Q / 261E, 149E / 179R / 261E, 149E / 25 3G, 149E / 253R, 149E / 261E, 160S, 178L, 179N / 253G, 179N / 253G / 260Q, 179R / 214A / 253G, 179R / 253G, 179R / 253G / 260Q, 179R / 253G / 260Q / 261E, 179R / 253G / 261E, 179R / 260Q / 261E, 179R / 261E, 253G, 253G / 260Q / 261E, 253G / 261E and 255P, wherein the positions are numbered with reference to SEQ ID NO:908.In some embodiments, the engineered polypeptide comprises at least one substitution or a set of substitutions selected from the group consisting of: R21K / K42T / V149E, R21K / K42T / V149E / P214A / S253G, R21K / K42T / K179R / P214A / L244F / S253G, R21K / K42T / S253G / R260Q / G261E, R21K / V149E, R21K / V149E / K179N, R21K / V149E / K179N / S253G, R21K / V149E / K179R, R21K / V149 E / K179R / S253G / R260Q / G261E, R21K / V149E / K179R / S253R / R260Q, R21K / V149E / R260Q / G261E, R21K / V149E / G261E, R21K / K179N / S253G, R21K / K17 9R / P214A / S253G, R21K / K179R / S253G, R21K / K179R / G261E, R21K / P214A / S253G, R21K / S253G, S40T, K42T / V149E / K179N / R260Q / G261E, K42T / V149E / K179R / S253G / R260Q, K42T / V149E / P214A / S253G, K42T / K179N / S253G / G 261E, K42T / S253G, M104L, K111L, V149E, V149E / K179N / S253G, V149E / K1 79N / S253G / R260Q / G261E, V149E / K179R, V149E / K179R / S253G, V149E / K1 79R / R260Q, V149E / K179R / R260Q / G261E, V149E / K179R / G261E, V149E / S2 53G, V149E / S253R, V149E / G261E, A160S, S178L, K179N / S253G, K179N / S253G / R260Q, K179R / P214A / S253G, K179R / S253G, K179R / S253G / R260Q, K179R / S253G / R260Q / G261E, K179R / S253G / G261E, K179R / R260Q / G261E, K179R / G261E, S253G, S253G / R260Q / G261E, S253G / G261E and S255P, wherein the positions are numbered with reference to SEQ ID NO:908.In some additional embodiments, the engineered polypeptide comprises an amino acid sequence having at least 80% sequence identity to any even-numbered sequence set forth in SEQ ID NO:956 to SEQ ID NO:1060.

[0017] The present invention provides an engineered polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the reference sequence SEQ ID NO: 956, comprising at least one substitution or a set of substitutions at one or more positions selected from the group consisting of: 3, 21 / 104 / 160 / 178 / 179, 21 / 104 / 160 / 255 / 258, 21 / 104 / 178 / 179 / 255, 21 / 104 / 178 / 179 / 255. 4 / 178 / 255 / 258, 21 / 104 / 255, 21 / 104 / 255 / 258, 21 / 178 / 179 / 255 / 260, 21 / 255 / 260, 31, 32, 40, 40 / 104 / 178 / 179, 40 / 160 / 178, 46 / 82 / 259, 103, 104 / 149 / 255 / 258 / 260, 104 / 149 / 258, 104 / 160, 104 / 160 / 178, 104 / 160 / 178 / 179 / 255, 104 / 160 / 178 / 179 / 260, 104 / 160 / 178 / 255 / 260, 104 / 160 / 179, 104 / 178 / 179 / 255 / 258, 104 / 178 / 255 / 258 / 260, 104 / 178 / 255 / 260, 104 / 179, 104 / 255 / 260, 104 / 25 8 / 260, 111 / 149 / 255 / 258, 111 / 149 / 255 / 260, 111 / 160 / 178 / 179 / 255 / 258 / 260, 111 / 160 / 255 / 260, 154, 156, 160, 160 / 178 / 179, 174, 194, 197, 205, 205 / 229, 222, 253, 257, 258 / 260 and 260, wherein the positions are numbered with reference to SEQ ID NO:956.In some additional embodiments, the engineered polypeptide comprises at least one substitution or a set of substitutions selected from the group consisting of: 3C, 3T, 21K / 104L / 160S / 178L / 179R, 21K / 104L / 160S / 255A / 258P, 21K / 104L / 178R / 179R / 255F, 21K / 104L / 178R / 255F / 258P, 21K / 104L / 255A, 21K / 104L / 255F / 258P, 21K / 178R / 179R / 255A / 260Q、21K / 255A / 260Q、31H、32M、40K、40T / 104L / 178R / 179R、40T / 160S / 178L、46I / 82Q / 259N、103K、104L / 149E / 25 5F / 258P / 260Q, 104L / 149E / 258P, 104L / 160S, 104L / 160S / 178A / 255A / 260Q, 104L / 160S / 178K / 179R / 260Q, 104L / 160 S / 178L, 104L / 160S / 178R / 179R / 255A, 104L / 160S / 179R, 104L / 178A / 179R / 255A / 258P, 104L / 178K / 255F / 260Q, 104 L / 178L / 255F / 258P / 260Q, 104L / 179R, 104L / 255F / 260Q, 104L / 258P / 260Q, 111E / 149E / 255F / 258P, 111E / 149E / 255 F / 260Q, 111I / 160S / 255F / 260Q, 111L / 160S / 178A / 179R / 255A / 258P / 260Q, 154H, 154K, 154Q, 154W, 156A, 156T, 160S, 160S / 178L / 179R, 174A, 194L, 197K, 205I / 229S, 205L, 205M, 205T, 222F, 253Y, 257R, 258P / 260Q and 260Q, wherein the positions are numbered with reference to SEQ ID NO:956.In some embodiments, the engineered polypeptide comprises at least one substitution or a set of substitutions selected from the group consisting of P3C, P3T, R21K / M104L / A160S / S178L / N179R, R21K / M104L / A160S / S255A / A258P, R21K / M104L / S178R / N179R / S255F, R21K / M104L / S178R / S255F / A258P, R21K / M104L / S255A, R21K / M104L / S255F / A258P, R21K / M104L / S255A , R21K / S255A / R260Q, Q31H, A32M, S40K, S40T / M104L / S178R / N179R, S40T / A160S / S178L, N46I / E82Q / G259N, E103K, M104L / V149E / S255F / A258P / R260Q, M104L / V149E / A258P, M104L / A160S, M104L / A160S / S178A / S255A / R260Q, M104L / A160S / S178K / N179R / R260Q, M104L / A160S / S178L, M104L / A160S / S178R / N179R / S255A, M104L / A160S / N179R, M104L / S178A / N179R / S255A / A258P, M104L / S178K / S255F / R260Q, M104L / S178L / S255F / A258P / R260Q, M104L / N179R, M104L / S255F / R260Q, M104L / A258P / R260Q, K111E / V149E / S255F / A258P, K111E / V149E / S255 F / R260Q, K111I / A160S / S255F / R260Q, K111L / A160S / S178A / N179R / S255A / A258P / R260Q, L154H, L154K, L154Q, L154W, V156A, V156T, A160S, A160S / S178L / N179R, L174A, T194L, A197K, Q205I / A229S, Q205L, Q205M, Q205T, E222F, G253Y, E257R, A258P / R260Q and R260Q, wherein the positions are numbered with reference to SEQ ID NO:956. In some additional embodiments, the engineered polypeptide comprises an amino acid sequence having at least 80% sequence identity to any even-numbered sequence set forth in SEQ ID NO:1062 to SEQ ID NO:1170.

[0018] The present invention provides an engineered polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the reference sequence SEQ ID NO: 1062, comprising at least one substitution or a set of substitutions at one or more positions selected from the group consisting of: 3 / 31 / 154 / 205 / 257, 3 / 31 / 205, 3 / 154 / 205, 31 / 55 / 154 / 205, 31 / 55 / 205, 31 / 154 / 205, 31 / 154 / 205 / 257, 31 / 205, 31 / 205 / 205, 40 / 55 / 205, 40 / 154 / 205 / 257. 05, 40 / 154 / 205 / 257, 40 / 174 / 205 / 229, 40 / 205 / 229, 54 / 205 / 253, 63, 63 / 149 / 154 / 174 / 178 / 253, 63 / 149 / 154 / 174 / 205 / 253, 63 / 149 / 156 / 178 / 205 / 253, 63 / 149 / 205, 63 / 149 / 205 / 229 / 253, 63 / 149 / 205 / 253, 63 / 154, 63 / 154 / 178 / 205 / 253, 63 / 154 / 205, 63 / 154 / 205 / 2 29、63 / 154 / 205 / 229 / 253、63 / 154 / 205 / 253、63 / 156、63 / 156 / 205、63 / 205、63 / 205 / 253、63 / 253、67、98、149 / 154、149 / 154 / 205、149 / 156 / 253、149 / 205、149 / 205 / 253、154、154 / 160、154 / 160 / 205、154 / 160 / 205 / 222 / 257、154 / 160 / 253、154 / 174、154 / 174 / 178 / 205、154 / 174 / 205 / 229, 154 / 205, 154 / 205 / 222, 154 / 205 / 222 / 257, 154 / 205 / 229, 154 / 205 / 253, 154 / 205 / 257, 154 / 229, 154 / 253, 156 / 205, 156 / 205 / 253, 156 / 229 / 253, 160, 160 / 205, 160 / 205 / 229, 194, 205, 205 / 222, 205 / 229, 205 / 229 / 253, 205 / 253, 208, 226 and 253, wherein the positions are numbered with reference to SEQ ID NO:1062. In some additional embodiments, the engineered polypeptide comprises at least one substitution or a set of substitutions selected from: 3C / 31H / 154K / 205M / 257R,3L / 154W / 205L、3T / 31H / 205L、31H / 55W / 154K / 205T、31H / 55W / 154Q / 205T、31H / 55W / 154W / 205T、31H / 55W / 205I、31H / 154Q / 205L、31H / 154W / 205I、31H / 154W / 205I / 257R、31H / 154W / 205L、31H / 154W / 205L / 257R、31H / 205I / 222Q、31H / 205L、40K / 55W / 205L、40T / 154Q / 205T、40T / 154Q / 205T / 257R、40T / 174A / 205I / 229S、40T / 205T / 229S、54E / 205M / 253Y、63M、63M / 149E / 154K / 174A / 178K / 253Y、63M / 149E / 154W / 174A / 205T / 253Y、63M / 149E / 156T / 178K / 205L / 253Y、63M / 149E / 205L、63M / 149E / 205L / 229S / 253Y、63M / 149E / 205L / 253Y、63M / 149E / 205T / 253Y、63M / 154H / 178K / 205I / 253Y、63M / 154H / 205L / 229S、63M / 154K / 205L、63M / 154K / 205T / 253Y、63M / 154Q / 205I / 229S / 253Y、63M / 154W、63M / 154W / 178K / 205L / 253Y、63M / 154W / 205M、63M / 154W / 205T、63M / 156A、63M / 156A / 205T、63M / 205L、63M / 205M、63M / 205M / 253Y、63M / 205T、63M / 205T / 253Y、63M / 253Y、67S、98Q、149E / 154S / 205L、149E / 154W、149E / 156A / 253Y、149E / 205M、149E / 205M / 253Y、149E / 205T、149E / 205T / 253D、154H、154H / 174A、154H / 205L / 253Y、154H / 205T、154K、154K / 160A、154K / 160A / 205L、154K / 160A / 205M、154K / 160A / 253Y、154K / 174A / 205L / 229S、154K / 205I、154K / 205M、154K / 253Y、154Q / 160A / 205L / 222F / 257R、154Q / 205I / 253Y、154Q / 205L / 222R / 257R, 154Q / 205M / 257R, 154W, 154W / 174A / 178K / 205T, 154W / 205I, 154W / 205I / 222G, 154W / 205I / 257R, 154W / 2 05L、154W / 205L / 222R、154W / 205M、154W / 205T、154W / 205T / 222F / 257R、154W / 205T / 222Y、154W / 205T / 229S、154W / 205T / 253Y、15 156A / 205M / 253Y, 156A / 229S / 253Y, 156T / 205T, 160A, 160A / 205L, 160A / 205L / 229S, 160A / 205M / 229S, 194H, 205L, 205L / 222G, 205M, 205T, 205T / 222Y, 205T / 229S, 205T / 229S / 253Y, 205T / 253Y, 208L, 208R, 208T, 226C and 253Y, wherein the positions are numbered with reference to SEQ ID NO: 1062. In some embodiments, the engineered polypeptide comprises at least one substitution or a set of substitutions selected from the group consisting of: P3C / Q31H / L154K / Q205M / E257R, P3L / L154W / Q205L, P3T / Q31H / Q205L, Q31H / A55W / L154K / Q205T, Q31H / A55W / L154Q / Q205 T, Q31H / A55W / L154W / Q205T, Q31H / A55W / Q205I, Q31H / L154Q / Q205L, Q31H / L154W / Q205I、Q31H / L154W / Q205I / E257R、Q31H / L154W / Q205L、Q31H / L154W / Q205L / E257 R, Q31H / Q205I / E222Q, Q31H / Q205L, S40K / A55W / Q205L, S40T / L154Q / Q205T, S40T / L154Q / Q205T / E257R, S40T / L174A / Q205I / A229S, S40T / Q205T / A229S, K54E / Q205 M / G253Y, Q63M, Q63M / V149E / L154K / L174A / S178K / G253Y, Q63M / V149E / L154W / L1 74A / Q205T / G253Y, Q63M / V149E / V156T / S178K / Q205L / G253Y, Q63M / V149E / Q205L,Q63M / V149E / Q205L / A229S / G253Y、Q63M / V149E / Q205L / G253Y、Q63M / V149E / Q205T / G253Y、Q63M / L154H / S178K / Q205I / G253Y、Q63M / L154H / Q205L / A229S、Q63M / L154K / Q205L、Q63M / L154K / Q205T / G253Y、Q63M / L154Q / Q205I / A229S / G253Y、Q63M / L154W、Q63M / L154W / S178K / Q205L / G253Y、Q63M / L154W / Q205M、Q63M / L154W / Q205T、Q63M / V156A、Q63M / V156A / Q205T、Q63M / Q205L、Q63M / Q205M、Q63M / Q205M / G253Y、Q63M / Q205T、Q63M / Q205T / G253Y、Q63M / G253Y、T67S、P98Q、V149E / L154S / Q205L、V149E / L154W、V149E / V156A / G253Y、V149E / Q205M、V149E / Q205M / G253Y、V149E / Q205T、V149E / Q205T / G253D、L154H、L154H / L174A、L154H / Q205L / G253Y、L154H / Q205T、L154K、L154K / S160A、L154K / S160A / Q205L、L154K / S160A / Q205M、L154K / S160A / G253Y、L154K / L174A / Q205L / A229S、L154K / Q205I、L154K / Q205M、L154K / G253Y、L154Q / S160A / Q205L / E222F / E257R、L154Q / Q205I / G253Y、L154Q / Q205L / E222R / E257R、L154Q / Q205M / E257R、L154W、L154W / L174A / S178K / Q205T、L154W / Q205I、L154W / Q205I / E222G、L154W / Q205I / E257R、L154W / Q205L、L154W / Q205L / E222R、L154W / Q205M、L154W / Q205T、L154W / Q205T / E222F / E257R、L154W / Q205T / E222Y、L154W / Q205T / A229S、L154W / Q205T / G253Y、L154W / A229S、L154W / G253Y、V156A / Q205M / G253Y, V156A / A229S / G253Y, V156T / Q205T, S160A, S160A / Q205L, S160A / Q205L / A229S, S160A / Q205M / A229S, T194H, Q205L, Q205L / E222G, Q205M, Q205T, Q205T / E222Y, Q205T / A229S, Q205T / A229S / G253Y, Q205T / G253Y, D208L, D208R, D208T, A226C and G253Y, where the positions are numbered with reference to SEQ ID NO: 1062. In some additional embodiments, the engineered polypeptide comprises an amino acid sequence having at least 80% sequence identity to any even-numbered sequence set forth in SEQ ID NO: 1172 to SEQ ID NO: 1384.

[0019] The present invention also provides engineered polynucleotides encoding at least one engineered polypeptide described in the above paragraphs. In some embodiments, the engineered polynucleotides include the odd-numbered sequences listed in SEQ ID NO:5 to SEQ ID NO:1383.

[0020] The present invention also provides a vector comprising at least one engineered polynucleotide described above. In some embodiments, the vector further comprises at least one control sequence.

[0021] The present invention also provides a host cell comprising a vector provided herein. In some embodiments, the host cell produces at least one engineered polypeptide provided herein.

[0022] The present invention also provides a method for producing an engineered glucose dehydrogenase polypeptide having imine reductase activity, the method comprising the step of culturing a host cell provided herein under conditions such that the engineered polynucleotide is expressed and the engineered polypeptide is produced. In some embodiments, the method further comprises the step of recovering the engineered polypeptide.

[0023] Description of the invention

[0024] Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as those of ordinary skill in the art to which the present invention belongs. Generally, the nomenclature used herein and the experimental procedures in cell culture, molecular genetics, microbiology, organic chemistry, analytical chemistry and nucleic acid chemistry described below are those well known in the art and generally adopted. Such technology is well known and described in many textbooks and reference works well known to those skilled in the art. Standard techniques or their modified forms have been used for chemical synthesis and chemical analysis. All patents, patent applications, articles and publications mentioned herein (both above and below) are hereby expressly incorporated herein by reference.

[0025] Although any suitable methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, some methods and materials are described herein. It should be understood that the present invention is not limited to the specific methods, protocols and reagents described, as these can be changed according to the circumstances in which they are used by those skilled in the art. Therefore, the terms that will be defined below are more fully described by reference to the present invention as a whole.

[0026] It should be understood that the general description above and the detailed description below are only exemplary and illustrative, rather than limiting the present invention. The section titles used herein are only for organizational purposes and are not to be construed as limiting the subject matter described. Numerical ranges include numbers that limit the range. Therefore, each numerical range disclosed herein is intended to include each narrower numerical range that falls within such a wider numerical range, as such a narrower numerical range is clearly written out in full herein. It is also intended that each maximum (or minimum) numerical limit disclosed herein includes each lower (or higher) numerical limit, as such lower (or higher) numerical limits are clearly written out herein.

[0027] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polypeptide" includes more than one polypeptide. Similarly, "comprise, comprises, comprising", "include, includes, and including" are interchangeable and are not intended to be limiting.

[0028] It is to be understood that when the description of various embodiments uses the term "comprising / including", those skilled in the art will understand that in some specific cases, alternatively, the language "consisting essentially of" or "consisting of" may be used to describe the embodiments. It is also to be understood that where the terms "optional" or "optionally" are used in the description of various embodiments, the event or situation described subsequently may or may not occur, and the description includes situations where the event or situation occurs and situations where it does not occur. It is to be understood that both the general description above and the detailed description below are exemplary and illustrative only and are not intended to limit the present disclosure. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0029] abbreviation:

[0030] The abbreviations used for the genetically encoded amino acids are conventional and are as follows:

[0031] Amino Acids Three letter abbreviation Single letter abbreviation Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamate Glu E Glutamine Gln Q Glycine Gly G Histidine His H

[0032] Amino Acids Three letter abbreviation Single letter abbreviation Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

[0033] When a three-letter abbreviation is used, unless specifically preceded by "L" or "D" or clear from the context in which the abbreviation is used, the amino acid may be referred to with respect to the α-carbon (C α ) is in the L-configuration or the D-configuration. For example, "Ala" represents alanine without specifying the configuration about the α-carbon, while "D-Ala" and "L-Ala" represent D-alanine and L-alanine, respectively.

[0034] When single-letter abbreviations are used, capital letters represent amino acids in the L-configuration about the α-carbon, and lowercase letters represent amino acids in the D-configuration about the α-carbon. For example, "A" represents L-alanine and "a" represents D-alanine. When a polypeptide sequence is presented as a string of single-letter or three-letter abbreviations (or mixtures thereof), the sequence is presented in the amino (N) to carboxyl (C) direction according to common convention.

[0035] Abbreviations for genetically encoded nucleosides are conventional and are as follows: adenosine (A); guanosine (G); cytidine (C); thymidine (T); and uridine (U). Unless specifically described, abbreviated nucleosides can be ribonucleosides or 2'-deoxyribonucleosides. Nucleosides can be designated as ribonucleosides or 2'-deoxyribonucleosides on an individual or overall basis. When a nucleic acid sequence is presented as a single-letter abbreviation string, the sequence is presented in a 5' to 3' direction according to conventional practice, and the phosphate is not shown.

[0036] definition:

[0037] With reference to the present invention, technical and scientific terms used in the description herein shall have the meanings commonly understood by one of ordinary skill in the art, unless explicitly defined otherwise. Accordingly, the following terms are intended to have the following meanings.

[0038] "EC" numbers refer to the enzyme nomenclature of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB). The IUBMB biochemical classification is a numerical classification system for enzymes based on the chemical reactions that the enzymes catalyze.

[0039] "ATCC" refers to the American Type Culture Collection, whose biological collection includes genes and strains.

[0040] "NCBI" refers to the National Center for Biological Information and the sequence databases provided therein.

[0041] "Protein," "polypeptide," and "peptide" are used interchangeably herein to refer to a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modifications (e.g., glycosylation, phosphorylation, lipidation, myristylation, ubiquitination, etc.). Included in this definition are D- and L-amino acids, as well as mixtures of D- and L-amino acids, and polymers comprising mixtures of D- and L-amino acids and D- and L-amino acids.

[0042] "Amino acids" are referred to herein by either their commonly known three letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Likewise, nucleotides may be referred to by their commonly accepted single-letter codes.

[0043] As used herein, "polynucleotide" and "nucleic acid" refer to two or more nucleosides covalently linked together. Polynucleotides can completely contain ribonucleotides (i.e., RNA), completely contain 2' deoxyribonucleotides (i.e., DNA), or contain a mixture of ribonucleotides and 2' deoxyribonucleotides. Although nucleosides are typically connected together via standard phosphodiester connections, polynucleotides can include one or more non-standard connections. Polynucleotides can be single-stranded or double-stranded, or can include both single-stranded regions and double-stranded regions. In addition, although polynucleotides are typically composed of naturally occurring coding nucleoside bases (i.e., adenine, guanine, uracil, thymine and cytosine), it can include one or more modified and / or synthetic nucleoside bases, such as, for example, inosine, xanthine, hypoxanthine, etc. In some embodiments, such modified or synthetic nucleoside bases are nucleoside bases encoding amino acid sequences.

[0044] As used herein, "imine" refers to an organic compound or functional group containing a nitrogen-carbon double bond, wherein the nitrogen is bonded to a hydrogen or an organic group.

[0045] As used herein, "imine reductase activity" refers to an enzyme activity in which, as illustrated in Scheme 1, a carbonyl group of a ketone or aldehyde and an amino group of a primary or secondary amine (wherein the carbonyl group and the amino group may be on separate compounds or on the same compound) are converted into a secondary or tertiary amine product compound in the presence of the cofactor NAD(P)H.

[0046] As used herein, "imine reductase" or "IRED" refers to an enzyme having imine reductase activity. It should be understood that imine reductase is not limited to engineered polypeptides derived from wild-type imine reductase, but can include other enzymes having imine reductase activity, including engineered polypeptides derived from glucose dehydrogenase. As used herein, imine reductase includes naturally occurring (wild-type) imine reductase as well as non-naturally occurring engineered polypeptides produced by human manipulation.

[0047] "Glucose dehydrogenase" or "GDH" refers to the original member of the short-chain dehydrogenase reductase family. GDH is well known for its dependence on NAD (P) to oxidize D-glucose to gluconolactone. Although the GDH enzyme of the present invention is derived from Bacillus subtilis, the present invention is not limited thereto, and the GDH enzyme can be derived from any suitable organism or synthetically produced. Glucose dehydrogenase as used herein includes naturally occurring (wild type) glucose dehydrogenase and non-naturally occurring engineered polypeptides produced by human manipulation.

[0048] "Coding sequence" refers to a portion of a nucleic acid (eg, a gene) that encodes the amino acid sequence of a protein.

[0049] "Naturally occurring" or "wild-type" refers to the form found in nature. For example, a naturally occurring or wild-type polypeptide or polynucleotide sequence is a sequence that is present in an organism that can be isolated from a source in nature and that has not been intentionally modified by human manipulation.

[0050] As used herein, "recombinant," "engineered," and "non-naturally occurring" when used with respect to cells, nucleic acids, or polypeptides refer to materials that have been modified in a manner that does not otherwise occur in nature or to materials that correspond to the natural or native form of a material. In some embodiments, the cell, nucleic acid, or polypeptide is identical to a naturally occurring cell, nucleic acid, or polypeptide, but is produced or derived from synthetic materials and / or manipulated through the use of recombinant techniques. Non-limiting examples include, among others, recombinant cells that express genes not found in the natural (non-recombinant) form of the cell or that express natural genes that are otherwise expressed at different levels.

[0051] "Percentage of sequence identity" and "percentage of homology" are used interchangeably herein to refer to a comparison between a polynucleotide or polypeptide, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may include additions or deletions (i.e., gaps) compared to the reference sequence for the optimal alignment of the two sequences. The percentage can be calculated by determining the number of positions where the same nucleic acid base or amino acid residue appears in the two sequences to produce the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Alternatively, the percentage can be calculated by determining the number of positions where the same nucleic acid base or amino acid residue appears in the two sequences or where the nucleic acid base or amino acid residue is aligned with a gap to produce the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Those skilled in the art will appreciate that there are many established algorithms that can be used to compare two sequences. Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman (Smith and Waterman, Adv. Appl. Math., 2:482

[1981] ), by the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch, J. Mol. Biol., 48:443

[1970] ), by the search for similarity method of Pearson and Lipman (Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444

[1988] ), by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin software package), or by visual inspection as is known in the art. Examples of algorithms suitable for determining percent sequence identity and sequence similarity include, but are not limited to, BLAST and BLAST 2.0 algorithms, which are described by Altschul et al. (see, respectively, Altschul et al., J. Mol. Biol., 215:403-410

[1990] ; and Altschul et al., Nucl. Acids Res., 3389-3402

[1977] ). Software for performing BLAST analysis is available to the public through the website of the National Center for Biotechnology Information. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or satisfy a certain positive threshold score T when aligned with a word of the same length in the database sequence.T is called the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence to the point where the cumulative alignment score cannot be increased. For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for matching residue pairs; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is stopped when: the cumulative alignment score falls by an amount X from its maximum achieved value; the cumulative score reaches 0 or less due to the accumulation of one or more negative scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses the following as defaults: word length (W) of 11, expectation (E) of 10, M=5, N=-4, and comparison of both chains. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (See, Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915

[1989] ). Exemplary determinations of sequence alignments and % sequence identity can be made using the BESTFIT or GAP programs in the GCG Wisconsin software package (Accelrys, Madison WI) using the default parameters provided.

[0052] "Reference sequence" refers to a designated sequence used as a basis for sequence comparison. A reference sequence can be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence. Typically, a reference sequence is at least 20 nucleotides or amino acid residues in length, at least 25 residues in length, at least 50 residues in length, or the full length of a nucleic acid or polypeptide. Since two polynucleotides or polypeptides can each (1) contain a sequence similar to the two sequences (i.e., a portion of the complete sequence), and (2) can also contain a sequence different from the two sequences, a sequence comparison between two (or more) polynucleotides or polypeptides is typically performed by comparing the sequences of the two polynucleotides or polypeptides on a "comparison window" to identify and compare local regions of sequence similarity. In some embodiments, a "reference sequence" can be based on a primary amino acid sequence, wherein the reference sequence is a sequence that can have one or more changes in the primary sequence. For example, "a reference sequence based on SEQ ID NO:4 having valine at a residue corresponding to X14" or X14V refers to a reference sequence in which the corresponding residue (tyrosine) at X14 in SEQ ID NO:4 has been changed to valine.

[0053] "Comparison window" refers to a conceptual segment of at least about 20 consecutive nucleotide positions or amino acid residues, wherein a sequence can be compared to a reference sequence of at least 20 consecutive nucleotides or amino acids, and wherein the portion of the sequence in the comparison window can contain 20% or less additions or deletions (i.e., gaps) compared to a reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. The comparison window can be longer than 20 consecutive residues, and optionally includes windows of 30, 40, 50, 100 or more.

[0054] As used herein, "substantial identity" refers to a polynucleotide or polypeptide sequence having at least 80% sequence identity, at least 85% identity, at least 89% to 95% sequence identity, or more generally at least 99% sequence identity in a comparison window of at least 20 residue positions, usually in a window of at least 30-50 residues, compared to a reference sequence, wherein the percentage of sequence identity is calculated by comparing the reference sequence in the comparison window with a sequence comprising a deletion or addition totaling 20% ​​or less of the reference sequence. In some specific embodiments applied to polypeptides, the term "substantial identity" means that when optimally aligned, such as by the program GAP or BESTFIT using default gap weights, two polypeptide sequences have at least 80% sequence identity, preferably at least 89% sequence identity, at least 95% sequence identity or more (e.g., 99% sequence identity). In some embodiments, residue positions that are not identical in the compared sequences differ due to conservative amino acid substitutions.

[0055] When used in the context of numbering a given amino acid or polynucleotide sequence, "corresponding to", "reference to", and "relative to" refer to the numbering of the residues of a given amino acid or polynucleotide sequence when the given amino acid or polynucleotide sequence is compared to a specified reference sequence. In other words, the residue numbers or residue positions of a given polymer are specified with respect to a reference sequence, rather than being specified by the actual numerical positions of the residues within a given amino acid or polynucleotide sequence. For example, a given amino acid sequence, such as an engineered glucose dehydrogenase, can be compared to a reference sequence by introducing spaces to optimize the residue matching between the two sequences. In these cases, despite the presence of spaces, the residues in a given amino acid or polynucleotide sequence are numbered with respect to the reference sequence to which it is compared.

[0056] "Amino acid difference" or "residue difference" refers to the change of the amino acid residue at one position of a polypeptide sequence relative to the amino acid residue at the corresponding position in the reference sequence. The position of the amino acid difference is generally referred to as "Xn" herein, where n refers to the corresponding position in the reference sequence on which the residue difference is based. For example, "the residue difference at position X25 compared to SEQ ID NO:2" refers to the change of the amino acid residue at the polypeptide position corresponding to position 25 of SEQ ID NO:2. Therefore, if the reference polypeptide SEQ ID NO:2 has valine at position 25, "the residue difference at position X25 compared to SEQ ID NO:2" refers to the amino acid substitution of any residue except valine at the position of the polypeptide corresponding to position 25 of SEQ ID NO:2. In most examples herein, the specific amino acid residue difference at one position is indicated as "XnY", where "Xn" specifies the corresponding position as described above, and "Y" is the single-letter identifier of the amino acid found in the engineered polypeptide (i.e., the residue different from the reference polypeptide). In some embodiments, more than one amino acid may appear at a specified residue position (i.e., the substituted amino acids may be listed in the form of XnY / Z, where Y and Z represent substituted amino acid residues). In some cases (e.g., in Table 5.1, Table 6.1, Table 7.1, Table 8.1, Table 9.1, Table 10.1, Table 11.1, and Table 12.1), the present invention also provides specific amino acid differences represented by the conventional symbol "AnB", where A is a single letter identifier for a residue in a reference sequence, "n" is the number of the residue position in the reference sequence, and B is a single letter identifier for a residue substitution in the sequence of the engineered polypeptide. In addition, in some cases, the polypeptides of the present invention may include one or more amino acid residue differences relative to a reference sequence, which are indicated by a list of designated positions that have been changed relative to the reference sequence. In some additional embodiments, the present invention provides engineered polypeptide sequences comprising both conservative amino acid substitutions and non-conservative amino acid substitutions.

[0057] As used herein, "conservative amino acid substitution" refers to replacing a residue with a different residue having a similar side chain, and thus generally includes replacing an amino acid in a polypeptide with an amino acid in the same or similar amino acid definition class. For example, but not limited to, an amino acid with an aliphatic side chain is replaced by another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid with a hydroxyl side chain is replaced by another amino acid with a hydroxyl side chain (e.g., serine and threonine); an amino acid with an aromatic side chain is replaced by another amino acid with an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid with a basic side chain is replaced by another amino acid with a basic side chain (e.g., lysine and arginine); an amino acid with an acidic side chain is replaced by another amino acid with an acidic side chain (e.g., aspartic acid or glutamic acid); and / or a hydrophobic amino acid or a hydrophilic amino acid is replaced by another hydrophobic amino acid or a hydrophilic amino acid, respectively. Exemplary conservative substitutions are provided in Table 1 below.

[0058]

[0059] "Non-conservative substitutions" refer to substitutions of amino acids in a polypeptide with amino acids having significantly different side chain properties. Non-conservative substitutions may use amino acids between defined groups rather than within defined groups and affect (a) the structure of the peptide backbone in the area of ​​the substitution (e.g., substitution of glycine with proline), (b) charge or hydrophobicity, or (c) the bulk of the side chain. For example, but not limited to, exemplary non-conservative substitutions may be substitutions of acidic amino acids with basic or aliphatic amino acids; substitutions of aromatic amino acids with small amino acids; and substitutions of hydrophilic amino acids with hydrophobic amino acids.

[0060] "Deletion" refers to a polypeptide modification by removing one or more amino acids from a reference polypeptide. Deletion can include removal of 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total amino acids that make up the reference enzyme, or up to 20% of the total amino acids, while retaining enzymatic activity and / or retaining the improved properties of engineered glucose dehydrogenase. Deletion can involve internal portions and / or terminal portions of a polypeptide. In various embodiments, deletion can include continuous segments or can be discontinuous.

[0061] "Insertion" refers to the modification of a polypeptide by adding one or more amino acids to a reference polypeptide. In some embodiments, the improved engineered glucose dehydrogenase comprises inserting one or more amino acids into a naturally occurring polypeptide having imine reductase activity, and inserting one or more amino acids into other improved glucose dehydrogenase polypeptides. The insertion may be in an internal portion of the polypeptide or may be an insertion into the carboxyl or amino terminus. Insertion as used herein includes fusion proteins as known in the art. The insertion may be a continuous segment of amino acids, or may be separated by one or more amino acids in a naturally occurring polypeptide.

[0062] As used herein, "fragment" refers to a polypeptide having an amino-terminal and / or carboxyl-terminal deletion but the remaining amino acid sequence is identical to the corresponding position in the sequence. A fragment can be at least 14 amino acids long, at least 20 amino acids long, at least 50 amino acids long or longer, and up to 70%, 80%, 90%, 95%, 98% and 99% of the engineered glucose dehydrogenase provided in the even-numbered sequence of SEQ ID NO: 6-1384, for example, a full-length glucose dehydrogenase polypeptide.

[0063] "Isolated polypeptide" refers to a polypeptide that is substantially separated from other contaminants such as proteins, lipids, and polynucleotides that are naturally associated with it. The term includes polypeptides that have been removed or purified from their naturally occurring environment or expression system (e.g., host cells or in vitro synthesis). The engineered glucose dehydrogenase can be present in cells, in cell culture media, or prepared in various forms, such as lysates or isolated preparations. Therefore, in some embodiments, the engineered glucose dehydrogenase can be an isolated polypeptide.

[0064] "Substantially pure polypeptide" refers to a composition in which the polypeptide species is the predominant species present (i.e., on a molar or weight basis, it is more abundant than any other individual macromolecular species in the composition), and is generally a substantially pure composition when the target species constitutes at least about 50% of the macromolecular species present by mole or % weight. Typically, a substantially pure glucose dehydrogenase composition will contain about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, and about 98% or more of all macromolecular species present in the composition by mole or % weight. In some embodiments, the target substance is purified to substantially homogeneity (i.e., contaminant substances cannot be detected in the composition by conventional detection methods), wherein the composition is substantially composed of a single macromolecular substance. Solvent substances, small molecules (<500 Daltons), and elemental ion substances are not considered macromolecular substances. In some embodiments, the engineered glucose dehydrogenase polypeptide separated is a substantially pure polypeptide composition.

[0065] "Stereoselectivity" refers to the preferential formation of one stereoisomer relative to another stereoisomer in a chemical or enzymatic reaction. Stereoselectivity can be partial, in which case the formation of one stereoisomer is preferred over the other stereoisomer, or stereoselectivity can be complete, in which case only one stereoisomer is formed. When the stereoisomers are enantiomers, stereoselectivity is referred to as enantioselectivity, i.e., the fraction of one enantiomer in the sum of the two (usually reported as a percentage). Alternatively, it is generally reported in the art as enantiomeric excess (ee) (usually as a percentage) calculated therefrom according to the following formula: [major enantiomer-minor enantiomer] / [major enantiomer+minor enantiomer]. When the stereoisomers are diastereomers, stereoselectivity is referred to as diastereoselectivity, i.e., the fraction of one diastereomer in a mixture of two diastereomers (usually reported as a percentage), usually optionally reported as diastereomeric excess (de). Enantiomeric excess and diastereomeric excess are types of stereoisomer excess.

[0066] "Highly stereoselective" refers to a chemical or enzymatic reaction that is capable of converting one or more substrates (eg, substrate compounds (2) and (3)) to the corresponding amine product (eg, compound (1)) with at least about 85% stereoisomeric excess.

[0067] As used herein, "improved enzyme properties" refers to at least one improved property of an enzyme. In some embodiments, the invention provides engineered glucose dehydrogenase polypeptides, which present an improvement in any enzyme property compared to a reference glucose dehydrogenase polypeptide and / or a wild-type glucose dehydrogenase polypeptide and / or another engineered glucose dehydrogenase polypeptide. For engineered glucose dehydrogenase polypeptides described herein, comparisons are usually made for a wild-type enzyme derived from glucose dehydrogenase, although in some embodiments, the reference enzyme can be another improved engineered glucose dehydrogenase. Therefore, the level of "improvement" can be determined and compared between various glucose dehydrogenase polypeptides (including wild-type) and engineered glucose dehydrogenases. Improved properties include, but are not limited to, properties such as: enzyme activity (which can be represented by the percentage of substrate conversion), thermostability, solvent stability, pH activity spectrum, cofactor requirements, tolerance to inhibitors (refractoriness) (such as substrate or product inhibition), stereospecificity and stereoselectivity (including enantioselectivity).

[0068] "Increased enzyme activity" refers to an improved property of an engineered glucose dehydrogenase polypeptide, which can be represented by an increase in specific activity (e.g., product produced / time / weight protein) or an increase in the percentage of substrate converted to product (e.g., the percentage of conversion of a starting amount of substrate to product over a specified period of time using a specified amount of glucose dehydrogenase) compared to a reference glucose dehydrogenase. Exemplary methods for determining enzyme activity are provided in the Examples. Any property associated with enzyme activity can be affected, including classical enzyme properties. m 、V max or k cat , their changes can lead to increased enzymatic activity.The improvement of enzymatic activity can be up to 2 times, 5 times, 10 times, 20 times, 25 times, 50 times or more of the enzymatic activity of about 1.2 times of the corresponding wild-type enzyme to the naturally occurring or another engineered glucose dehydrogenase activity of the glucose dehydrogenase polypeptide derived therefrom.Glucose dehydrogenase activity can be measured by any standard assay, such as by monitoring the change of the property of substrate, cofactor or product.In some embodiments, the amount of the product produced can be measured by liquid chromatography-mass spectrometry (LC-MS).The comparison of enzyme activity is carried out using specified enzyme products, the specified determination under the set conditions and one or more specified substrates, as described in further detail herein.Usually, when comparing lysates, determine the number of cells and the amount of the protein measured, and use the same expression system and the same host cell to minimize the change of the amount of the enzyme produced by the host cell and present in the lysate.

[0069] "Conversion" refers to the enzymatic conversion of a substrate into a corresponding product. "Conversion percentage" refers to the percentage of substrate converted into a product within a certain period of time under specified conditions. Therefore, the "enzyme activity" or "activity" of a glucose dehydrogenase polypeptide can be expressed as the "conversion percentage" of a substrate into a product.

[0070] "Thermostable" refers to a glucose dehydrogenase polypeptide that retains similar activity (e.g., greater than 60% to 80%) after exposure to the same elevated temperature (e.g., 40°C-80°C) for a certain period of time (e.g., 0.5 hour-24 hours) compared to a wild-type enzyme exposed to the same elevated temperature.

[0071] "Solvent stable" refers to a glucose dehydrogenase polypeptide that retains similar activity (e.g., more than 60% to 80%) after exposure to the same solvent at the same concentration for a certain period of time (e.g., 0.5 hour to 24 hours) compared to the wild-type enzyme exposed to different concentrations (e.g., 5%-99%) of solvents (ethanol, isopropanol, dimethyl sulfoxide (DMSO), tetrahydrofuran, 2-methyltetrahydrofuran, acetone, toluene, butyl acetate, methyl tert-butyl ether, etc.).

[0072] "Thermostable and solvent stable" refers to a glucose dehydrogenase polypeptide that is both thermostable and solvent stable.

[0073] The term "stringent hybridization conditions" is used herein to refer to conditions under which nucleic acid hybrids are stable. As known to those skilled in the art, the stability of the hybrid is reflected in the melting temperature (T m ). In general, the stability of the hybrid is a function of ionic strength, temperature, G / C content, and the presence of chaotropic agents. m Values ​​can be calculated using known methods for predicting melting temperatures (see, e.g., Baldino et al., Meth. Enzymol., 168:761-777

[1989] ; Bolton et al., Proc. Natl. Acad. Sci. USA 48:1390

[1962] ; Bresslauer et al., Proc. Natl. Acad. Sci. USA 83:8893-8897

[1986] ; Freier et al., Proc. Natl. Acad. Sci. USA 83:9373-9377

[1986] ; Kierzek et al., Biochem., 25:7840-7846

[1986] ; Rychlik et al., 1990, Nucl. Acids Res., 18:6409-6412

[1990] (Corrigendum, Nucl. Acids Res., 19:698

[1991] ); Sambrook et al., supra); Suggs et al., 1981, Developmental Biology Using Purified Genes , Brown et al. [Eds.], pp. 683-693, Academic Press, Cambridge, MA

[1981] ; and Wetmur, Crit. Rev. Biochem. Mol. Biol., 26:227-259

[1991] ). In some embodiments, the polynucleotide encodes a polypeptide disclosed herein and hybridizes under defined conditions, such as moderately stringent or highly stringent conditions, to the complement of a sequence encoding an engineered glucose dehydrogenase of the invention.

[0074] "Hybridization stringency" refers to the hybridization conditions, such as washing conditions, in nucleic acid hybridization. Typically, hybridization reactions are performed under conditions of lower stringency, followed by washes of different but higher stringency. The term "moderate stringency hybridization" refers to conditions that allow target DNA to bind to complementary nucleic acids that have about 60% identity, preferably about 75% identity, about 85% identity, and greater than about 90% identity with the target polynucleotide. Exemplary moderate stringency conditions are conditions equivalent to hybridization at 42°C in 50% formamide, 5×Denhart solution, 5×SSPE, 0.2% SDS, followed by washing at 42°C in 0.2×SSPE, 0.2% SDS. "High stringency hybridization" generally refers to conditions with a thermal melting temperature T as determined for a defined polynucleotide sequence under solution conditions. m Conditions that differ by about 10°C or less. In some embodiments, high stringency conditions refer to conditions that allow only hybridization of those nucleic acid sequences that form stable hybrids in 0.018M NaCl at 65°C (i.e., if the hybrid is unstable in 0.018M NaCl at 65°C, it is unstable under high stringency conditions as contemplated herein). High stringency conditions can be provided, for example, by hybridizing in conditions equivalent to 42°C, 50% formamide, 5×Denhart solution, 5×SSPE, 0.2%SDS, followed by washing in 0.1×SSPE and 0.1%SDS at 65°C. Another high stringency condition is to hybridize in 5X SSC containing 0.1% (w:v) SDS at 65°C, and wash in conditions of 0.1×SSC containing 0.1% SDS at 65°C. Other high stringency hybridization conditions as well as medium stringency conditions are described in the references cited above.

[0075] A "heterologous" polynucleotide refers to any polynucleotide that is introduced into a host cell by laboratory techniques and includes polynucleotides removed from a host cell, subjected to laboratory manipulations, and then reintroduced into the host cell.

[0076] "Codon optimization" refers to that the codons of the polynucleotides encoding proteins are changed to those codons preferentially used in a specific organism so that the encoded protein is effectively expressed in the organism of interest. Although the genetic code is degenerate, i.e., most amino acids are represented by several codons referred to as "synonyms" or "synonymous" codons, it is well known that the codon usage of a specific organism is non-random and biased for specific codon triplets. With respect to the aggregated protein coding regions of a given gene, a gene with a common function or ancestral origin, a highly expressed protein versus a low copy number protein and an organism's genome, this codon usage bias may be higher. In some embodiments, the polynucleotides encoding glucose dehydrogenase can be codon optimized for optimal production from a host organism selected for expression.

[0077] As used herein, "preferred, optimal, high codon usage bias codons" interchangeably refer to codons that are used more frequently in protein coding regions than other codons encoding the same amino acid. Preferred codons can be determined based on codon usage in a single gene, a group of genes of a common function or origin, highly expressed genes, codon frequencies in aggregating protein coding regions of an entire organism, codon frequencies in aggregating protein coding regions of related organisms, or a combination thereof. Codons whose frequencies increase with the level of gene expression are generally the best codons for expression. A variety of methods are known for determining the frequency of codons (e.g., codon usage, relative synonymous codon usage) and codon preference in a particular organism, including multivariate analysis, such as using cluster analysis or correlation analysis and the effective number of codons used in a gene (see, e.g., GCG Codon Preference, Genetics Computer Group Wisconsin Package; Codon W, Peden, University of Nottingham; McInerney, Bioinform., 14:372-73

[1998] ; Stenico et al., Nucl. Acids Res., 222437-46

[1994] ; Wright, Gene 87:23-29

[1990] ). Codon usage tables are available for many different organisms (see, e.g., Wada et al., Nucl. Acids Res., 20:2111-2118

[1992] ; Nakamura et al., Nucl. Acids Res., 28:292

[2000] ; Duret, et al., supra; Henaut and Danchin, et al., supra). Escherichia coli and Salmonella, Neidhardt, et al. (eds.), ASM Press, Washington DC, pp. 2047-2066

[1996] ). The data source for obtaining codon usage can rely on any available nucleotide sequence that can encode a protein. These data sets include nucleic acid sequences that are actually known to encode expressed proteins (e.g., complete protein coding sequences - CDS), expressed sequence tags (ESTS), or predicted coding regions of genomic sequences (see, e.g., Mount, Bioinformatics:Sequence and Genome Analysis , Chapter 8, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

[2001] ; Uberbacher, Meth. Enzymol., 266:259-281

[1996] ; and Tiwari et al., Comput. Appl. Biosci., 13:263-270

[1997] ).

[0078] "Control sequences" are defined herein to include all components that are necessary or advantageous for the expression of the polynucleotides and / or polypeptides of the present invention. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, leader sequences, polyadenylation sequences, propeptide sequences, promoters, signal peptide sequences, and transcription terminators. At a minimum, control sequences include promoters and transcription and translation termination signals. Control sequences may be provided with linkers for the purpose of introducing specific restriction sites that facilitate connection of the control sequences to the coding region of the nucleic acid sequence encoding the polypeptide.

[0079] "Operably linked" is defined herein as a configuration in which a control sequence is appropriately placed (i.e., in a functional relationship) at a position relative to a polynucleotide of interest such that the control sequence directs or regulates expression of the polynucleotide and / or polypeptide of interest.

[0080] "Promoter sequence" refers to a nucleic acid sequence that is recognized by a host cell for expression of a polynucleotide of interest, such as a coding sequence. The promoter sequence comprises a transcriptional control sequence that mediates the expression of the polynucleotide of interest. The promoter can be any nucleic acid sequence that exhibits transcriptional activity in a selected host cell, including mutants, truncated and hybrid promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to the host cell.

[0081] "Suitable reaction conditions" refer to those conditions in a biocatalytic reaction solution (e.g., ranges of enzyme loading, substrate loading, cofactor loading, temperature, pH, buffer, cosolvent, etc.), under which the imine reductase polypeptide of the present invention is able to convert one or more substrate compounds into product compounds (e.g., converting compound (2) and compound (3) into compound (1)). Exemplary "suitable reaction conditions" are provided and illustrated by the examples herein.

[0082] A "composition" refers to a mixture or combination of one or more substances, wherein each substance or component of the composition retains its individual properties. As used herein, a biocatalytic composition refers to a combination of one or more substances that can be used for biocatalysis.

[0083] "Cofactor regeneration system" or "cofactor recycling system" refers to the reactions involved in reducing the oxidized form of a cofactor (e.g., NADP + to NADPH). The oxidized cofactor of the reductive amination reaction of the keto substrate catalyzed by the imine reductase is regenerated in reduced form by a cofactor regeneration system. The cofactor regeneration system includes a stoichiometric reductant that is a source of reducing hydrogen equivalents and is capable of reducing the oxidized form of the cofactor. The cofactor regeneration system may also include a catalyst, such as an enzyme catalyst that catalyzes the reduction of the oxidized form of the cofactor by the reductant. + or NADP + Cofactor regeneration systems that regenerate NADH or NADPH are known in the art and can be used in the methods described herein.

[0084] "Phosphate dehydrogenase" and "PDH" are used interchangeably herein to refer to enzymes that catalyze the dehydrogenation of phosphite and NAD. + or NADP + NAD converted to carbon dioxide and NADH or NADPH + or NADP + Dependent enzyme.

[0085] "Loading" (such as in "compound loading" or "enzyme loading" or "cofactor loading") refers to the concentration or amount of a component in the reaction mixture at the start of the reaction.

[0086] In the context of a biocatalyst-mediated process, "substrate" refers to a compound or molecule on which the biocatalyst acts. For example, in the glucose dehydrogenase biocatalyst used in the reductive amination method disclosed herein, there is a ketone (or aldehyde) substrate of formula (V), and an amine substrate of formula (VI).

[0087] In the context of a biocatalyst-mediated process, "product" refers to a compound or molecule produced by the action of a biocatalyst. For example, an exemplary product of the glucose dehydrogenase biocatalyst used in the methods disclosed herein is a secondary or tertiary amine compound, such as a compound of formula (IV).

[0088] "Alkyl" refers to a saturated hydrocarbon group having from 1 to 18 carbon atoms (inclusive), linear or branched, more preferably 1 to 8 carbon atoms (inclusive), and most preferably 1 to 6 carbon atoms (inclusive). Alkyl groups having a specified number of carbon atoms are indicated in brackets (e.g., (C1-C6) alkyl refers to an alkyl group of 1 to 6 carbon atoms).

[0089] "Alkenyl" refers to a hydrocarbon group having from 2 to 12 carbon atoms (inclusive), straight or branched, containing at least one double bond but optionally containing more than one double bond.

[0090] "Alkynyl" refers to a hydrocarbon group having from 2 to 12 carbon atoms (inclusive), straight or branched, containing at least one triple bond but optionally more than one triple bond, and further optionally containing one or more double bonded moieties.

[0091] "Alkylene" refers to a linear or branched divalent hydrocarbon group having from 1 to 18 carbon atoms (inclusive), more preferably 1 to 8 carbon atoms (inclusive), and most preferably 1 to 6 carbon atoms (inclusive), optionally substituted with one or more suitable substituents. Exemplary "alkylene" include, but are not limited to, methylene, ethylene, propylene, butylene, etc.

[0092] "Alkenylene" refers to a straight or branched divalent hydrocarbon group having 2 to 12 carbon atoms (inclusive) and one or more carbon-carbon double bonds, more preferably 2 to 8 carbon atoms (inclusive), and most preferably 2 to 6 carbon atoms (inclusive), optionally substituted by one or more suitable substituents.

[0093] "Heteroalkyl", "heteroalkenyl" and "heteroalkynyl" refer to alkyl, alkenyl and alkynyl groups, respectively, as defined herein, in which one or more carbon atoms are each independently replaced by the same or different heteroatoms or heteroatom groups. Heteroatoms and / or heteroatom groups that may replace carbon atoms include, but are not limited to, -O-, -S-, -SO-, -NR γ -, -PH-, -S(O)-, -S(O)2-, -S(O)NR γ -、-S(O)2NR γ - etc., including combinations thereof, where each R γ are independently selected from hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl.

[0094] "Aryl" refers to an unsaturated aromatic carbocyclic group having 6 to 12 carbon atoms (including endpoints) with a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthracenyl). Exemplary aryl groups include phenyl, pyridyl, naphthyl, and the like.

[0095] "Arylalkyl" refers to an alkyl group substituted with an aryl group (i.e., an "aryl-alkyl-" group), preferably having 1 to 6 carbon atoms (inclusive) in the alkyl portion and 6 to 12 carbon atoms (inclusive) in the aryl portion. Such arylalkyl groups are exemplified by benzyl, phenethyl, and the like.

[0096] "Aryloxy" refers to -OR λ Group, where R λ is an aryl group which may be optionally substituted.

[0097] "Cycloalkyl" refers to a cyclic alkyl group having 3 to 12 carbon atoms (inclusive), having a single cyclic ring or multiple condensed rings, which may be optionally substituted by 1 to 3 alkyl groups. Exemplary cycloalkyl groups include, but are not limited to, monocyclic structures and polycyclic structures, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, 1-methylcyclopropyl, 2-methylcyclopentyl, 2-methylcyclooctyl, etc., and polycyclic structures including bridged ring systems such as adamantyl, etc.

[0098] "Cycloalkylalkyl" refers to an alkyl group substituted with a cycloalkyl group (i.e., a "cycloalkyl-alkyl-" group), preferably having from 1 to 6 carbon atoms (inclusive) in the alkyl portion and from 3 to 12 carbon atoms (inclusive) in the cycloalkyl portion. Such cycloalkylalkyl groups are exemplified by cyclopropylmethyl, cyclohexylethyl, and the like.

[0099] "Amino" refers to the group -NH2. Substituted amino refers to the group -NHR ε NR ε R ε and NR ε R ε R ε , where each R ε Independently selected from substituted or unsubstituted alkyl, cycloalkyl, cycloheteroalkyl, alkoxy, aryl, heteroaryl, heteroarylalkyl, acyl, alkoxycarbonyl, sulfhydryl, sulfinyl, sulfonyl, etc. Typical amino groups include, but are not limited to, dimethylamino, diethylamino, trimethylammonium, triethylammonium, methylsulfonylamino, furanyl-oxy-sulfonylamino, etc.

[0100] "Aminoalkyl" refers to an alkyl group in which one or more hydrogen atoms are replaced by one or more amino groups (including substituted amino groups).

[0101] "Aminocarbonyl" refers to -C(O)NH2. Substituted aminocarbonyl refers to -C(O)NR ε R ε , where the amino group NR ε R ε As defined herein.

[0102] "Oxy" refers to a divalent group -O-, which may have various substituents to form different oxy groups including ethers and esters.

[0103] "Alkoxy" or "alkyloxy" are used interchangeably herein to refer to the group -OR δ , where R δ is an alkyl group, including an optionally substituted alkyl group.

[0104] "Carboxyl" refers to -COOH.

[0105] "Carbonyl" refers to -C(O)-, which can have a variety of substituents to form different carbonyl groups including acids, acid halides, aldehydes, amides, esters and ketones.

[0106] "Carboxyalkyl" refers to an alkyl group in which one or more hydrogen atoms are replaced with one or more carboxyl groups.

[0107] "Aminocarbonylalkyl" means an alkyl group substituted with an aminocarbonyl group as defined herein.

[0108] "Halogen" or "halo" refers to fluorine, chlorine, bromine and iodine.

[0109] "Haloalkyl" refers to an alkyl group in which one or more hydrogen atoms are replaced by halogen. Therefore, the term "haloalkyl" is meant to include monohaloalkyl, dihaloalkyl, trihaloalkyl, etc. up to perhaloalkyl. For example, the expression "(C1-C2)haloalkyl" includes 1-fluoromethyl, difluoromethyl, trifluoromethyl, 1-fluoroethyl, 1,1-difluoroethyl, 1,2-difluoroethyl, 1,1,1-trifluoroethyl, perfluoroethyl, etc.

[0110] "Hydroxyl" refers to -OH.

[0111] "Hydroxyalkyl" refers to an alkyl group in which one or more hydrogen atoms are replaced with one or more hydroxy groups.

[0112] "Thiol" or "sulfanyl" refers to -SH. Substituted thiol or sulfanyl refers to -SR ε , where R ε is an alkyl group, an aryl group or other suitable substituents.

[0113] "Alkylthio" refers to -SR δ , where R δ is an alkyl group which may be optionally substituted. Typical alkylthio groups include, but are not limited to, methylthio, ethylthio, n-propylthio, and the like.

[0114] "Alkylthioalkyl" refers to an alkylthio group -SR δ Substituted alkyl, wherein R δ is an alkyl group which may be optionally substituted.

[0115] "Sulfonyl" refers to -SO2-. A substituted sulfonyl refers to -SO2-R ε , where R ε is an alkyl group, an aryl group or other suitable substituents.

[0116] "Alkylsulfonyl" refers to -SO2-R δ , where R δ is an alkyl group which may be optionally substituted. Typical alkylsulfonyl groups include, but are not limited to, methylsulfonyl, ethylsulfonyl, n-propylsulfonyl, and the like.

[0117] "Alkylsulfonylalkyl" refers to an alkylsulfonyl group -SO2-R δ Substituted alkyl, wherein R δ is an alkyl group which may be optionally substituted.

[0118] "Heteroaryl" refers to an aromatic heterocyclic group having 1 to 10 carbon atoms (inclusive) and 1 to 4 heteroatoms (inclusive) selected from oxygen, nitrogen and sulfur in the ring. Such heteroaryl groups can have a single ring (e.g., pyridyl or furanyl) or more than one condensed ring (e.g., indolizinyl or benzothienyl).

[0119] "Heteroarylalkyl" refers to an alkyl group substituted with a heteroaryl group (i.e., a "heteroaryl-alkyl-" group), preferably having 1 to 6 carbon atoms (inclusive) in the alkyl portion and 5 to 12 ring atoms (inclusive) in the heteroaryl portion. Such heteroarylalkyl groups are exemplified by pyridylmethyl and the like.

[0120] "Heterocycle", "heterocyclic" and interchangeably "heterocycloalkyl" refer to saturated or unsaturated groups having a single ring or multiple fused rings, having 2 to 10 carbon ring atoms (inclusive) and 1 to 4 heterocyclic atoms (inclusive) selected from nitrogen, sulfur or oxygen within the ring. Such heterocyclic groups can have a single ring (e.g., piperidinyl or tetrahydrofuranyl) or more than one fused ring (e.g., indolinyl, dihydrobenzofuran or quinuclidinyl). Examples of heterocycles include, but are not limited to, furan, thiophene, thiazole, oxazole, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, pyrrolidine, indoline, and the like.

[0121] "Heterocycloalkylalkyl" refers to an alkyl group substituted with a heterocycloalkyl (i.e., a "heterocycloalkyl-alkyl-" group), preferably having from 1 to 6 carbon atoms (inclusive) in the alkyl portion and from 3 to 12 ring atoms (inclusive) in the heterocycloalkyl portion.

[0122] "Membered ring" is intended to include any cyclic structure. The number before the term "membered" indicates the number of backbone atoms that make up the ring. Thus, for example, cyclohexyl, pyridine, pyran, and thiopyran are 6-membered rings, and cyclopentyl, pyrrole, furan, and thiophene are 5-membered rings.

[0123] As used herein, "fused bicyclic ring" refers to unsubstituted and substituted carbocyclic and / or heterocyclic ring moieties having from 5 to 8 atoms in each ring, the rings having 2 atoms in common.

[0124] As used herein, "optionally substituted" with respect to the above chemical groups means that the position of the chemical group occupied by hydrogen may be replaced by another atom (unless otherwise specified) such as, but not limited to, carbon, oxygen, nitrogen or sulfur, or a chemical group such as, but not limited to, hydroxy, oxo, nitro, methoxy, ethoxy, alkoxy, substituted alkoxy, trifluoromethoxy, haloalkoxy, fluorine, chlorine, bromine, iodine, halogen, methyl, ethyl, propyl, butyl, alkyl, alkenyl, alkynyl, substituted alkyl, trifluoromethyl, haloalkyl, hydroxyalkyl, alkoxyalkyl, thio, alkylthio, acyl, carboxyl, alkoxycarbonyl, carboxamido, substituted carboxamido, alkylsulfonyl, alkylsulfinyl, alkyl The invention also includes but is not limited to alkyl, alkylsulfonylamino, sulfonylamino, substituted sulfonylamino, cyano, amino, substituted amino, alkylamino, dialkylamino, aminoalkyl, acylamino, amidino, amidoximino, hydroxamoyl, phenyl, aryl, substituted aryl, aryloxy, arylalkyl, arylalkenyl, arylalkynyl, pyridinyl, imidazolyl, heteroaryl, substituted heteroaryl, heteroaryloxy, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, substituted cycloalkyl, cycloalkyloxy, pyrrolidinyl, piperidinyl, morpholino, heterocyclyl, (heterocyclyl)oxy and (heterocyclyl)alkyl; wherein preferred heteroatoms are oxygen, nitrogen and sulfur. In addition, where there are open valences on these substituted chemical groups, they can be further substituted by alkyl, cycloalkyl, aryl, heteroaryl and / or heterocyclic groups, where these open valences are present on carbon, they can be further substituted by halogens and by oxygen-, nitrogen- or sulfur-bonded substituents, and where there are more than one such open valences, these groups can be connected to form a ring by directly forming a bond or by forming a bond with a new heteroatom (preferably oxygen, nitrogen or sulfur). It is also envisioned that the above substitutions can be made, provided that replacing hydrogen with a substituent does not bring unacceptable instability to the molecules of the present invention, and is otherwise chemically reasonable. It will be understood by those of ordinary skill in the art that for any chemical group described as optionally substituted, only sterically achievable and / or synthetically feasible chemical groups are intended to be included. "Optionally substituted" as used herein refers to all subsequent modifiers in a term or a series of chemical groups. For example, in the term "optionally substituted arylalkyl", the "alkyl" portion and the "aryl" portion of the molecule may or may not be substituted, and for a series of "optionally substituted alkyl, cycloalkyl, aryl and heteroaryl", the alkyl, cycloalkyl, aryl and heteroaryl groups independently of each other may or may not be substituted.

[0125] As used herein, "reaction" refers to one or more processes in which one or more substances or compounds or substrates are converted into one or more different substances, compounds.

[0126] Conversion of imines to secondary amines

[0127] Compound (1) is a substituted carbamoylmethylaminoacetic acid derivative whose chemical synthesis is a multi-step process. The low yield of this multi-step chemical process makes the use of an enzyme with imine reductase activity an attractive alternative because several steps can be removed from the synthetic sequence.

[0128] Glucose dehydrogenase (GDH), the original member of the short-chain dehydrogenase reductase family, is well known for its NAD(P)-dependent oxidation of D-glucose to gluconolactone. It has been widely used for the regeneration of nicotinamide cofactor. Recently, the multifunctional imine reductase activity of GDH was reported, with several GDH enzymes from different organisms asymmetric reduction of cyclic imine salts to cyclic chiral amines (Roth et al., ChemBioChem 2017, 18, 1703–1706). This preliminary report raised the question of whether GDH enzymes, similar to IREDs, could be used to generate a range of chiral secondary or tertiary amines, including, inter alia, acyclic amines, amino acid esters, and amino acids with alkyl chains (Scheme 1).

[0129] Initial reports of the multifunctionality of GDH, while intriguing, focused only on a small subset of imine reductase reactions using stable cyclic imine salts, which have limited utility in complex multistep processes that can be used to synthesize a range of chiral secondary and tertiary amines. The natural substrates of GDH are cyclic, but biocatalysts with imine reductase activity can be used on a range of substrates, including acyclic substrates, substrates containing other functional groups present, or substrates with alkyl chains (including branches). In addition, enzymes that catalyze several process steps are particularly useful in the preparation of industrial and pharmaceutical compounds and intermediates.

[0130] Solution 1

[0131]

[0132] The present invention provides novel biocatalysts and related methods for synthesizing chiral secondary amines by direct reductive amination of substrates containing carbonyls and amines. The biocatalysts of the present disclosure are engineered polypeptide variants of wild-type genes from Bacillus subtilis, which encode glucose dehydrogenase having the amino acid sequence of SEQ ID NO:2. A variant of wild-type glucose dehydrogenase (SEQ ID NO:4) containing the following residue differences compared to SEQ ID NO:2: E170K, Q252L, I165M, P194T, is used as a starting point for protein engineering. These engineered polypeptides are capable of catalyzing the conversion of carbonyl compounds and amines into secondary amines. The general imine reductase activity of IRED and GDH is shown in Scheme 1 above.

[0133] Solution 2

[0134]

[0135] The engineered glucose dehydrogenase polypeptides of the present invention can accept a range of substrates. Therefore, in the biocatalytic reaction of Scheme 2, the R1 group of the substrate is selected from a hydrogen atom or an optionally substituted alkyl, alkenyl, alkynyl, alkoxy, arylalkoxy, hydroxyalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocyclic hydrocarbon, heteroaryl and heteroarylalkyl; the R2 group of the substrate is independently selected from alkyl, alkenyl, alkynyl, hydroxyalkyl, carboxyl, aminocarbonyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carboxylalkyl, alkylamino, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocyclic hydrocarbon, heteroaryl and heteroarylalkyl; and the R3 group of the substrate is independently selected from methyl, d3-methyl and ethyl.

[0136] As further described herein, engineered glucose dehydrogenase polypeptides having imine reductase activity exhibit stereoselectivity; thus, the imine reductase reaction of Scheme 2 can be used to establish one or more chiral centers of a product in a single biocatalytic reaction.

[0137] In some embodiments, the invention provides an engineered polypeptide comprising an amino acid sequence having at least 80% sequence identity to the amino acid reference sequence of SEQ ID NO: 4, and further comprising one or more amino acid residue differences compared to the reference amino acid sequence, wherein the engineered glucose dehydrogenase polypeptide has imine reductase activity. In some embodiments of the engineered glucose dehydrogenase polypeptide, the imine reductase activity is the activity of Scheme 2.

[0138] In particular, the engineered glucose dehydrogenase polypeptides of the present disclosure have been engineered to efficiently convert the ketoester substrate 3-[4-(3-chlorophenyl)phenyl]-2-oxo-propionic acid (referred to herein as chlorobiphenylpyruvic acid and "compound (2)") and the amine substrate L-alanine ethyl ester (referred to herein as "compound (3)") into the corresponding chiral amine product compound 3-[4-(3-chlorophenyl)phenyl]-2-[(2-ethoxy-1-methyl-2-oxo-ethyl)amino]propionic acid (referred to herein as "compound (1)") under aqueous conditions, as shown in Scheme 3.

[0139] Solution 3

[0140]

[0141] Engineered glucose dehydrogenase peptide

[0142] The present invention provides a glucose dehydrogenase polypeptide having imine reductase activity, a polynucleotide encoding the polypeptide, a method for preparing the polypeptide, and a method for using the polypeptide. Where the description relates to a polypeptide, it should be understood that it may describe a polynucleotide encoding the polypeptide.

[0143] Suitable reaction conditions for carrying out the desired reaction by which the improved properties of the engineered polypeptides described above are achieved can be determined taking into account the concentration or amount of the polypeptide, substrate, co-substrate, buffer, solvent, pH, conditions including temperature and reaction time, and / or conditions under which the polypeptide is immobilized on a solid support, as further described below and in the Examples.

[0144] In some embodiments, exemplary engineered glucose dehydrogenase polypeptides having imine reductase activity with improved properties, particularly in converting compounds of formula (2) and (3) to compounds of formula (1), comprise an amino acid sequence having one or more residue differences at the residue positions shown in Table 5.1 compared to SEQ ID NO:4.

[0145] In some embodiments, exemplary engineered glucose dehydrogenase polypeptides having imine reductase activity with improved properties, particularly in converting compounds of formula (2) and (3) to compounds of formula (1), comprise an amino acid sequence having one or more residue differences at the residue positions shown in Table 6.1 compared to SEQ ID NO: 14.

[0146] In some embodiments, exemplary engineered glucose dehydrogenase polypeptides having imine reductase activity with improved properties, particularly in converting compounds of formula (2) and (3) to compounds of formula (1), comprise an amino acid sequence having one or more residue differences at the residue positions shown in Table 7.1 compared to SEQ ID NO: 34.

[0147] In some embodiments, exemplary engineered glucose dehydrogenase polypeptides having imine reductase activity with improved properties, particularly in converting compounds of formula (2) and (3) to compounds of formula (1), comprise an amino acid sequence having one or more residue differences at the residue positions shown in Table 8.1 compared to SEQ ID NO: 112.

[0148] In some embodiments, exemplary engineered glucose dehydrogenase polypeptides having imine reductase activity with improved properties, particularly in converting compounds of formula (2) and (3) to compounds of formula (1), comprise an amino acid sequence having one or more residue differences at the residue positions shown in Table 9.1 compared to SEQ ID NO: 736.

[0149] In some embodiments, exemplary engineered glucose dehydrogenase polypeptides having imine reductase activity with improved properties, particularly in converting compounds of formula (2) and (3) to compounds of formula (1), comprise an amino acid sequence having one or more residue differences at the residue positions shown in Table 10.1 compared to SEQ ID NO: 908.

[0150] In some embodiments, exemplary engineered glucose dehydrogenase polypeptides having imine reductase activity with improved properties, particularly in converting compounds of formula (2) and (3) to compounds of formula (1), comprise an amino acid sequence having one or more residue differences at the residue positions shown in Table 11.1 compared to SEQ ID NO: 956.

[0151] In some embodiments, exemplary engineered glucose dehydrogenase polypeptides having imine reductase activity with improved properties, particularly in converting compounds of formula (2) and (3) to compounds of formula (1), comprise an amino acid sequence having one or more residue differences at the residue positions shown in Table 12.1 compared to SEQ ID NO: 1062.

[0152] The structural and functional information of exemplary non-naturally occurring (or engineered) polypeptides of the present invention is based on the conversion of compounds of formula (2) and (3) to compounds of formula (1), the results of which are shown in Tables 5.1, 6.1, 7.1, 8.1, 9.1, 10.1, 11.1 and 12.1 below, and further described in the Examples. The odd-numbered sequence identifiers (i.e., SEQ ID NOs) in these tables refer to the nucleotide sequences encoding the amino acid sequences provided by the even-numbered SEQ ID NOs in these tables. Exemplary sequences are provided in the electronic sequence listing file accompanying the present invention, which is hereby incorporated by reference herein. Amino acid residue differences are based on comparisons with reference sequences SEQ ID NOs: 4, 14, 34, 112, 736, 908, 956 and / or 1062, as shown.

[0153] Using the amino acid sequence of the naturally occurring Bacillus subtilis glucose dehydrogenase (SEQ ID NO: 2) as a starting enzyme, a GDH (SEQ ID NO: 4) with improved glucose dehydrogenase activity was developed (see U.S. Pat. Nos. 7,816,111 and 7,939,309). SEQ ID NO: 3 is a codon-optimized polynucleotide synthesized based on the polypeptide sequence of SEQ ID NO: 4 for expression in Escherichia coli. The engineered glucose dehydrogenase of SEQ ID NO: 4 was found to have multifunctional imine reductase activity.

[0154] The activity of each engineered glucose dehydrogenase polypeptide relative to the reference polypeptide SEQ ID NO: 4, 14, 34, 112, 736, 908, 956 and / or 1062 is determined by the conversion of the substrate described in the examples herein. In some embodiments, shake flask powder (SFP) is used as a secondary screen to evaluate the properties of the engineered glucose dehydrogenase, the results of which are provided in the examples. In some embodiments, the SFP format provides a purer powder preparation of the engineered polypeptide and can contain up to about 30% of the total protein of the engineered polypeptide.

[0155] In some embodiments, specific enzyme properties are associated with residue differences at the residue positions set forth herein compared to SEQ ID NO: 4, 14, 34, 112, 736, 908, 956, and / or 1062. In some embodiments, residue differences that affect polypeptide expression can be used to increase expression of an engineered glucose dehydrogenase.

[0156] In view of the guidance provided herein, it is also contemplated that any of the exemplary engineered polypeptides comprising even-numbered sequences of SEQ ID NOs: 6-1384 can be used as starting amino acid sequences for synthesizing other engineered glucose dehydrogenase polypeptides by, for example, subsequent rounds of evolution incorporating new combinations of multiple amino acid differences from other polypeptides in Table 5.1, Table 6.1, Table 7.1, Table 8.1, Table 9.1, Table 10.1, Table 11.1, and Table 12.1 and other residue positions described herein. Additional improvements can be generated by including amino acid differences at residue positions that remain unchanged throughout earlier rounds of evolution.

[0157] In some embodiments, the engineered glucose dehydrogenase polypeptide having imine reductase activity comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO:4 and one or more residue differences compared to SEQ ID NO:4 selected from the group consisting of: 496, 96 / 118, 147, 155, 155 / 253, 195, 200, and 256. In some embodiments, the engineered glucose dehydrogenase polypeptide having imine reductase activity comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO:4 and one or more residue differences compared to SEQ ID NO:4 selected from the group consisting of: 96A, 96C / 118M, 147A, 147I, 147S, 155A / 253H, 155N, 195G, 200W, 256A, 256S, 256T, and 256V. In some embodiments, the engineered glucose dehydrogenase polypeptide having imine reductase activity comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO:4 and one or more residue differences compared to SEQ ID NO:4 selected from the group consisting of: E96A, E96C / T118M, H147A, H147I, H147S, F155A / Y253H, F155N, I195G, F200W, F256A, F256S, F256T, and F256V.

[0158] In some embodiments, the engineered glucose dehydrogenase polypeptide having imine reductase activity comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 14 and an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: NO: 14 has one or more residue differences selected from the group consisting of: 17, 95 / 96, 95 / 96 / 118, 95 / 96 / 118 / 156 / 200 / 253, 95 / 96 / 147, 95 / 96 / 147 / 200, 95 / 96 / 147 / 200 / 253, 95 / 96 / 155 / 156, 95 / 96 / 155 / 195 / 200, 95 / 96 / 159 / 195 / 253, 95 / 155 / 156, 95 / 155 / 159 / 200, 95 / 155 / 200, 96, 96 / 147 / 195 / 200 / 253, 96 / 155, 96 / 155 / 159, 96 / 155 / 159 / 195, 96 / 155 / 159 / 200, 96 / 156 / 159 / 195, 96 / 156 / 159 / 195 / 200, 96 / 195, 147, 147 / 155, 147 / 155 / 156, 147 / 155 / 156 / 200, 147 / 155 / 159, 147 / 195 / 200 / 253, 155, 155 / 156, 155 / 156 / 195 / 200 / 253, 155 / 159 / 200 and 253.In some embodiments, the engineered glucose dehydrogenase polypeptide having imine reductase activity comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 14 and one or more residue differences compared to SEQ ID NO: 14 selected from the group consisting of 17T, 95V / 96A / 118M / 156T / 200W / 253H, 95V / 96A / 147Q / 200W / 253H, 95V / 96C, 95V / 96C / 118M, 95V / 96C / 147Q, 95V / 96C / 147Q / 200W, 95V / 96C / 1 55A / 195G / 200W, 95V / 96C / 155N / 156Q, 95V / 96C / 159T / 195G / 253H, 95V / 155N / 156A, 95V / 155N / 159C / 200W, 95V / 155N / 200W, 96A / 147Q / 195G / 200W / 253H, 96C / 155A, 96C / 155N / 159C, 96C / 155N / 159C / 195G, 96C / 155N / 159C / 200W, 96C / 155N / 159T, 96C / 156 A / 159T / 195G / 200W, 96C / 156L / 159T / 195G, 96C / 195G, 96S, 147Q / 155N, 147Q / 155N / 1 56A / 200W, 147Q / 155N / 156T, 147Q / 155N / 159C, 147Q / 195G / 200W / 253H, 147T, 155N, 155N / 156A, 155N / 156T / 195G / 200W / 253H, 155N / 159C / 200W, 155Q, 155R, 253G, and 253S.In some embodiments, the engineered glucose dehydrogenase polypeptide having imine reductase activity comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 14 and an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 14. ID NO: 14 and one or more residue differences selected from the group consisting of: S17T, L95V / E96A / T118M / V156T / F200W / Y253H, L95V / E96A / I147Q / F200W / Y253H, L95V / E96C, L95V / E96C / T118M, L95V / E96C / I147Q, L95V / E96C / I147Q / F200W, L95V / E96C / F 155A / I195G / F200W, L95V / E96C / F155N / V156Q, L95V / E96C / A159T / I195G / Y253H, L95V / F155N / V156A, L95V / F155N / A159C / F200W, L95V / F155N / F200W, E96A / I147Q / I195G / F200W / Y253H, E96C / F155A, E96C / F155N / A159C, E96C / F155N / A159C / I195G, E96C / F155N / A159C / F200W, E96C / F155N / A159T, E96C / V15 6A / A159T / I195G / F200W, E96C / V156L / A159T / I195G, E96C / I195G, E96S, I147Q / F155N, I147Q / F155N / 39C / F200W, F155N, F155N / V156A, F155N / V156T, I147Q / F155N / A159C, I147Q / I195G / F200W / Y253H, I147T, F155N, F155N / V156A, F155N / V156T / I195G / F200W / Y253H, F155N / A159C / F200W, F155Q, F155R, Y253G, and Y253S.

[0159] In some embodiments, the engineered glucose dehydrogenase polypeptide having imine reductase activity comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 34 and an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: NO:34 differs from one or more residues selected from the group consisting of: 17, 17 / 21, 17 / 21 / 42, 17 / 21 / 42 / 155 / 260, 17 / 21 / 49, 17 / 21 / 49 / 51, 17 / 21 / 49 / 51 / 96, 17 / 21 / 49 / 51 / 260, 17 / 21 / 49 / 96 / 155 / 260, 17 / 21 / 49 / 260, 17 / 21 / 51 / 96, 17 / 21 / 51 / 195, 17 / 21 / 96, 17 / 21 / 96 / 155, 17 / 21 / 96 / 195 / 260, 17 / 21 / 9 6 / 253、17 / 21 / 147 / 155 / 253、17 / 21 / 155 / 253、17 / 21 / 195、17 / 21 / 253、17 / 42、17 / 42 / 49 / 195 / 260、17 / 42 / 96、17 / 44、17 / 44 / 47 / 51 / 96、17 / 44 / 47 / 51 / 96 / 114 / 147 / 195 / 199 / 253、17 / 44 / 47 / 51 / 96 / 114 / 147 / 199、17 / 44 / 47 / 51 / 96 / 114 / 195 / 199、17 / 44 / 47 / 51 / 96 / 147 / 253、17 / 44 / 47 / 51 / 178 / 195、17 / 44 / 47 / 195 / 199、17 / 44 / 51 / 114 / 195 / 253、17 / 46 / 47 / 51 / 96 / 147、17 / 46 / 47 / 51 / 96 / 195 / 253、17 / 46 / 47 / 51 / 155、17 / 46 / 47 / 51 / 155 / 195 / 199、17 / 46 / 47 / 96 / 195、17 / 46 / 47 / 178、17 / 46 / 96 / 253、17 / 47、17 / 47 / 51、17 / 47 / 51 / 9 6 / 114 / 147 / 195、17 / 47 / 51 / 96 / 114 / 155 / 178 / 253、17 / 47 / 51 / 96 / 195、17 / 47 / 51 / 96 / 195 / 199、17 / 47 / 96 / 114 / 155 / 253、17 / 47 / 96 / 147 / 155 / 195 / 199、17 / 47 / 96 / 147 / 195 / 199 / 253、17 / 47 / 96 / 155、17 / 47 / 96 / 195 / 199、17 / 47 / 114、17 / 47 / 114 / 155 / 195 / 199 / 253、17 / 47 / 147 / 195 / 199 / 253、17 / 49、17 / 49 / 51、17 / 49 / 51 / 96 / 195 / 253、17 / 49 / 51 / 155 / 253、17 / 49 / 51 / 155 / 253 / 260、17 / 49 / 51 / 155 / 260、17 / 49 / 51 / 253、17 / 49 / 96、17 / 49 / 96 / 155 / 253、17 / 49 / 96 / 195、17 / 49 / 260、17 / 51、17 / 51 / 96 / 114、17 / 51 / 96 / 155 / 178、17 / 51 / 96 / 155 / 195 / 199 / 253、17 / 51 / 96 / 155 / 199、17 / 51 / 96 / 155 / 253、17 / 51 / 96 / 178 / 195 / 199、17 / 51 / 96 / 195、17 / 51 / 96 / 260、17 / 51 / 114、17 / 51 / 114 / 253、17 / 51 / 147、17 / 51 / 155 / 195、17 / 51 / 178 / 195 / 253、17 / 51 / 253、17 / 96、17 / 96 / 114 / 147 / 155、17 / 96 / 114 / 147 / 155 / 178 / 195 / 199、17 / 96 / 147、17 / 96 / 147 / 155、17 / 96 / 155 / 178、17 / 96 / 155 / 178 / 253、17 / 96 / 155 / 195、17 / 96 / 155 / 195 / 199、17 / 96 / 178、17 / 96 / 178 / 195、17 / 96 / 195、17 / 96 / 195 / 199、17 / 96 / 195 / 199 / 253、17 / 96 / 253、17 / 114、17 / 114 / 253、17 / 147 / 155 / 253、17 / 147 / 178、17 / 147 / 199、17 / 155、17 / 155 / 178、17 / 155 / 178 / 195、17 / 155 / 195、17 / 155 / 195 / 253、17 / 155 / 199、17 / 155 / 253、17 / 178、17 / 178 / 195 / 253、17 / 195、17 / 195 / 199、17 / 195 / 199 / 253、17 / 195 / 253、17 / 199、17 / 253、17 / 260、21 / 49 / 51、21 / 49 / 96 / 195、21 / 49 / 96 / 253、21 / 49 / 195、21 / 51 / 96 / 155、21 / 96、21 / 96 / 195、21 / 96 / 253、44 / 46 / 47 / 51 / 114 / 178 / 253、44 / 46 / 47 / 51 / 155 / 178 / 253、44 / 46 / 47 / 96 / 155 / 195、44 / 46 / 51 / 96 / 147 / 178 / 195 / 199、44 / 46 / 51 / 96 / 155 / 195 / 199 / 253、44 / 46 / 51 / 96 / 195、44 / 46 / 51 / 147 / 155 / 178 / 195 / 253、44 / 47 / 51 / 96 / 114、44 / 47 / 51 / 96 / 177 / 178 / 199 / 253、44 / 47 / 51 / 114 / 178 / 253、44 / 47 / 96 / 114、44 / 47 / 96 / 195 / 253、44 / 47 / 147 / 155、44 / 47 / 147 / 155 / 199、44 / 96 / 155 / 178、46 / 47 / 51 / 96 / 114 / 178、46 / 47 / 51 / 114 / 147 / 195 / 199、46 / 47 / 51 / 155 / 195 / 199、46 / 47 / 96 / 114 / 195 / 199、46 / 47 / 96 / 155 / 178 / 195 / 253、46 / 47 / 155 / 195 / 199、46 / 114 / 147 / 155 / 178 / 195 / 199、46 / 114 / 195 / 199 / 253、47 / 51 / 96、47 / 51 / 96 / 114、47 / 51 / 96 / 147 / 155 / 195 / 199、47 / 51 / 96 / 147 / 195 / 199、47 / 51 / 96 / 178 / 195 / 199 / 253、47 / 51 / 96 / 195、47 / 51 / 96 / 195 / 199 / 253、47 / 51 / 114 / 253、47 / 51 / 155 / 178 / 195、47 / 51 / 155 / 195 / 253、47 / 96 / 114、47 / 96 / 114 / 253、47 / 96 / 155 / 178 / 195、47 / 96 / 155 / 195、47 / 96 / 155 / 195 / 199、47 / 96 / 178 / 195 / 253、47 / 96 / 178 / 253、47 / 96 / 195 / 199 / 253、47 / 96 / 195 / 253、47 / 114、47 / 114 / 155 / 195 / 199、47 / 114 / 178、47 / 147 / 195、47 / 155 / 253、47 / 178 / 195、47 / 178 / 195 / 199、47 / 195 / 199、49 / 51 / 96、49 / 96、49 / 96 / 253、51 / 96、51 / 96 / 114 / 155 / 195 / 199 / 253、51 / 96 / 155 / 178、51 / 96 / 155 / 178 / 195 / 253、51 / 96 / 155 / 195、51 / 96 / 155 / 195 / 253、51 / 96 / 195 / 253、51 / 96 / 199、51 / 96 / 253、51 / 114 / 155 / 195 / 199、51 / 114 / 195 / 199、51 / 147 / 253、51 / 155 / 195 / 199、51 / 195、51 / 253、96、96 / 114 / 147 / 155 / 195 / 199、96 / 114 / 155 / 178 / 195 / 199 / 253、96 / 114 / 155 / 195 / 253、96 / 114 / 199, 96 / 147, 96 / 147 / 155 / 178 / 195 / 253, 96 / 147 / 178 / 195 / 199, 96 / 147 / 195, 96 / 147 / 195 / 199, 96 / 147 / 253, 96 / 155 / 178 / 199, 96 / 155 / 195, 96 / 155 / 199, 96 / 155 / 253, 96 / 178 / 195, 96 / 178 / 195 / 199、96 / 195、96 / 195 / 199、96 / 195 / 253、96 / 199、96 / 199 / 253、96 / 253、114、114 / 147 / 155 / 253、114 / 178 / 195 / 199 / 253、114 / 195、114 / 195 / 199 / 253、132、147 / 155、149、154、155 / 178, 155 / 178 / 195 / 199, 155 / 195, 155 / 195 / 199, 155 / 195 / 199 / 253, 155 / 195 / 253, 155 / 253, 157, 167, 170, 178, 178 / 195, 178 / 199, 195, 196, 198, 214, 218, 251, 252, 256, 257, 259 and 260. In some embodiments, the engineered glucose dehydrogenase polypeptide having imine reductase activity comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 34 and an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: NO:34 has one or more residue differences selected from the group consisting of: 17T, 17T / 21R, 17T / 21R / 42T, 17T / 21R / 42T / 155R / 260G, 17T / 21R / 49A, 17T / 21R / 49A / 51L, 17T / 21R / 49A / 51L / 96S, 17T / 21R / 49A / 51L / 260G, 17T / 21R / 49A / 96S / 155R / 260G, 17T / 21R / 49A / 260G, 17T / 21R / 51L / 96S, 17T / 21R / 51L / 195N, 17T / 21R / 96S, 17T / 21R / 96S / 155R,17T / 21R / 96S / 195N / 260G、17T / 21R / 96S / 253L、17T / 21R / 147T / 155R / 253L、17T / 21R / 155R / 253L、17T / 21R / 195N、17T / 21R / 253L、17T / 42T、17T / 42T / 49A / 195N / 260G、17T / 42T / 96S、17T / 44N、17T / 44N / 47A / 51A / 96A、17T / 44N / 47A / 195N / 199R、17T / 44N / 47L / 51A / 96A / 114A / 147T / 195G / 199R / 253G、17T / 44N / 47L / 51A / 96S / 114A / 147T / 199R、17T / 44N / 47L / 51A / 96T / 114A / 195G / 199R、17T / 44N / 47S / 51A / 96T / 147T / 253G、17T / 44N / 47S / 51A / 178S / 195N、17T / 44N / 47S / 195G / 199R、17T / 44N / 51A / 114A / 195N / 253G、17T / 46W / 47A / 96C / 195G、17T / 46W / 47A / 178S、17T / 46W / 47L / 51A / 96C / 195G / 253R、17T / 46W / 47L / 51A / 96T / 147T、17T / 46W / 47L / 51A / 155Q、17T / 46W / 47L / 51A / 155R / 195G / 199R、17T / 46W / 96C / 253G、17T / 47A / 51A、17T / 47A / 51A / 96T / 114A / 147T / 195G、17T / 47A / 51A / 96T / 114A / 155R / 178S / 253R、17T / 47A / 96A / 155Q、17T / 47A / 96T / 147T / 195G / 199R / 253G、17T / 47A / 147T / 195G / 199R / 253S、17T / 47L / 51A / 96T / 195G、17T / 47L / 51A / 96T / 195G / 199R、17T / 47L / 96A / 114A / 155Q / 253G、17T / 47L / 114A、17T / 47L / 114A / 155R / 195G / 199R / 253S、17T / 47S、17T / 47S / 96C / 147T / 155Q / 195G / 199R、17T / 47S / 96C / 195N / 199R、17T / 49A、17T / 49A / 51L、17T / 49A / 51L / 96S / 195N / 253L、17T / 49A / 51L / 155R / 253L、17T / 49A / 51L / 155R / 253L / 260G、17T / 49A / 51L / 155R / 260G、17T / 49A / 51L / 253L、17T / 49A / 96S、17T / 49A / 96S / 155R / 253L、17T / 49A / 96S / 195N、17T / 49A / 260G、17T / 51A / 96C / 155Q / 195G / 199R / 253G、17T / 51A / 96C / 155R / 178S、17T / 51A / 96C / 195N、17T / 51A / 96S / 114A、17T / 51A / 96S / 155Q / 199R、17T / 51A / 96T / 155R / 195G / 199R / 253S、17T / 51A / 96T / 178S / 195G / 199R、17T / 51A / 114A、17T / 51A / 114A / 253R、17T / 51A / 147T、17T / 51A / 155R / 195G、17T / 51A / 178S / 195G / 253G、17T / 51L、17T / 51L / 96S / 155R / 253L、17T / 51L / 96S / 260G、17T / 51L / 253L、17T / 96A / 147T / 155R、17T / 96A / 155Q / 195G / 199R、17T / 96A / 155R / 178S / 253G、17T / 96A / 195N / 199R、17T / 96C、17T / 96C / 114A / 147T / 155Q、17T / 96C / 178S / 195G、17T / 96C / 195G / 199R、17T / 96S / 114A / 147T / 155Q / 178S / 195G / 199R、17T / 96S / 155R / 178S、17T / 96S / 155R / 195N、17T / 96S / 178S、17T / 96S / 195N、17T / 96S / 253L、17T / 96T、17T / 96T / 147T、17T / 96T / 195G / 199R、17T / 96T / 195G / 199R / 253R、17T / 96T / 253R、17T / 114A、17T / 114A / 253R、17T / 147T / 155Q / 253G、17T / 147T / 178S、17T / 147T / 199R、17T / 155Q、17T / 155Q / 178S、17T / 155Q / 195G / 253S、17T / 155Q / 199R、17T / 155R、17T / 155R / 178S、17T / 155R / 178S / 195G、17T / 155R / 195G、17T / 155R / 253G、17T / 155R / 253L、17T / 178S、17T / 178S / 195N / 253S、17T / 195G、17T / 195G / 199R、17T / 195G / 199R / 253S、17T / 195N、17T / 195N / 199R、17T / 195N / 253L、17T / 199R、17T / 253L、17T / 260G、21R / 49A / 51L、21R / 49A / 96S / 195N、21R / 49A / 96S / 253L、21R / 49A / 195N、21R / 51L / 96S / 155R、21R / 96S、21R / 96S / 195N、21R / 96S / 253L、44N / 46W / 47A / 51A / 155R / 178S / 253G、44N / 46W / 47L / 51A / 114A / 178S / 253G、44N / 46W / 47L / 96C / 155R / 195G、44N / 46W / 51A / 96A / 147T / 178S / 195G / 199R、44N / 46W / 51A / 96T / 155R / 195G / 199R / 253G、44N / 46W / 51A / 96T / 195G、44N / 46W / 51A / 147T / 155R / 178S / 195G / 253G、44N / 47A / 51A / 96T / 177T / 178S / 199R / 253S、44N / 47L / 96T / 114A、44N / 47S / 51A / 96S / 114A、44N / 47S / 51A / 114A / 178S / 253S、44N / 47S / 96T / 195N / 253G、44N / 47S / 147T / 155Q、44N / 47S / 147T / 155Q / 199R、44N / 96C / 155R / 178S、46W / 47A / 51A / 114A / 147T / 195G / 199R、46W / 47A / 51A / 155R / 195G / 199R、46W / 47L / 51A / 96T / 114A / 178S、46W / 47L / 96C / 155R / 178S / 195G / 253S、46W / 47L / 96T / 114A / 195N / 199R、46W / 47L / 155R / 195N / 199R、46W / 114A / 147T / 155Q / 178S / 195G / 199R、46W / 114A / 195G / 199R / 253S、47A / 51A / 96C / 178S / 195G / 199R / 253G、47A / 51A / 96T / 195G、47A / 51A / 155R / 195G / 253G、47A / 96S / 114A、47A / 96S / 195N / 253S、47A / 96T / 155Q / 178S / 195G、47A / 96T / 155Q / 195N / 199R、47A / 96T / 178S / 195N / 253R、47A / 114A、47A / 147T / 195G、47A / 178S / 195N / 199R、47L / 51A / 96C / 147T / 195G / 199R、47L / 51A / 96C / 195G / 199R / 253G、47L / 51A / 96S / 147T / 155Q / 195G / 199R、47L / 51A / 155Q / 195G / 253S、47L / 96A / 195G / 199R / 253S、47L / 96T / 114A / 253R、47L / 96T / 178S / 253G、47L / 114A / 155R / 195G / 199R、47L / 114A / 178S、47L / 195N / 199R、47S / 51A / 96A、47S / 51A / 96C / 114A、47S / 51A / 96C / 195G、47S / 51A / 114A / 253S、47S / 51A / 155R / 178S / 195N、47S / 96S / 155R / 195G、47S / 96T / 195G / 253S、47S / 155R / 253G、47S / 178S / 195G、49A / 51L / 96S、49A / 96S、49A / 96S / 253L、51A / 96C / 114A / 155Q / 195G / 199R / 253G、51A / 96C / 155Q / 195G / 253G、51A / 96C / 155R / 178S、51A / 96C / 155R / 178S / 195G / 253R、51A / 96S / 155Q / 195G、51A / 96S / 199R、51A / 96T / 195G / 253G、51A / 96T / 199R、51A / 96T / 253R、51A / 114A / 155Q / 195G / 199R、51A / 114A / 195G / 199R、51A / 147T / 253S、51A / 155Q / 195N / 199R、51A / 195G、51A / 195N、51A / 253G、51L / 96S、51L / 195N、96A、96A / 114A / 155R / 178S / 195G / 199R / 253G、96A / 147T、96A / 147T / 155R / 178S / 195G / 253G、96A / 147T / 195G、96A / 147T / 195G / 199R、96A / 147T / 253R、96A / 195G、96A / 195G / 199R、96C、96C / 114A / 147T / 155R / 195G / 199R、96C / 147T / 195G / 199R、96C / 155R / 195G、96C / 178S / 195G / 199R, 96C / 195G / 199R, 96C / 199R, 96C / 253S, 96G, 96K, 96N, 96S, 96S / 114A / 155Q / 195N / 253S, 96S / 147T, 96S / 155R / 253L, 96S / 195N, 96S / 195N / 253L, 96S / 253G, 96S / 253S, 96T, 96T / 114A / 199R, 96T / 147T / 178S / 195 N / 199R, 96T / 155Q / 178S / 199R, 96T / 155Q / 199R, 96T / 178S / 195G, 96T / 178S / 195N, 96T / 195G, 96T / 195G / 253G, 96T / 195G / 253R, 96T / 195N / 199R, 96T / 199R, 96T / 199R / 253S, 96T / 253G, 114A, 114A / 147T / 155Q / 253S, 114A / 178S / 195G / 199R / 253S, 114A / 195G, 114A / 195G / 199R / 253G, 132L, 147T / 155R, 149D, 149E, 154E, 155Q / 178S, 155Q / 178S / 195G / 199R、155Q / 178S / 195N / 199R、155Q / 195G / 253G、155Q / 195N、155Q / 195N / 199R、155R / 195G / 199R / 253G、155R / 195N 199R, 199R / 253R, 199R / 253R, 199R / 253R, 199R / 253N / 253L, 199R / 253S, 199R / 253R, 199R / 253N / 253L, 199R / 253S, 199R / 253R, 199R / 253N / 253L, 199R / 253S, 199N, 199G, 199R, 199R / 253R, 199R / 253N / 253L, 199R / 253S, 199N, 199G, 199R, 199R / 253R, 199R / 253N / 253L, 199R / 253S In some embodiments, the engineered glucose dehydrogenase polypeptide having imine reductase activity comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 34 and one or more residue differences compared to SEQ ID NO: 34 selected from the group consisting of S17T, S17T / K21R, S17T / K21R / K42T, S17T / K21R / K42T / N155R / R260G, S17T / K21R / K49A,S17T / K21R / K49A / E51L、S17T / K21R / K49A / E51L / E96S、S17T / K21R / K49A / E51L / R260G、S17T / K21R / K49A / E96S / N155R / R260G、S17T / K21R / K49A / R260G、S17T / K21R / E51L / E96S、S17T / K21R / E51L / I195N、S17T / K21R / E96S、S17T / K21R / E96S / N155R、S17T / K21R / E96S / I195N / R260G、S17T / K21R / E96S / Y253L、S17T / K21R / I147T / N155R / Y253L、S17T / K21R / N155R / Y253L、S17T / K21R / I195N、S17T / K21R / Y253L、S17T / K42T、S17T / K42T / K49A / I195N / R260G、S17T / K42T / E96S、S17T / D44N、S17T / D44N / E47A / E51A / E96A、S17T / D44N / E47A / I195N / K199R、S17T / D44N / E47L / E51A / E96A / G114A / I147T / I195G / K199R / Y253G、S17T / D44N / E47L / E51A / E96S / G114A / I147T / K199R、S17T / D44N / E47L / E51A / E96T / G114A / I195G / K199R、S17T / D44N / E47S / E51A / E96T / I147T / Y253G、S17T / D44N / E47S / E51A / P178S / I195N、S17T / D44N / E47S / I195G / K199R、S17T / D44N / E51A / G114A / I195N / Y253G、S17T / N46W / E47A / E96C / I195G、S17T / N46W / E47A / P178S、S17T / N46W / E47L / E51A / E96C / I195G / Y253R、S17T / N46W / E47L / E51A / E96T / I147T、S17T / N46W / E47L / E51A / N155Q、S17T / N46W / E47L / E51A / N155R / I195G / K199R、S17T / N46W / E96C / Y253G、S17T / E47A / E51A、S17T / E47A / E51A / E96T / G114A / I147T / I195G、<h2 style=";text-align:left;direction:ltr">S17T / E47A / E51A / E96T / G114A / N155R / P178S / Y253R、S17T / E47A / E96A / N155Q、S17T / E47A / E96T / I147T / I195G / K199R / Y253G、S17T / E47A / I147T / I19 5G / K199R / Y253S、S17T / E47L / E51A / E96T / I195G、S17T / E47L / E51A / E96T / I 195G / K199R、S17T / E47L / E96A / G114A / N155Q / Y253G、S17T / E47L / G114A、S1 7T / E47L / G114A / N155R / I195G / K199R / Y253S、S17T / E47S、S17T / E47S / E96C / I147T / N155Q / I195G / K199R、S17T / E47S / E96C / I195N / K199R、S17T / K49A、 S17T / K49A / E51L, S17T / K49A / E51L / E96S / I195N / Y253L, S17T / K49A / E51L / N155R / Y253L, S17T / K49A / E51L / N155R / Y253L / R260G, S17T / K49A / E51L / N1 55R / R260G, S17T / K49A / E51L / Y253L, S17T / K49A / E96S, S17T / K49A / E96S / N155R / Y253L, S17T / K49A / E96S / I195N, S17T / K49A / R260G, S17T / E51A / E96 C / N155Q / I195G / K199R / Y253G、S17T / E51A / E96C / N155R / P178S、S17T / E51A / E96C / I195N、S17T / E51A / E96S / G114A、S17T / E51A / E96S / N155Q / K199R、S1 7T / E51A / E96T / N155R / I195G / K199R / Y253S、S17T / E51A / E96T / P178S / I195 G / K199R、S17T / E51A / G114A、S17T / E51A / G114A / Y253R、S17T / E51A / I147T、 S17T / E51A / N155R / I195G、S17T / E51A / P178S / I195G / Y253G、S17T / E51L、S17T / E51L / E96S / N155R / Y253L、S17T / E51L / E96S / R260G、S17T / E51L / Y253L、S17T / E96A / I147T / N155R、S17T / E96A / N155Q / I195G / K199R、S17T / E96A / N155R / P178S / Y253G、S17T / E96A / I195N / K199R、S17T / E96C、S17T / E96C / G114A / I147T / N155Q、S17T / E96C / P178S / I195G、S17T / E96C / I195G / K199R、S17T / E96S / G114A / I147T / N155Q / P178S / I195G / K199R、S17T / E96S / N155R / P178S、S17T / E96S / N155R / I195N、S17T / E96S / P178S、S17T / E96S / I195N、S17T / E96S / Y253L、S17T / E96T、S17T / E96T / I147T、S17T / E96T / I195G / K199R、S17T / E96T / I195G / K199R / Y253R、S17T / E96T / Y253R、S17T / G114A、S17T / G114A / Y253R、S17T / I147T / N155Q / Y253G、S17T / I147T / P178S、S17T / I147T / K199R、S17T / N155Q、S17T / N155Q / P178S、S17T / N155Q / I195G / Y253S、S17T / N155Q / K199R、S17T / N155R、S17T / N155R / P178S、S17T / N155R / P178S / I195G、S17T / N155R / I195G、S17T / N155R / Y253G、S17T / N155R / Y253L、S17T / P178S、S17T / P178S / I195N / Y253S、S17T / I195G、S17T / I195G / K199R、S17T / I195G / K199R / Y253S、S17T / I195N、S17T / I195N / K199R、S17T / I195N / Y253L、S17T / K199R、S17T / Y253L、S17T / R260G、K21R / K49A / E51L、K21R / K49A / E96S / I195N、K21R / K49A / E96S / Y253L、K21R / K49A / I195N、K21R / E51L / E96S / N155R、K21R / E96S、K21R / E96S / I195N、K21R / E96S / Y253L、D44N / N46W / E47A / E51A / N155R / P178S / Y253G、D44N / N46W / E47L / E51A / G114A / P178S / Y253G、D44N / N46W / E47L / E96C / N155R / I195G、D44N / N46W / E51A / E96A / I147T / P178S / I195G / K199R、D44N / N46W / E51A / E96T / N155R / I195G / K199R / Y253G、D44N / N46W / E51A / E96T / I195G、D44N / N46W / E51A / I147T / N155R / P178S / I195G / Y253G、D44N / E47A / E51A / E96T / A177T / P178S / K199R / Y253S、D44N / E47L / E96T / G114A、D44N / E47S / E51A / E96S / G114A、D44N / E47S / E51A / G114A / P178S / Y253S、D44N / E47S / E96T / I195N / Y253G、D44N / E47S / I147T / N155Q、D44N / E47S / I147T / N155Q / K199R、D44N / E96C / N155R / P178S、N46W / E47A / E51A / G114A / I147T / I195G / K199R、N46W / E47A / E51A / N155R / I195G / K199R、N46W / E47L / E51A / E96T / G114A / P178S、N46W / E47L / E96C / N155R / P178S / I195G / Y253S、N46W / E47L / E96T / G114A / I195N / K199R、N46W / E47L / N155R / I195N / K199R、N46W / G114A / I147T / N155Q / P178S / I195G / K199R、N46W / G114A / I195G / K199R / Y253S、E47A / E51A / E96C / P178S / I195G / K199R / Y253G、E47A / E51A / E96T / I195G、E47A / E51A / N155R / I195G / Y253G、E47A / E96S / G114A、E47A / E96S / I195N / Y253S、E47A / E96T / N155Q / P178S / I195G、E47A / E96T / N155Q / I195N / K199R、E47A / E96T / P178S / I195N / Y253R、E47A / G114A、E47A / I147T / I195G、E47A / P178S / I195N / K199R、E47L / E51A / E96C / I147T / I195G / K199R、E47L / E51A / E96C / I195G / K199R / Y253G、E47L / E51A / E96S / I147T / N155Q / I195G / K199R、E47L / E51A / N155Q / I195G / Y253S、E47L / E96A / I195G / K199R / Y253S、E47L / E96T / G114A / Y253R、E47L / E96T / P178S / Y253G、E47L / G114A / N155R / I195G / K199R、E47L / G114A / P178S、E47L / I195N / K199R、E47S / E51A / E96A、E47S / E51A / E96C / G114A、E47S / E51A / E96C / I195G、E47S / E51A / G114A / Y253S、E47S / E51A / N155R / P178S / I195N、E47S / E96S / N155R / I195G、E47S / E96T / I195G / Y253S、E47S / N155R / Y253G、E47S / P178S / I195G、K49A / E51L / E96S、K49A / E96S、K49A / E96S / Y253L、E51A / E96C / G114A / N155Q / I195G / K199R / Y253G、E51A / E96C / N155Q / I195G / Y253G、E51A / E96C / N155R / P178S、E51A / E96C / N155R / P178S / I195G / Y253R、E51A / E96S / N155Q / I195G、E51A / E96S / K199R、E51A / E96T / I195G / Y253G、E51A / E96T / K199R、E51A / E96T / Y253R、E51A / G114A / N155Q / I195G / K199R、E51A / G114A / I195G / K199R、E51A / I147T / Y253S、E51A / N155Q / I195N / K199R、E51A / I195G、E51A / I195N、E51A / Y253G、E51L / E96S、E51L / I195N、E96A、E96A / G114A / N155R / P178S / I195G / K199R / Y253G、E96A / I147T、E96A / I147T / N155R / P178S / I195G / Y253G、E96A / I147T / I195G、E96A / I147T / I195G / K199R、E96A / I147T / Y253R、E96A / I195G、E96A / I195G / K199R、E96C、E96C / G114A / I147T / N155R / I195G / K199R、E96C / I147T / I195G / K199R、E96C / N155R / I195G、E96C / P178S / I195G / K199R、E96C / I195G / K199R、E96C / K199R、E96C / Y253S、E96G、E96K、E96N、E96S、E96S / G114A / N155Q / I195N / Y253S、E96S / I147T、E96S / N155R / Y253L、E96S / I195N、E96S / I195N / Y253L、E96S / Y253G、E96S / Y253S、E96T、E96T / G114A / K199R、E96T / I147T / P178S / I195N / K199R、E96T / N155Q / P178S / K199R、E96T / N155Q / K199R、E96T / P178S / I195G、E96T / P178S / I195N、E96T / I195G、E96T / I195G / Y253G、E96T / I195G / Y253R、E96T / I195N / K199R、E96T / K199R、E96T / K199R / Y253S、E96T / Y253G、G114A、G114A / I147T / N155Q / Y253S、G114A / P178S / I195G / K199R / Y253S、G114A / I195G、G114A / I195G / K199R / Y253G、V132L、I147T / N155R、V149D、V149E、L154E、N155Q / P178S、N155Q / P178S / I195G / K199R、N155Q / P178S / I195N / K199R、N155Q / I195G / Y253G、N155Q / I195N、N155Q / I195N / K199R、N155R / I195G / K199R / Y253G、N155R / I195N / K199R / Y253R、N155R / I195N / Y253L、N155R / Y253S、H157N、L167G、L167Q、K170Q、P178A、P178G、P178K、P178S、P178S / I195N、P178S / K199R、I195G、I195N、I195R、I195S、N196G、N196H, E198H, P214G, I218P, T251A, T251L, L252A, L252T, F256I, Q257E, Q257W, G259D, G259E, and R260E.

[0160] In some embodiments, the engineered glucose dehydrogenase polypeptide having imine reductase activity comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 112 and an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: NO: 112 has one or more residue differences selected from the group consisting of: 21 / 42 / 96 / 178 / 195 / 253, 21 / 42 / 96 / 195, 21 / 96, 21 / 96 / 147 / 195, 21 / 96 / 149, 21 / 96 / 155 / 195 / 253, 21 / 96 / 167, 21 / 96 / 167 / 195, 21 / 96 / 178, 21 / 96 / 179 / 195 / 251 / 253, 21 / 96 / 195, 21 / 147 / 149, 21 / 147 / 253, 21 / 149 / 178 / 195, 21 / 149 / 178 / 195 1 / 155 / 178 / 253 / 256、21 / 178 / 195 / 256、42 / 96 / 149 / 253 / 256、94、96、96 / 114 / 155 / 198 / 218 / 253 / 256 / 257、96 / 114 / 155 / 252 / 253 / 257、96 / 114 / 155 / 253、96 / 114 / 196 / 214 / 253 / 256、96 / 114 / 214 / 253 / 256、96 / 147、96 / 147 / 149、96 / 147 / 149 / 155、96 / 147 / 167 / 195, 96 / 149 / 155 / 167 / 178 / 253, 96 / 149 / 155 / 195, 96 / 149 / 178 / 179 / 251 / 256, 96 / 149 / 178 / 253, 96 / 149 / 198 / 252 / 253 / 257, 96 / 155, 96 / 155 / 167, 96 / 155 / 167 / 178, 96 / 155 / 195, 96 / 155 / 196 / 218 / 253 / 256 / 257, 96 / 155 / 196 / 252 / 253, 96 / 155 / 198, 96 / 155 5 / 252 / 253、96 / 167、96 / 178、96 / 178 / 179 / 195、96 / 195、96 / 196、96 / 196 / 198、96 / 196 / 201 / 256、96 / 251 / 253 / 256、96 / 253 / 257、96 / 256 / 257、98、103、114 / 253 / 256 / 257、147、147 / 149、149 / 167、149 / 195、149 / 218、149 / 253、149 / 257、155 / 167、155 / 178 / 195、155 / 178 / 195 / 251 / 253, 155 / 195 / 251 / 253 / 256, 155 / 195 / 253 / 256, 155 / 196 / 198 / 253, 167, 167 / 195, 167 / 195 / 251 / 253, 196, 196 / 253 / 256 / 257, 198 / 253 / 256, 198 / 256, 205, 214 / 253 / 256, 251 and 257. In some embodiments, the engineered glucose dehydrogenase polypeptide having imine reductase activity comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 112 and an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: NO: 112 has one or more residue differences selected from the group consisting of: 21R / 42T / 96C / 178S / 195N / 253G, 21R / 42T / 96S / 195R, 21R / 96C / 147T / 195R, 21R / 96C / 149E, 21R / 96C / 178S, 21R / 96C / 195R, 21R / 96G / 155N / 195R / 253G, 21R / 96G / 167Q / 195N, 21R / 96K / 167Q, 21R / 96S, 21R / 96S / 178S, 21R / 96S / 179N / 195N / 251A / 253G, 21R / 147T / 149E, 21R / 147T / 253G , 21R / 149E / 178S / 195N, 21R / 155N / 178S / 253G / 256I, 21R / 178S / 195R / 256I, 42T / 96C / 149E / 253G / 256I, 94A, 96A / 149E / 155N / 167Q / 178S / 253G, 96A / 155N / 167Q, 96C, 96C / 114G / 155N / 252A / 253G / 257W, 96C / 1 14G / 155N / 253R, 96C / 114G / 196G / 214G / 253G / 256I, 96C / 147T, 96C / 147T / 149E / 155C, 96C / 147T / 167Q / 1 95N、96C / 149D / 198H / 252A / 253R / 257E、96C / 149E / 155N / 195N、96C / 149E / 155N / 195R、96C / 155N / 196G / 218P / 253G / 256I / 257E, 96C / 155N / 196G / 252T / 253G, 96C / 155N / 198H, 96C / 155N / 252T / 253R, 96C / 195N,96C / 196G / 198H, 96C / 196G / 201P / 256I, 96G / 149E / 178S / 253G, 96G / 155N / 167Q, 96G / 155N / 195R, 96K / 155N, 96K / 178S, 96S, 96S / 114G / 155N / 198H / 218P / 253R / 256I / 257E, 96S / 114G / 214G / 253G / 256I, 96S / 147T / 149E, 96S / 149E / 178S / 179N / 251A / 256I, 96S / 155N, 96S / 155N / 167Q / 178S, 96S / 167Q, 96S / 178 S, 96S / 178S / 179N / 195N, 96S / 195N, 96S / 196G, 96S / 251A / 253G / 256I, 96S / 253G / 257E, 96S / 256I / 257 E, 98A, 103L, 114G / 253G / 256I / 257E, 147A, 147R, 147T, 147T / 149E, 149D / 257E, 149E / 167Q, 149E / 195 N, 149E / 218P, 149E / 253G, 155N / 167Q, 155N / 178S / 195R, 155N / 178S / 195R / 251A / 253G, 155N / 195N / 25 1A / 253G / 256I, 155N / 195N / 253G / 256I, 155N / 196G / 198H / 253G, 167Q, 167Q / 195N, 167Q / 195N / 251A / 253G, 196A, 196G / 253G / 256I / 257W, 198H / 253G / 256I, 198H / 256I, 205E, 214G / 253G / 256I, 251L, and 257E. In some embodiments, the engineered glucose dehydrogenase polypeptide having imine reductase activity comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 112 and an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: NO: 112 has one or more residue differences selected from the group consisting of K21R / K42T / E96C / P178S / G195N / S253G, K21R / K42T / E96S / G195R, K21R / E96C / I147T / G195R, K21R / E96C / V149E, K21R / E96C / P178S, K21R / E96C / G195R, K21R / E96G / R155N / G195R / S253G,K21R / E96G / L167Q / G195N、K21R / E96K / L167Q、K21R / E96S、K21R / E96S / P178S、K21R / E96S / K179N / G195N / T251A / S253G、K21R / I147T / V149E、K21R / I147T / S253G、K21R / V149E / P178S / G195N、K21R / R155N / P178S / S253G / F256I、K21R / P178S / G195R / F256I、K42T / E96C / V149E / S253G / F256I、G94A、E96A / V149E / R155N / L167Q / P178S / S253G、E96A / R155N / L167Q、E96C、E96C / A114G / R155N / L252A / S253G / Q257W、E96C / A114G / R155N / S253R、E96C / A114G / N196G / P214G / S253G / F256I、E96C / I147T、E96C / I147T / V149E / R155C、E96C / I147T / L167Q / G195N、E96C / V149D / E198H / L252A / S253R / Q257E、E96C / V149E / R155N / G195N、E96C / V149E / R155N / G195R、E96C / R155N / N196G / I218P / S253G / F256I / Q257E、E96C / R155N / N196G / L252T / S253G、E96C / R155N / E198H、E96C / R155N / L252T / S253R、E96C / G195N、E96C / N196G / E198H、E96C / N196G / A201P / F256I、E96G / V149E / P178S / S253G、E96G / R155N / L167Q、E96G / R155N / G195R、E96K / R155N、E96K / P178S、E96S、E96S / A114G / R155N / E198H / I218P / S253R / F256I / Q257E、E96S / A114G / P214G / S253G / F256I、E96S / I147T / V149E、E96S / V149E / P178S / K179N / T251A / F256I、E96S / R155N、E96S / R155N / L167Q / P178S、E96S / L167Q、E96S / P178S、E96S / P178S / K179N / G195N、E96S / G195N、E96S / N196G, E96S / T251A / S253G / F256I, E96S / S253G / Q257E, E96S / F2 56I / Q257E, P98A, E103L, A114G / S253G / F256I / Q257E, I147A, I147R, I1 47T, I147T / V149E, V149D / Q257E, V149E / L167Q, V149E / G195N, V149E / I218P, V149E / S253G, R155N / L167Q, R155N / P178S / G195R, R155N / P178S / G195R / T251A / S253G, R155N / G195N / T251A / S253G / F256I, R155N / G195N / S253G / F256I, R155N / N196G / E198H / S253G, L167Q, L167Q / G195N, L167Q / G195N / T251A / S253G, N196A, N196G / S253G / F256I / Q257W, E198H / S253G / F256I, E198H / F256I, Q205E, P214G / S253G / F256I, T251L, and Q257E.

[0161] In some embodiments, the engineered glucose dehydrogenase polypeptide having imine reductase activity comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 736 and one or more residue differences compared to SEQ ID NO: 736 selected from the group consisting of: 21 / 47 / 96 / 114 / 155 / 251, 21 / 47 / 149 / 257, 21 / 47 / 155 / 178 / 257, 21 / 47 / 178 / 251 / 256 / 257, 21 / 96 / 114 / 155 / 178, 21 / 47 / 178 / 251 / 256 / 257. 5 / 251 / 257, 21 / 114 / 178 / 179, 21 / 155 / 178 / 209 / 256, 25 / 155, 25 / 178 / 256, 42 / 47 / 155 / 178, 47 / 114 / 149 / 178 / 251, 47 / 114 / 178 / 257, 47 / 149, 47 / 178, 47 / 178 / 179 / 25 1 / 257, 47 / 178 / 251 / 257, 96 / 114 / 155 / 198, 96 / 114 / 155 / 253 / 257, 96 / 253, 114 / 149 / 155 / 251 / 256, 114 / 149 / 178 / 256 / 257, 114 / 155 / 195 / 198 / 256, 114 / 209 / 256 / 257 , 149, 149 / 256 / 257, 155, 155 / 178 / 179, 155 / 178 / 198 / 252 / 253, 155 / 195 / 198, 155 / 195 / 198 / 253 / 256 / 257, 155 / 256 / 257, 178 / 253, 178 / 256, 179 / 256 / 257, 251 and 253.In some embodiments, the engineered glucose dehydrogenase polypeptide having imine reductase activity comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 736 and an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 736. NO:736 has one or more residue differences selected from the group consisting of: 21R / 47A / 96A / 114G / 155N / 251A, 21R / 47A / 149E / 257E, 21R / 47A / 155N / 178S / 257E, 21R / 47A / 178S, 21R / 47A / 178S / 251A / 256I / 257W, 21R / 96A / 114G / 155N / 178S, 21R / 114G / 155N / 257E 1A / 257E, 21R / 114G / 178S / 179N, 21R / 155N / 178S / 209L / 256I, 25L / 155N, 25L / 178S / 256I, 42T / 47A / 155N / 178S、47A / 114G / 149E / 178S / 251A、47A / 114G / 178S / 257E、47A / 149E、47A / 178S、47A / 178S / 179N / 251A / 2 57E, 47A / 178S / 251A / 257E, 96T / 114G / 155N / 198L, 96T / 114G / 155N / 253G / 257W, 96T / 253G, 114G / 149E / 1 55N / 251A / 256I, 114G / 149E / 178S / 256I / 257E, 114G / 155N / 195N / 198L / 256I, 114G / 209L / 256I / 257E, 14 9E, 149E / 256I / 257E, 155N, 155N / 178S / 179N, 155N / 178S / 198H / 252T / 253G, 155N / 195N / 198H / 253G / 256 I / 257W, 155N / 195N / 198L, 155N / 256I / 257E, 178S / 253G, 178S / 256I, 179N / 256I / 257E, 251I, 251L and 253G.In some embodiments, the engineered glucose dehydrogenase polypeptide having imine reductase activity comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 736 and an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 736. NO:736 has one or more residue differences selected from the group consisting of K21R / L47A / E96A / A114G / R155N / T251A, K21R / L47A / V149E / Q257E, K21R / L47A / R155N / P178S / Q257E, K21R / L47A / P178S, K21R / L47A / P178S / T251A / F256I / Q257W, K21R / E96A / A114G / R155N / P178S, K21R / A114G / R155N / T251A / Q257E、K21R / A114G / P178S / K179N、K21R / R155N / P178S / V209L / F256I、I25L / R155N、I25L / P178S / F256I、K42T / L47A / R155N / P178S, L47A / A114G / V149E / P178S / T251A, L47A / A114G / P178S / Q257E, L47A / V149E, L47A / P178S, L47A / P178S / K179N / T251A / Q 257E, L47A / P178S / T251A / Q257E, E96T / A114G / R155N / E198L, E96T / A114G / R155N / S253G / Q257W, E96T / S253G, A114G / V149E / R155N / T251A / F256I, A114G / V149E / P178S / F256I / Q257E, A114G / R155N / G195N / E198L / F256I, A114G / V209L / F256I / Q257E, V1 49E, V149E / F256I / Q257E, R155N, R155N / P178S / K179N, R155N / P178S / E198H / L252T / S253G, R155N / G195N / E198H / S253G / F256I / Q257W, R155N / G195N / E198L, R155N / F256I / Q257E, P178S / S253G, P178S / F256I, K179N / F256I / Q257E, T251I, T251L, and S253G.

[0162] In some embodiments, the engineered glucose dehydrogenase polypeptide having imine reductase activity comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 908 and one or more residue differences compared to SEQ ID NO: 908 selected from the group consisting of: 21 / 42 / 149, 21 / 42 / 149 / 214 / 253, 21 / 42 / 179 / 214 / 244 / 253, 21 / 42 / 253 / 260 / 261, 21 / 149 / 179, 21 / 149 / 179 / 253, 21 / 149 / 179 / 253 / 261. 60, 21 / 149 / 179 / 253 / 260 / 261, 21 / 149 / 179 / 261, 21 / 149 / 260 / 261, 21 / 149 / 261, 21 / 179 / 214 / 253, 21 / 179 / 253, 21 / 179 / 261, 21 / 214 / 253, 21 / 253, 40, 42 / 149 / 179 / 253 / 2 60, 42 / 149 / 179 / 260 / 261, 42 / 149 / 214 / 253, 42 / 179 / 253 / 261, 42 / 253, 104, 111, 149, 149 / 179, 149 / 179 / 253, 149 / 179 / 253 / 260 / 261, 149 / 179 / 260, 149 / 179 / 260 / 261, 14 9 / 179 / 261, 149 / 253, 149 / 261, 160, 178, 179 / 214 / 253, 179 / 253, 179 / 253 / 260, 179 / 253 / 260 / 261, 179 / 253 / 261, 179 / 260 / 261, 179 / 261, 253, 253 / 260 / 261, 253 / 261 and 255. In some embodiments, the engineered glucose dehydrogenase polypeptide having imine reductase activity comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 908 and an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 908.NO:908 has one or more residue differences selected from the group consisting of: 21K / 42T / 149E, 21K / 42T / 149E / 214A / 253G, 21K / 42T / 179R / 214A / 244F / 253G, 21K / 42T / 253G / 260Q / 261E, 21K / 149E, 21K / 149E / 179N, 21K / 149E / 179N / 253G, 21K / 149E / 179N / 261E, 21K / 149E / 179R, 21K / 149E / 179N / 253G. 79R / 253G / 260Q / 261E, 21K / 149E / 179R / 253R / 260Q, 21K / 149E / 260Q / 261E, 21K / 149E / 261E, 21K / 179N / 253G, 21K / 179R / 214A / 253G, 21K / 179R / 253G, 21K / 179R / 261E, 21K / 214A / 253G, 21K / 253G, 40T, 42T / 149E / 179N / 260Q / 261E, 42T / 149E / 179R / 253G / 260Q、42T / 149E / 214A / 253G、42T / 179N / 253G / 261E、42T / 253G、104L、111L、149E、149E / 179N / 253G、149E / 179N / 253G / 260Q / 261E, 149E / 179R, 149E / 179R / 253G, 149E / 179R / 260Q, 149E / 179R / 260Q / 261E, 149E / 179R / 261E, 149 E / 253G, 149E / 253R, 149E / 261E, 160S, 178L, 179N / 253G, 179N / 253G / 260Q, 179R / 214A / 253G, 179R / 253G, 179R / 253G / 2 60Q, 179R / 253G / 260Q / 261E, 179R / 253G / 261E, 179R / 260Q / 261E, 179R / 261E, 253G, 253G / 260Q / 261E, 253G / 261E and 255P. In some embodiments, the engineered glucose dehydrogenase polypeptide having imine reductase activity comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 908 and an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 908.NO:908 has one or more residue differences selected from the group consisting of: R21K / K42T / V149E, R21K / K42T / V149E / P214A / S253G, R21K / K42T / K179R / P214A / L244F / S253G, R21K / K42T / S253G / R260Q / G261E, R21K / V149E, R21K / V149E / K179N, R21K / V149E / K179N / S253G, R21K / V149E / K179R, R21K / V149E / K179R / P214A / L244F / S253G 9R / S253G / R260Q / G261E, R21K / V149E / K179R / S253R / R260Q, R21K / V149 E / R260Q / G261E, R21K / V149E / G261E, R21K / K179N / S253G, R21K / K179R / P214A / S253G, R21K / K179R / S253G, R21K / K179R / G261E, R21K / P214A / S2 53G, R21K / S253G, S40T, K42T / V149E / K179N / R260Q / G261E, K42T / V149E / K179R / S253G / R260Q、K42T / V149E / P214A / S253G、K42T / K179N / S253G / G261E, K42T / S253G, M104L, K111L, V149E, V149E / K179N / S253G, V149E / K179N / S253G / R260Q / G261E, V149E / K179R, V149E / K179R / S253G, V149E / K179R / R260Q, V149E / K179R / R260Q / G261E, V149E / K179R / G261E, V149 E / S253G, V149E / S253R, V149E / G261E, A160S, S178L, K179N / S253G, K17 9N / S253G / R260Q, K179R / P214A / S253G, K179R / S253G, K179R / S253G / R2 60Q, K179R / S253G / R260Q / G261E, K179R / S253G / G261E, K179R / R260Q / G 261E, K179R / G261E, S253G, S253G / R260Q / G261E, S253G / G261E and S255P.

[0163] In some embodiments, the engineered glucose dehydrogenase polypeptide having imine reductase activity comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 956 and one or more residue differences compared to SEQ ID NO: 956 selected from the group consisting of: 3, 21 / 104 / 160 / 178 / 179, 21 / 104 / 160 / 255 / 258, 21 / 104 / 178 / 179 / 255, 21 / 104 / 178 / 255 / 258, 21 / 104 / 178 / 255 / 258. 5 / 260, 21 / 255 / 260, 31, 32, 40, 40 / 104 / 178 / 179, 40 / 160 / 178, 46 / 82 / 259, 103, 104 / 149 / 255 / 258 / 260, 104 / 149 / 258, 104 / 160, 104 / 160 / 178, 104 / 160 / 178 / 179 / 255, 104 / 160 / 178 / 179 / 260、104 / 160 / 178 / 255 / 260、104 / 160 / 179、104 / 178 / 179 / 255 / 258、104 / 178 / 255 / 258 / 260、104 / 178 / 255 / 260、104 / 179、104 / 255 / 260、104 / 258 / 260、111 / 14 9 / 255 / 258, 111 / 149 / 255 / 260, 111 / 160 / 178 / 179 / 255 / 258 / 260, 111 / 160 / 255 / 260, 154, 156, 160, 160 / 178 / 179, 174, 194, 197, 205, 205 / 229, 222, 253, 257, 258 / 260 and 260. In some embodiments, the engineered glucose dehydrogenase polypeptide having imine reductase activity comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 956 and an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 956.NO:956 has one or more residue differences selected from the group consisting of: 3C, 3T, 21K / 104L / 160S / 178L / 179R, 21K / 104L / 160S / 255A / 258P, 21K / 104L / 178R / 179R / 255F, 21K / 104L / 178R / 255F / 258P, 21K / 104L / 255A, 21K / 104L / 255F / 258P, 21K / 178R / 179R / 255A / 260Q, 21K / 104L / 160S / 178L / 179R K / 255A / 260Q, 31H, 32M, 40K, 40T / 104L / 178R / 179R, 40T / 160S / 178L, 46I / 82Q / 259N, 103K, 104L / 149E / 255F / 25 8P / 260Q, 104L / 149E / 258P, 104L / 160S, 104L / 160S / 178A / 255A / 260Q, 104L / 160S / 178K / 179R / 260Q, 104L / 160S / 178L, 104L / 160S / 178R / 179R / 255A, 104L / 160S / 179R, 104L / 178A / 179R / 255A / 258P, 104L / 178K / 255F / 260Q, 10 4L / 178L / 255F / 258P / 260Q, 104L / 179R, 104L / 255F / 260Q, 104L / 258P / 260Q, 111E / 149E / 255F / 258P, 111E / 149E / 174A, 194L, 197K, 205I / 229S, 205L, 205M, 205T, 222F, 253Y, 257R, 258P / 260Q, and 260Q. In some embodiments, the engineered glucose dehydrogenase polypeptide having imine reductase activity comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 956 and an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 956.NO:956 has one or more residue differences selected from the group consisting of: P3C, P3T, R21K / M104L / A160S / S178L / N179R, R21K / M104L / A160S / S255A / A258P, R21K / M104L / S178R / N179R / S255F, R21K / M104L / S178R / S255F / A258P, R21K / M104L / S255A, R21K / M104L / S255F / A258P, R21K / S178R / N179R / S255A / R260Q, R21K / S255A / R260Q, Q31H, A32M, S40K, S40T / M104L / S178R / N179R, S40T / A160S / S178L, N46I / E82Q / G259N, E103K, M104L / V149E / S255F / A25 8P / R260Q, M104L / V149E / A258P, M104L / A160S, M104L / A160S / S178A / S255A / R260Q, M104L / A160S / S178K / N179R / R260Q, M104L / A160S / S178L, M104L / A160S / S178R / N179R / S255A, M104L / A160S / N179R, M104L / S178A / N179R / S255A / A258P, M104L / S178K / S255F / R260Q, M10 4L / S178L / S255F / A258P / R260Q, M104L / N179R, M104L / S255F / R260Q, M104L / A258P / R260Q, K111E / V149E / S255F / A258P, K111E / V149E / , S255F / R260Q, K111I / A160S / S255F / R260Q, K111L / A160S / S178A / N179R / S255A / A258P / R260Q, L154H, L154K, L154Q, L154W, V156A, V156T, A160S, A160S / S178L / N179R, L174A, T194L, A197K, Q205I / A229S, Q205L, Q205M, Q205T, E222F, G253Y, E257R, A258P / R260Q, and R260Q.

[0164] In some embodiments, the engineered glucose dehydrogenase polypeptide having imine reductase activity comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 1062 and an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1062. NO:1062 has one or more residue differences selected from the group consisting of: 3 / 31 / 154 / 205 / 257, 3 / 31 / 205, 3 / 154 / 205, 31 / 55 / 154 / 205, 31 / 55 / 205, 31 / 154 / 205, 31 / 154 / 205 / 257, 31 / 205, 31 / 205 / 222, 40 / 55 / 205, 40 / 154 / 205, 40 / 154 / 205 / 257, 40 / 174 / 205 / 229, 40 / 205 / 229, 54 / 205 / 253、63、63 / 149 / 154 / 174 / 178 / 253、63 / 149 / 154 / 174 / 205 / 253、63 / 149 / 156 / 178 / 205 / 253、63 / 149 / 205、63 / 149 / 205 / 229 / 253、63 / 149 / 205 / 253、63 / 149 / 205 / 253、63 / 154、63 / 154 / 178 / 205 / 253、63 / 154 / 205、63 / 154 / 205 / 229、63 / 154 / 205 / 229 / 253、63 / 154 / 205 / 253, 63 / 156, 63 / 156 / 205, 63 / 205, 63 / 205 / 253, 63 / 253, 67, 98, 149 / 154, 149 / 154 / 205, 149 / 156 / 253, 149 / 205, 149 / 205 / 253, 154, 154 / 160, 154 / 160 / 205, 154 / 160 / 205 / 222 / 257, 154 / 160 / 253, 154 / 174, 154 / 174 / 178 / 205, 154 / 174 / 2 05 / 229、154 / 205、154 / 205 / 222、154 / 205 / 222 / 257、154 / 205 / 229、154 / 205 / 253、154 / 205 / 257、154 / 229、154 / 253、156 / 205、156 / 205 / 253、156 / 229 / 253、160、160 / 205、160 / 205 / 229、194、205、205 / 222、205 / 229、205 / 229 / 253、205 / 253、208、226 and 253. In some embodiments, the engineered glucose dehydrogenase polypeptide having imine reductase activity comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 1062 and an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1062. NO: 1062 has one or more residue differences selected from the group consisting of: 3C / 31H / 154K / 205M / 257R, 3L / 154W / 205L, 3T / 31H / 205L, 31H / 55W / 154K / 205T, 31H / 55W / 154Q / 205T, 31H / 55W / 154W / 205T, 31H / 55W / 205I, 31H / 154Q / 205L, 31H / 154W / 205I, 31H / 154W / 205I / 257R, 31H / 154W / 205L, 31H / 154W / 205 L / 257R, 31H / 205I / 222Q, 31H / 205L, 40K / 55W / 205L, 40T / 154Q / 205T, 40T / 154Q / 205T / 257R, 40T / 174A / 205I / 229S, 40T / 205T / 22 9S、54E / 205M / 253Y、63M、63M / 149E / 154K / 174A / 178K / 253Y、63M / 149E / 154W / 174A / 205T / 253Y、63M / 149E / 156T / 178K / 205L / 253 Y, 63M / 149E / 205L, 63M / 149E / 205L / 229S / 253Y, 63M / 149E / 205L / 253Y, 63M / 149E / 205T / 253Y, 63M / 154H / 178K / 205I / 253Y, 63M / 154H / 205L / 229S, 63M / 154K / 205L, 63M / 154K / 205T / 253Y, 63M / 154Q / 205I / 229S / 253Y, 63M / 154W, 63M / 154W / 178K / 205L / 253Y, 6 3M / 154W / 205M, 63M / 154W / 205T, 63M / 156A, 63M / 156A / 205T, 63M / 205L, 63M / 205M, 63M / 205M / 253Y, 63M / 205T, 63M / 205T / 253Y, 6 3M / 253Y, 67S, 98Q, 149E / 154S / 205L, 149E / 154W, 149E / 156A / 253Y, 149E / 205M, 149E / 205M / 253Y, 149E / 205T, 149E / 205T / 253D,154H, 154H / 174A, 154H / 205L / 253Y, 154H / 205T, 154K, 154K / 160A, 154K / 160A / 205L, 154K / 160A / 205M, 154K / 160A / 253Y, 154K / 174A / 205L / 229S, 154K / 205I, 154K / 205M, 154K / 253Y, 1 54Q / 160A / 205L / 222F / 257R, 154Q / 205I / 253Y, 154Q / 205L / 222R / 257R, 154Q / 205M / 257R, 1 54W, 154W / 174A / 178K / 205T, 154W / 205I, 154W / 205I / 222G, 154W / 205I / 257R, 154W / 205L, 15 4W / 205L / 222R, 154W / 205M, 154W / 205T, 154W / 205T / 222F / 257R, 154W / 205T / 222Y, 154W / 20 5T / 229S, 154W / 205T / 253Y, 154W / 229S, 154W / 253Y, 156A / 205M / 253Y, 156A / 229S / 253Y, 156 3Y, 205T, 160A, 160A / 205L, 160A / 205L / 229S, 160A / 205M / 229S, 194H, 205L, 205L / 222G, 205M, 205T, 205T / 222Y, 205T / 229S, 205T / 229S / 253Y, 205T / 253Y, 208L, 208R, 208T, 226C and 253Y. In some embodiments, the engineered glucose dehydrogenase polypeptide having imine reductase activity comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 1062 and an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1062. NO: 1062 has one or more residue differences selected from the group consisting of P3C / Q31H / L154K / Q205M / E257R, P3L / L154W / Q205L, P3T / Q31H / Q205L, Q31H / A55W / L154K / Q205T, Q31H / A55W / L154Q / Q205T, Q31H / A55W / L154W / Q205T, Q31H / A55W / Q205I, Q31H / L154Q / Q205L, Q31H / L154W / Q205I, Q31H / L154W / Q205I / E257R, Q31H / L154W / Q205L,Q31H / L154W / Q205L / E257R、Q31H / Q205I / E222Q、Q31H / Q205L、S40K / A55W / Q205L、S40T / L154Q / Q205T、S40T / L154Q / Q205T / E257R、S40T / L174A / Q205I / A229S、S40T / Q205T / A229S、K54E / Q205M / G253Y、Q63M、Q63M / V149E / L154K / L174A / S178K / G253Y、Q63M / V149E / L154W / L174A / Q205T / G253Y、Q63M / V149E / V156T / S178K / Q205L / G253Y、Q63M / V149E / Q205L、Q63M / V149E / Q205L / A229S / G253Y、Q63M / V149E / Q205L / G253Y、Q63M / V149E / Q205T / G253Y、Q63M / L154H / S178K / Q205I / G253Y、Q63M / L154H / Q205L / A229S、Q63M / L154K / Q205L、Q63M / L154K / Q205T / G253Y、Q63M / L154Q / Q205I / A229S / G253Y、Q63M / L154W、Q63M / L154W / S178K / Q205L / G253Y、Q63M / L154W / Q205M、Q63M / L154W / Q205T、Q63M / V156A、Q63M / V156A / Q205T、Q63M / Q205L、Q63M / Q205M、Q63M / Q205M / G253Y、Q63M / Q205T、Q63M / Q205T / G253Y、Q63M / G253Y、T67S、P98Q、V149E / L154S / Q205L、V149E / L154W、V149E / V156A / G253Y、V149E / Q205M、V149E / Q205M / G253Y、V149E / Q205T、V149E / Q205T / G253D、L154H、L154H / L174A、L154H / Q205L / G253Y、L154H / Q205T、L154K、L154K / S160A、L154K / S160A / Q205L、L154K / S160A / Q205M、L154K / S160A / G253Y、L154K / L174A / Q205L / A229S、L154K / Q205I、L154K / Q205M、L154K / G253Y、L154Q / S160A / Q205L / E222F / E257R、L154Q / Q205I / G253Y, L154Q / Q205L / E222R / E257R, L154Q / Q205M / E257R, L154 W, L154W / L174A / S178K / Q205T, L154W / Q205I, L154W / Q205I / E222G, L154W / Q20 5I / E257R, L154W / Q205L, L154W / Q205L / E222R, L154W / Q205M, L154W / Q205T, L 154W / Q205T / E222F / E257R, L154W / Q205T / E222Y, L154W / Q205T / A229S, L154W / Q205T / G253Y, L154W / A229S, L154W / G253Y, V156A / Q205M / G253Y, V156A / A229 S / G253Y, V156T / Q205T, S160A, S160A / Q205L, S160A / Q205L / A229S, S160A / Q20 5M / A229S, T194H, Q205L, Q205L / E222G, Q205M, Q205T, Q205T / E222Y, Q205T / A229S, Q205T / A229S / G253Y, Q205T / G253Y, D208L, D208R, D208T, A226C, and G253Y.

[0165] As will be appreciated by those skilled in the art, in some embodiments, one or a combination of the above residue differences selected can be kept constant (i.e. maintained) as a core feature in the engineered glucose dehydrogenase, and additional residue differences at other residue positions can be incorporated into the sequence to produce additional engineered glucose dehydrogenase polypeptides with improved properties. Therefore, it should be understood that for any engineered glucose dehydrogenase containing one or a subset of the above residue differences, the present invention contemplates other engineered glucose dehydrogenases comprising one or a subset of the residue differences and additionally comprising one or more residue differences at other residue positions disclosed herein.

[0166] As mentioned above, the engineered glucose dehydrogenase polypeptide having imine reductase activity is also capable of converting substrates (e.g., compound (2) and compound (3)) into products (e.g., compound (1)). In some embodiments, the engineered glucose dehydrogenase polypeptide is capable of converting a substrate compound into a product compound with an activity at least 1.2 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times or more relative to the activity of the reference polypeptide SEQ ID NO: 4, 14, 34, 112, 736, 908, 956 and / or 1062.

[0167] In some embodiments, the engineered glucose dehydrogenase polypeptide capable of converting a substrate compound into a product compound with at least 2 times the activity of SEQ ID NO: 4, 14, 34, 112, 736, 908, 956 and / or 1062 comprises an amino acid sequence selected from the even-numbered sequences of SEQ ID NO: 6-1384.

[0168] In some embodiments, the engineered glucose dehydrogenase has an amino acid sequence comprising one or more residue differences compared to SEQ ID NO: 4, 14, 34, 112, 736, 908, 956 and / or 1062, which increases the expression of the engineered glucose dehydrogenase activity in a bacterial host cell, particularly in E. coli.

[0169] In some embodiments, the engineered glucose dehydrogenase polypeptide with improved properties has an amino acid sequence comprising a sequence selected from the even-numbered sequences within the range of SEQ ID NOs: 6-1384.

[0170] In some embodiments, the engineered glucose dehydrogenase polypeptide having imine reductase activity comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to one of the even-numbered sequences within the range of SEQ ID NOs: 6-1384, and the amino acid residue differences present in any of the even-numbered sequences within the range of SEQ ID NOs: 6-1384 compared to SEQ ID NOs: 4, 14, 34, 112, 736, 908, 956 and / or 1062, as provided in the Examples.

[0171] In addition to the residue positions specified above, any engineered glucose dehydrogenase polypeptide disclosed herein may also include other residue differences relative to SEQ ID NO: 4, 14, 34, 112, 736, 908, 956 and / or 1062 at other residue positions (i.e., residue positions other than those contained herein). Residue differences at these other residue positions can provide additional changes in the amino acid sequence without adversely affecting the ability of the polypeptide to convert substrates to products. Therefore, in some embodiments, in addition to the amino acid residue differences present in any one of the engineered glucose dehydrogenase polypeptides of even-numbered sequences within the range of SEQ ID NO: 6-1384, the sequence may also include residue differences with SEQ ID NO: 4, 14, 34, 112, 736, 908, 956 and / or 1062. 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40, 1-45 or 1-50 residue differences at other amino acid residue positions compared to ID NO: 4, 14, 34, 112, 736, 908, 956 and / or 1062. In some embodiments, the number of amino acid residue differences compared to the reference sequence can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45, or 50 residue positions. In some embodiments, the number of amino acid residue differences compared to the reference sequence can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24, or 25 residue positions. The residue differences at these other positions can be conservative changes or non-conservative changes. In some embodiments, the residue differences can include conservative substitutions and non-conservative substitutions compared to the glucose dehydrogenase polypeptides of SEQ ID NO: 4, 14, 34, 112, 736, 908, 956 and / or 1062.

[0172] In some embodiments, the present invention also provides engineered polypeptides comprising fragments of engineered glucose dehydrogenase polypeptides that retain the functional activity and / or improved properties of any engineered glucose dehydrogenase described herein. Therefore, in some embodiments, the present invention provides polypeptide fragments capable of converting substrates into products under suitable reaction conditions, wherein the fragment comprises at least about 90%, 95%, 96%, 97%, 98% or 99% of the full-length amino acid sequence of the engineered glucose dehydrogenase polypeptide of the present invention, the engineered glucose dehydrogenase polypeptide of the present invention such as the exemplary engineered glucose dehydrogenase polypeptides of the even-numbered sequences within the range of SEQ ID NO: 6-1384. In some embodiments, the engineered imine reductase polypeptide may have an amino acid sequence comprising a deletion in any one of the engineered glucose dehydrogenase polypeptide sequences described herein, the engineered glucose dehydrogenase polypeptides described herein such as the exemplary engineered polypeptides of the even-numbered sequences within the range of SEQ ID NO: 6-1384.

[0173] Thus, for various and each embodiments of the engineered glucose dehydrogenase polypeptides of the invention, the amino acid sequence can include a deletion of 1 or more amino acids, 2 or more amino acids, 3 or more amino acids, 4 or more amino acids, 5 or more amino acids, 6 or more amino acids, 8 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids in the glucose dehydrogenase polypeptide, up to 20% of the total number of amino acids, or up to 30% of the total number of amino acids, wherein the relevant functional activity and / or improved properties of the engineered glucose dehydrogenase described herein are maintained. In some embodiments, the deletion may include 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1-45, or 1-50 amino acid residues. In some embodiments, the number of deletions can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45, or 50 amino acid residues. In some embodiments, the deletion can include the deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24, or 25 amino acid residues.

[0174] In some embodiments, the engineered glucose dehydrogenase polypeptides herein can have an amino acid sequence comprising an insertion compared to any of the engineered glucose dehydrogenase polypeptides described herein, such as the exemplary engineered polypeptides of even-numbered sequences within the range of SEQ ID NO:6-1384. Therefore, for each and every embodiment of the glucose dehydrogenase polypeptides of the present invention, the insertion can comprise 1 or more amino acids, 2 or more amino acids, 3 or more amino acids, 4 or more amino acids, 5 or more amino acids, 6 or more amino acids, 8 or more amino acids, 10 or more amino acids, 15 or more amino acids, 20 or more amino acids, 30 or more amino acids, 40 or more amino acids or 50 or more amino acids, wherein the relevant functional activity and / or improved properties of the engineered glucose dehydrogenase described herein are maintained. The insertion can be at the amino terminal or carboxyl terminal of the glucose dehydrogenase polypeptide, or an internal portion.

[0175] In some embodiments, the engineered glucose dehydrogenase polypeptides herein can have an amino acid sequence comprising an even-numbered sequence selected from the group consisting of SEQ ID NO: 6-1384 and optionally one or more (e.g., up to 3, 4, 5, or up to 10) amino acid residues deleted, inserted, and / or substituted. In some embodiments, the amino acid sequence optionally has 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1-45, or 1-50 amino acid residues deleted, inserted, and / or substituted. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45 or 50 amino acid residue deletions, insertions and / or substitutions. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24 or 25 amino acid residue deletions, insertions and / or substitutions. In some embodiments, the substitutions may be conservative substitutions or non-conservative substitutions.

[0176] In the above embodiments, suitable reaction conditions for engineering polypeptides are provided in Table 5.1, Table 6.1, Table 7.1, Table 8.1, Table 9.1, Table 10.1, Table 11.1, and Table 12.1, and as described in the Examples herein.

[0177] In some embodiments, the polypeptides of the present invention are fusion polypeptides, wherein the engineered polypeptides are fused to other polypeptides, such as, for example but not limited to, antibody tags (e.g., myc epitopes), purification sequences (e.g., His tags for binding metals), and cell localization signals (e.g., secretion signals). Thus, the engineered polypeptides described herein can be used fused to other polypeptides or not.

[0178] It should be understood that the polypeptides described herein are not limited to genetically encoded amino acids. In addition to genetically encoded amino acids, the polypeptides described herein may contain naturally occurring and / or synthetic non-encoded amino acids in whole or in part. Some common non-encoded amino acids that the polypeptides described herein may contain include, but are not limited to: D-stereoisomers of genetically encoded amino acids; 2,3-diaminopropionic acid (Dpr); α-aminoisobutyric acid (Aib); ε-aminocaproic acid (Aha); δ-aminovaleric acid (Ava); N-methylglycine or sarcosine (MeGly or Sar); ornithine (Orn); citrulline (Cit); tert-butylalanine (Bua); tert-butylglycine (Bug); N-methylisoleucine (MeIle); phenylglycine (Phg); cyclohexylalanine (Cha); norleucine (Nle); naphthylalanine (Nal); 2-chlorophenylalanine Acid (Ocf); 3-chlorophenylalanine (Mcf); 4-chlorophenylalanine (Pcf); 2-fluorophenylalanine (Off); 3-fluorophenylalanine (Mff); 4-fluorophenylalanine (Pff); 2-bromophenylalanine (Obf); 3-bromophenylalanine (Mbf); 4-bromophenylalanine (Pbf); 2-methylphenylalanine (Omf); 3-methylphenylalanine (Mmf); 4-methylphenylalanine (Pmf); 2-nitrophenylalanine (Onf); 3-nitrophenylalanine (Mnf); 4-nitrophenylalanine (Pnf); 2-cyanophenylalanine (Ocf); 3-cyanophenylalanine (Mcf); 4-cyanophenylalanine (Pnf). phenylalanine (Pcf); 2-trifluoromethylphenylalanine (Otf); 3-trifluoromethylphenylalanine (Mtf); 4-trifluoromethylphenylalanine (Ptf); 4-aminophenylalanine (Paf); 4-iodophenylalanine (Pif); 4-aminomethylphenylalanine (Pamf); 2,4-dichlorophenylalanine (Opef); 3,4-dichlorophenylalanine (Mpcf); 2,4-difluorophenylalanine (Opff); 3,4-difluorophenylalanine (Mpff); pyridin-2-ylalanine (2pAla); pyridin-3-ylalanine (3pAla); pyridin-4-ylalanine (4pAla); naphth-1-ylalanine (1 nAla); naphthylalanine (2nAla); thiazolylalanine (taAla); benzothienylalanine (bAla); thienylalanine (tAla); furanylalanine (fAla); homophenylalanine (hPhe); homotyrosine (hTyr); homotryptophan (hTrp); pentafluorophenylalanine (5ff); styrylkalanine (sAla); anthrylalanine (aAla); 3,3-diphenylalanine (Dfa); 3-amino-5-phenylpentanoic acid (Afp); penicillamine (Pen); 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic);β-2-Thienylalanine (Thi); methionine sulfoxide (Mso); N(w)-nitroarginine (nArg); homolysine (hLys); phosphonomethylphenylalanine (pmPhe); phosphoserine (pSer); phosphothreonine (pThr); homoaspartic acid (hAsp); homoglutamic acid (hGlu); 1-aminocyclopentyl-(2 or 3)-ene-4-carboxylic acid; pipecolic acid (PA); azetidine-3-carboxylic acid (ACA); 1-aminocyclopentane-3-carboxylic acid; allylglycine (aGly ); propargylglycine (pgGly); homoalanine (hAla); norvaline (nVal); homoleucine (hLeu), homovaline (hVal); homoisoleucine (hlle); homoarginine (hArg); N-acetyl lysine (AcLys); 2,4-diaminobutyric acid (Dbu); 2,3-diaminobutyric acid (Dab); N-methyl valine (MeVal); homocysteine ​​(hCys); homoserine (hSer); hydroxyproline (Hyp) and homoproline (hPro). Additional non-encoded amino acids that the polypeptides described herein may contain will be apparent to those skilled in the art (see, e.g., Fasman,; CRC Practical Handbook of Biochemistry and Molecular Biology , CRC Press, Boca Raton, FL, pp. 3-70

[1989] and the references cited therein, all of which are incorporated by reference). These amino acids can be in the L-configuration or the D-configuration.

[0179] Those skilled in the art will recognize that amino acids or residues with side chain protecting groups may also constitute the polypeptides described herein. Non-limiting examples of such protected amino acids, which in this case belong to the aromatic class, include, but are not limited to (protecting groups are listed in parentheses): Arg (tos), Cys (methylbenzyl), Cys (nitropyridinesulfenyl), Glu (δ-benzyl ester), Gln (xanthene), Asn (N-δ-xanthene), His (bom), His (benzyl), His (tos), Lys (fmoc), Lys (tos), Ser (O-benzyl), Thr (O-benzyl) and Tyr (O-benzyl).

[0180] Conformational restricted non-encoded amino acids that can make up the polypeptides described herein include, but are not limited to, N-methyl amino acids (L-configuration), 1-aminocyclopent-(2 or 3)-ene-4-carboxylic acid, pipecolic acid, azetidine-3-carboxylic acid, homoproline (hPro), and 1-aminocyclopentane-3-carboxylic acid.

[0181] In some embodiments, the engineered polypeptide can be in various forms, for example, such as an isolated preparation, as a substantially pure enzyme, intact cells transformed with a gene encoding the enzyme, and / or as a cell extract and / or lysate of such cells. The enzyme can be lyophilized, spray dried, precipitated, or in the form of a crude paste, as discussed further below.

[0182] In some embodiments, the engineered polypeptide can be in the form of a biocatalytic composition. In some embodiments, the biocatalytic composition comprises (a) a means of converting carbonyl compounds and amine compounds into chiral secondary or tertiary amines by contacting with a glucose dehydrogenase polypeptide, and (b) a suitable cofactor. In some embodiments, the biocatalytic composition comprises a chiral secondary or tertiary amine that is acyclic, comprises one or more functional groups, or comprises an alkyl chain (including a branched chain). In some embodiments, the biocatalytic composition comprises a glucose dehydrogenase with imine reductase activity. In some other embodiments, the biocatalytic composition comprises a glucose dehydrogenase with imine reductase activity that additionally catalyzes one or more reactions in a multi-step reaction pathway. In some embodiments, the biocatalytic composition comprises an NAD+ (nicotinamide adenine dinucleotide) or NADP+ (nicotinamide adenine dinucleotide phosphate) cofactor.

[0183] In some embodiments, the engineered polypeptide can be provided on a solid support such as a film, a resin, a solid carrier or other solid phase material. The solid support can include organic polymers, such as polystyrene, polyethylene, polypropylene, polyfluoroethylene, polyethyleneoxy and polyacrylamide and their copolymers and grafts. The solid support can also be inorganic, such as glass, silica, controlled pore glass (CPG), reversed silica or metals such as gold or platinum. The configuration of the solid support can be in the form of beads, balls, microparticles, particles, gels, films or surfaces. The surface can be flat, substantially flat or non-flat. The solid support can be porous or non-porous and can have swelling or non-swelling properties. The solid support can be configured in the form of a hole, a depression or other container, a vessel, a feature or a position.

[0184] In some embodiments, the engineered glucose dehydrogenase polypeptides with imine reductase activity of the present invention can be fixed on a solid support so that they retain their improved activity and / or other improved properties relative to reference polypeptide SEQ ID NO: 4, 14, 34, 112, 736, 908, 956 and / or 1062. In such embodiments, the fixed polypeptide can promote the biocatalytic conversion of substrate compounds or other suitable substrates to products, and after the reaction is completed, it is easy to retain (e.g., by retaining the beads fixed with the polypeptide) and then reuse or recycle in subsequent reactions. Such immobilized enzyme methods allow further improvement in efficiency and cost reduction. Therefore, it is also envisioned that any method using the glucose dehydrogenase polypeptide of the present invention can be carried out using the same glucose dehydrogenase polypeptide bound or fixed on a solid support.

[0185] Methods for enzyme immobilization are well known in the art. Engineered polypeptides can be non-covalently or covalently bound. Various methods for conjugating and fixing enzymes to solid supports (e.g., resins, membranes, beads, glass, etc.) are well known in the art (see, e.g., Yi et al., Proc. Biochem., 42(5):895-898

[2007] ; Martin et al., Appl. Microbiol. Biotechnol., 76(4):843-851

[2007] ; Koszelewski et al., J. Mol. Cat. B: Enzymatic, 63:39-44

[2010] ; Truppo et al., Org. Proc. Res. Dev., online publication: dx.doi.org / 10.1021 / op200157c; Hermanson, Bioconjugate Techniques , 2nd ed., Academic Press, Cambridge, MA

[2008] ; Mateo et al., Biotechnol. Prog., 18(3):629-34

[2002] ; and “Bioconjugation Protocols: Strategies and Methods,” in Methods in Molecular Biology, Niemeyer (ed.), Humana Press, New York, NY

[2004] ; the disclosures of each reference are incorporated herein by reference). Solid supports that can be used to immobilize the engineered glucose dehydrogenase of the present invention include, but are not limited to, beads or resins comprising polymethacrylates with epoxide functional groups, polymethacrylates with aminoepoxide functional groups, styrene / DVB copolymers or polymethacrylates with octadecyl functional groups. Exemplary solid supports that can be used to immobilize the engineered glucose dehydrogenase peptides of the present invention include, but are not limited to, chitosan beads, Eupergit C, and SEPABEAD (Mitsubishi), including the following different types of SEPABEAD: EC-EP, EC-HFA / S, EXA252, EXE119, and EXE120.

[0186] In some embodiments, the polypeptides described herein are provided in the form of a kit. The enzyme in the kit may exist alone or as a plurality of enzymes. The kit may also include reagents for performing enzymatic reactions, substrates for evaluating the activity of the enzyme, and reagents for detecting the product. The kit may also include a reagent dispenser and instructions for use of the kit.

[0187] In some embodiments, kit of the present invention is included in the array that comprises a plurality of different glucose dehydrogenase polypeptides on different addressable positions, wherein different polypeptides are different variants of the enzyme property that each has at least one different improvement of reference sequence. In some embodiments, a plurality of polypeptides that are fixed on the solid support are configured on a plurality of positions of the array, for the automatic delivery of reagent or by detection method and / or instrument addressable. Array can be used to test the conversion of polypeptide to various substrate compounds. Such array comprising multiple engineered polypeptides and method for using thereof are known in the art (see, for example, WO2009 / 008908A2).

[0188] Polynucleotides encoding engineered glucose dehydrogenase, expression vectors and host cells

[0189] On the other hand, the invention provides polynucleotides encoding engineered glucose dehydrogenase polypeptides described herein. The polynucleotides can be operably linked to one or more heterologous regulatory sequences controlling gene expression to create a recombinant polynucleotide capable of expressing a polypeptide. An expression construct comprising a heterologous polynucleotide encoding an engineered glucose dehydrogenase is introduced into an appropriate host cell to express the corresponding glucose dehydrogenase polypeptide.

[0190] As will be apparent to the technician, the availability of protein sequences and the knowledge of the codons corresponding to various amino acids provide a description of all polynucleotides that can encode the subject polypeptide. The degeneracy of the genetic code (wherein the same amino acid is encoded by a selectable codon or a synonymous codon) allows the preparation of a very large number of nucleic acids, all of which encode improved glucose dehydrogenases. Therefore, knowing the specific amino acid sequence, those skilled in the art can prepare any number of different nucleic acids by simply changing the sequence of one or more codons in a manner that does not change the amino acid sequence of the protein. In this regard, the present invention specifically contemplates that each possible change of the polynucleotide encoding the polypeptide described herein can be prepared by selecting a combination based on possible codon selections, and for any polypeptide described herein, all such changes are considered to be specifically disclosed, including the amino acid sequences disclosed as even-numbered sequences in the sequence table presented in Table 5.1, Table 6.1, Table 7.1, Table 8.1, Table 9.1, Table 10.1, Table 11.1 and Table 12.1 and incorporated by reference into this paper as SEQ ID NO:6-1384 range.

[0191] In various embodiments, codons are preferably selected to adapt to the host cell in which the protein is produced. For example, the preferred codons used in bacteria are used to express genes in bacteria; the preferred codons used in yeast are used for expression in yeast; and the preferred codons used in mammals are used for expression in mammalian cells. In some embodiments, it is not necessary to replace all codons to optimize the codon usage of glucose dehydrogenase, because the native sequence will contain preferred codons and because it may not be necessary to use preferred codons for all amino acid residues. Therefore, the codon-optimized polynucleotide encoding glucose dehydrogenase can contain preferred codons at about 40%, 50%, 60%, 70%, 80% or more than 90% of the codon positions in the full-length coding region.

[0192] In some embodiments, the polynucleotide comprises a codon-optimized nucleotide sequence encoding a glucose dehydrogenase polypeptide amino acid sequence as represented by SEQ ID NO: 4, 14, 34, 112, 736, 908, 956 and / or 1062. In some embodiments, the polynucleotide has a nucleic acid sequence comprising at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a codon-optimized nucleic acid sequence encoding an even-numbered sequence within the range of SEQ ID NO: 6-1384. In some embodiments, the polynucleotide has a nucleic acid sequence comprising at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a codon-optimized nucleic acid sequence encoding an odd-numbered sequence within the range of SEQ ID NO: 5-1383. In some embodiments, the codon-optimized sequence of the odd-numbered sequences within the range of SEQ ID NO: 5-1383 enhances expression of the encoded glucose dehydrogenase, providing a preparation of the enzyme capable of converting a substrate to a product.

[0193] In some embodiments, the polynucleotide is capable of hybridizing under high stringency conditions to a reference sequence selected from an odd-numbered sequence among SEQ ID NOs: 5-1383 or a complementary sequence thereof, and encodes a glucose dehydrogenase polypeptide having imine reductase activity.

[0194] In some embodiments, as described above, the polynucleotide encodes an engineered glucose dehydrogenase polypeptide with imine reductase activity having improved properties compared to SEQ ID NO: 4, 14, 34, 112, 736, 908, 956 and / or 1062, wherein the polypeptide comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a reference sequence selected from SEQ ID NO: 4, 14, 34, 112, 736, 908, 956 and / or 1062, and one or more residue differences compared to SEQ ID NO: 4, 14, 34, 112, 736, 908, 956 and / or 1062, wherein the sequence is selected from the even numbered sequences within the range of SEQ ID NO: 6-1384. In some embodiments, the reference amino acid sequence is selected from the even-numbered sequences within the range of SEQ ID NO: 6 to 1384. In some embodiments, the reference amino acid sequence is SEQ ID NO: 4, while in some other embodiments, the reference sequence is SEQ ID NO: 14, while in some other embodiments, the reference sequence is SEQ ID NO: 34, while in some other embodiments, the reference sequence is SEQ ID NO: 112, while in some other embodiments, the reference sequence is SEQ ID NO: 736, while in some other embodiments, the reference sequence is SEQ ID NO: 908, while in some other embodiments, the reference sequence is SEQ ID NO: 956, while in some other embodiments, the reference sequence is SEQ ID NO: 1062, and in still some other embodiments, the reference sequence is SEQ ID NO: 1172.

[0195] In some embodiments, the polynucleotide encodes a glucose dehydrogenase polypeptide capable of converting one or more substrates into products with improved properties compared to SEQ ID NO: 4, 14, 34, 112, 736, 908, 956 and / or 1062, wherein the polypeptide comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO: 4, 14, 34, 112, 736, 908, 956 and / or 1062.

[0196] In some embodiments, the polynucleotide encoding the engineered glucose dehydrogenase comprises a polynucleotide sequence selected from the odd-numbered sequences within the range of SEQ ID NOs: 5-1383.

[0197] In some embodiments, the polynucleotide is capable of hybridizing under high stringency conditions to a reference polynucleotide sequence selected from an odd-numbered sequence within the range of SEQ ID NO: 5-1383, or its complementary sequence, and encodes a glucose dehydrogenase polypeptide having one or more improved properties described herein with imine reductase activity. In some embodiments, the polynucleotide capable of hybridizing under high stringency conditions encodes a glucose dehydrogenase polypeptide comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 4, the glucose dehydrogenase polypeptide having an amino acid sequence comprising one or more residue differences compared to SEQ ID NO: 4 at residue positions selected from the group consisting of: 96, 96 / 118, 147, 155, 155 / 253, 195, 200, and 256.

[0198] In some embodiments, the polynucleotide is capable of hybridizing under high stringency conditions to a reference polynucleotide sequence selected from an odd-numbered sequence within the range of SEQ ID NO: 5-1383, or a complementary sequence thereof, and encodes a glucose dehydrogenase polypeptide having imine reductase activity with one or more improved properties described herein. In some embodiments, the polynucleotide capable of hybridizing under high stringency conditions encodes a glucose dehydrogenase polypeptide comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 14, the glucose dehydrogenase polypeptide having an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 14. NO:14 an amino acid sequence that differs at one or more residues compared to a residue position selected from the group consisting of: 17, 95 / 96, 95 / 96 / 118, 95 / 96 / 118 / 156 / 200 / 253, 95 / 96 / 147, 95 / 96 / 147 / 200, 95 / 96 / 147 / 200 / 253, 95 / 96 / 155 / 156, 95 / 96 / 155 / 195 / 200, 95 / 96 / 159 / 195 / 253, 95 / 155 / 156, 95 / 155 / 159 / 200, 95 / 155 / 200, 96 / 147 / / 155 / 156, 155 / 156 / 195 / 200 / 253, 155 / 156, 155 / 156 / 195 / 200 / 253, 155 / 159 / 200 and 253.

[0199] In some embodiments, the polynucleotide capable of hybridizing under high stringency conditions encodes an engineered glucose dehydrogenase polypeptide having imine reductase activity with improved properties, the engineered glucose dehydrogenase polypeptide comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 34 and an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 34. NO:34 differs from one or more residues at a residue position selected from the group consisting of: 17, 17 / 21, 17 / 21 / 42, 17 / 21 / 42 / 155 / 260, 17 / 21 / 49, 17 / 21 / 49 / 51, 17 / 21 / 49 / 51 / 96, 17 / 21 / 49 / 51 / 260, 17 / 21 / 49 / 96 / 155 / 260, 17 / 21 / 49 / 260, 17 / 21 / 51 / 96, 17 / 21 / 51 / 195, 17 / 21 / 96 / 155 / 260. 5 / 260、17 / 21 / 96 / 253、17 / 21 / 147 / 155 / 253、17 / 21 / 155 / 253、17 / 21 / 195、17 / 21 / 253、17 / 42、17 / 42 / 49 / 195 / 260、17 / 42 / 96、17 / 44、17 / 44 / 47 / 51 / 96、17 / 44 / 47 / 51 / 96 / 114 / 147 / 195 / 199 / 253、17 / 44 / 47 / 51 / 96 / 114 / 147 / 199、17 / 44 / 47 / 51 / 96 / 114 / 19 5 / 199、17 / 44 / 47 / 51 / 96 / 147 / 253、17 / 44 / 47 / 51 / 178 / 195、17 / 44 / 47 / 195 / 199、17 / 44 / 51 / 114 / 195 / 253、17 / 46 / 47 / 51 / 96 / 147、17 / 46 / 47 / 51 / 96 / 195 / 253、17 / 46 / 47 / 51 / 155、17 / 46 / 47 / 51 / 155 / 195 / 199、17 / 46 / 47 / 96 / 195、17 / 46 / 47 / 178、17 / 46 / 96 / 253、17 / 47、17 / 47 / 51、17 / 47 / 51 / 96 / 114 / 147 / 195、17 / 47 / 51 / 96 / 114 / 155 / 178 / 253、17 / 47 / 51 / 96 / 195、17 / 47 / 51 / 96 / 195 / 199、17 / 47 / 96 / 114 / 155 / 253、17 / 47 / 96 / 147 / 155 / 195 / 199、17 / 47 / 96 / 147 / 195 / 199 / 253、17 / 47 / 96 / 155、17 / 47 / 96 / 195 / 199、17 / 47 / 114、17 / 47 / 114 / 155 / 195 / 199 / 253、17 / 47 / 147 / 195 / 199 / 253、17 / 49、17 / 49 / 51、17 / 49 / 51 / 96 / 195 / 253、17 / 49 / 51 / 155 / 253、17 / 49 / 51 / 155 / 253 / 260、17 / 49 / 51 / 155 / 260、17 / 49 / 51 / 253、17 / 49 / 96、17 / 49 / 96 / 155 / 253、17 / 49 / 96 / 195、17 / 49 / 260、17 / 51、17 / 51 / 96 / 114、17 / 51 / 96 / 155 / 178、17 / 51 / 96 / 155 / 195 / 199 / 253、17 / 51 / 96 / 155 / 199、17 / 51 / 96 / 155 / 253、17 / 51 / 96 / 178 / 195 / 199、17 / 51 / 96 / 195、17 / 51 / 96 / 260、17 / 51 / 114、17 / 51 / 114 / 253、17 / 51 / 147、17 / 51 / 155 / 195、17 / 51 / 178 / 195 / 253、17 / 51 / 253、17 / 96、17 / 96 / 114 / 147 / 155、17 / 96 / 114 / 147 / 155 / 178 / 195 / 199、17 / 96 / 147、17 / 96 / 147 / 155、17 / 96 / 155 / 178、17 / 96 / 155 / 178 / 253、17 / 96 / 155 / 195、17 / 96 / 155 / 195 / 199、17 / 96 / 178、17 / 96 / 178 / 195、17 / 96 / 195、17 / 96 / 195 / 199、17 / 96 / 195 / 199 / 253、17 / 96 / 253、17 / 114、17 / 114 / 253、17 / 147 / 155 / 253、17 / 147 / 178、17 / 147 / 199、17 / 155、17 / 155 / 178、17 / 155 / 178 / 195、17 / 155 / 195、17 / 155 / 195 / 253、17 / 155 / 199、17 / 155 / 253、17 / 178、17 / 178 / 195 / 253、17 / 195、17 / 195 / 199、17 / 195 / 199 / 253、17 / 195 / 253、17 / 199、17 / 253、17 / 260、21 / 49 / 51、21 / 49 / 96 / 195、21 / 49 / 96 / 253、21 / 49 / 195、21 / 51 / 96 / 155、21 / 96、21 / 96 / 195、21 / 96 / 253、44 / 46 / 47 / 51 / 114 / 178 / 253、44 / 46 / 47 / 51 / 155 / 178 / 253、44 / 46 / 47 / 96 / 155 / 195、44 / 46 / 51 / 96 / 147 / 178 / 195 / 199、44 / 46 / 51 / 96 / 155 / 195 / 199 / 253、44 / 46 / 51 / 96 / 195、44 / 46 / 51 / 147 / 155 / 178 / 195 / 253、44 / 47 / 51 / 96 / 114、44 / 47 / 51 / 96 / 177 / 178 / 199 / 253、44 / 47 / 51 / 114 / 178 / 253、44 / 47 / 96 / 114、44 / 47 / 96 / 195 / 253、44 / 47 / 147 / 155、44 / 47 / 147 / 155 / 199、44 / 96 / 155 / 178、46 / 47 / 51 / 96 / 114 / 178、46 / 47 / 51 / 114 / 147 / 195 / 199、46 / 47 / 51 / 155 / 195 / 199、46 / 47 / 96 / 114 / 195 / 199、46 / 47 / 96 / 155 / 178 / 195 / 253、46 / 47 / 155 / 195 / 199、46 / 114 / 147 / 155 / 178 / 195 / 199、46 / 114 / 195 / 199 / 253、47 / 51 / 96、47 / 51 / 96 / 114、47 / 51 / 96 / 147 / 155 / 195 / 199、47 / 51 / 96 / 147 / 195 / 199、47 / 51 / 96 / 178 / 195 / 199 / 253、47 / 51 / 96 / 195、47 / 51 / 96 / 195 / 199 / 253、47 / 51 / 114 / 253、47 / 51 / 155 / 178 / 195、47 / 51 / 155 / 195 / 253、47 / 96 / 114、47 / 96 / 114 / 253、47 / 96 / 155 / 178 / 195、47 / 96 / 155 / 195、47 / 96 / 155 / 195 / 199、47 / 96 / 178 / 195 / 253、47 / 96 / 178 / 253、47 / 96 / 195 / 199 / 253、47 / 96 / 195 / 253、47 / 114、47 / 114 / 155 / 195 / 199、47 / 114 / 178、47 / 147 / 195、47 / 155 / 253、47 / 178 / 195、47 / 178 / 195 / 199、47 / 195 / 199、49 / 51 / 96、49 / 96、49 / 96 / 253、51 / 96、51 / 96 / 114 / 155 / 195 / 199 / 253、51 / 96 / 155 / 178、51 / 96 / 155 / 178 / 195 / 253、51 / 96 / 155 / 195、51 / 96 / 155 / 195 / 253、51 / 96 / 195 / 253、51 / 96 / 199、51 / 96 / 253、51 / 114 / 155 / 195 / 199、51 / 114 / 195 / 199、51 / 147 / 253、51 / 155 / 195 / 199、51 / 195、51 / 253、96、96 / 114 / 147 / 155 / 195 / 199、96 / 114 / 155 / 178 / 195 / 199 / 253、96 / 114 / 155 / 195 / 253 、96 / 114 / 199、96 / 147、96 / 147 / 155 / 178 / 195 / 253、96 / 147 / 178 / 195 / 199、96 / 147 / 195、96 / 147 / 195 / 199、96 / 147 / 253、96 / 155 / 178 / 199、96 / 155 / 195、96 / 155 / 199、96 / 155 / 253、96 / 178 / 19 5. 96 / 178 / 195 / 199, 96 / 195, 96 / 195 / 199, 96 / 195 / 253, 96 / 199, 96 / 199 / 253, 96 / 253, 114, 114 / 147 / 155 / 253, 114 / 178 / 195 / 199 / 253, 114 / 195, 114 / 195 / 199 / 253, 132, 147 / 155, 149, 154, 155 / 178, 155 / 178 / 195 / 199, 155 / 195, 155 / 195 / 199, 155 / 195 / 199 / 253, 155 / 195 / 253, 155 / 253, 157, 167, 170, 178, 178 / 195, 178 / 199, 195, 196, 198, 214, 218, 251, 252, 256, 257, 259 and 260.

[0200] In some embodiments, the polynucleotide capable of hybridizing under high stringency conditions encodes an engineered glucose dehydrogenase polypeptide having imine reductase activity with improved properties, the engineered glucose dehydrogenase polypeptide comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 112 and an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 112. NO:112 differs from one or more residues at a residue position selected from the group consisting of: 21 / 42 / 96 / 178 / 195 / 253, 21 / 42 / 96 / 195, 21 / 96, 21 / 96 / 147 / 195, 21 / 96 / 149, 21 / 96 / 155 / 195 / 253, 21 / 96 / 167, 21 / 96 / 167 / 195, 21 / 96 / 178, 21 / 96 / 179 / 195 / 251 / 253, 21 / 96 / 195, 21 / 147 / 149, 21 / 147 / 253, 21 / 149 / 178 / 195、21 / 155 / 178 / 253 / 256、21 / 178 / 195 / 256、42 / 96 / 149 / 253 / 256、94、96、96 / 114 / 155 / 198 / 218 / 253 / 256 / 257、96 / 114 / 155 / 252 / 253 / 257、96 / 114 / 155 / 253、96 / 114 / 196 / 214 / 253 / 256、96 / 114 / 214 / 253 / 256、96 / 147、96 / 147 / 149、96 / 147 / 149 / 1 55, 96 / 147 / 167 / 195, 96 / 149 / 155 / 167 / 178 / 253, 96 / 149 / 155 / 195, 96 / 149 / 178 / 179 / 251 / 256, 96 / 149 / 178 / 253, 96 / 149 / 198 / 252 / 253 / 257, 96 / 155, 96 / 155 / 167, 96 / 155 / 167 / 178, 96 / 155 / 195, 96 / 155 / 196 / 218 / 253 / 256 / 257, 96 / 155 / 196 / 252 / 253, 96 / 155 / 198、96 / 155 / 252 / 253、96 / 167、96 / 178、96 / 178 / 179 / 195、96 / 195、96 / 196、96 / 196 / 198、96 / 196 / 201 / 256、96 / 251 / 253 / 256、96 / 253 / 257、96 / 256 / 257、98、103、114 / 253 / 256 / 257、147、147 / 149、149 / 167、149 / 195、149 / 218、149 / 253、149 / 257、155 / 167, 155 / 178 / 195, 155 / 178 / 195 / 251 / 253, 155 / 195 / 251 / 253 / 256, 155 / 195 / 253 / 256, 155 / 196 / 198 / 253, 167, 167 / 195, 167 / 195 / 251 / 253, 196, 196 / 253 / 256 / 257, 198 / 253 / 256, 198 / 256, 205, 214 / 253 / 256, 251 and 257.

[0201] In some embodiments, the polynucleotide capable of hybridizing under high stringency conditions encodes an engineered glucose dehydrogenase polypeptide having imine reductase activity with improved properties, the engineered glucose dehydrogenase polypeptide comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 736 and an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 736. NO:736 differs from one or more residues at a residue position selected from the group consisting of: 21 / 47 / 96 / 114 / 155 / 251, 21 / 47 / 149 / 257, 21 / 47 / 155 / 178 / 257, 21 / 47 / 178, 21 / 47 / 178 / 251 / 256 / 257, 21 / 96 / 114 / 155 / 178, 21 / 11 4 / 155 / 251 / 257、21 / 114 / 178 / 179、21 / 155 / 178 / 209 / 256、25 / 155、25 / 178 / 256、42 / 47 / 155 / 178、47 / 114 / 149 / 178 / 251、47 / 114 / 178 / 257、47 / 149、47 / 178、47 / 178 / 179 / 251 / 257、47 / 178 / 251 / 257、96 / 114 / 155 / 198、96 / 114 / 155 / 253 / 257、96 / 253、114 / 149 / 155 / 251 / 256、114 / 149 / 178 / 256 / 257、114 / 155 / 195 / 198 / 256、114 / 209 / 256 / 2 57, 149, 149 / 256 / 257, 155, 155 / 178 / 179, 155 / 178 / 198 / 252 / 253, 155 / 195 / 198, 155 / 195 / 198 / 253 / 256 / 257, 155 / 256 / 257, 178 / 253, 178 / 256, 179 / 256 / 257, 251 and 253.

[0202] In some embodiments, the polynucleotide capable of hybridizing under high stringency conditions encodes an engineered glucose dehydrogenase polypeptide having imine reductase activity with improved properties, the engineered glucose dehydrogenase polypeptide comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 908 and an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 908. NO:908 differs from one or more residues at a residue position selected from the group consisting of: 21 / 42 / 149, 21 / 42 / 149 / 214 / 253, 21 / 42 / 179 / 214 / 244 / 253, 21 / 42 / 253 / 260 / 261, 21 / 149, 21 / 149 / 179, 21 / 149 / 179 / 253, 21 / 149 / 179 / 253 / 260, 21 / 149 / 179 / 253 / 260 / 261, 21 / 149 / 179 / 261, 21 / 149 / 260 / 261, 21 / 149 / 261, 21 / 179 / 214 / 253, 21 / 179 / 253, 21 / 179 / 261, 21 / 214 / 253, 21 / 253, 40, 42 / 149 / 179 / 25 3 / 260、42 / 149 / 179 / 260 / 261、42 / 149 / 214 / 253、42 / 179 / 253 / 261、42 / 253、104、111、149、149 / 179、149 / 179 / 253、149 / 179 / 253 / 260 / 261、149 / 179 / 260、149 / 179 / 260 / 261、1 49 / 179 / 261, 149 / 253, 149 / 261, 160, 178, 179 / 214 / 253, 179 / 253, 179 / 253 / 260, 179 / 253 / 260 / 261, 179 / 253 / 261, 179 / 260 / 261, 179 / 261, 253, 253 / 260 / 261, 253 / 261 and 255.

[0203] In some embodiments, the polynucleotide capable of hybridizing under high stringency conditions encodes an engineered glucose dehydrogenase polypeptide having imine reductase activity with improved properties, the engineered glucose dehydrogenase polypeptide comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 956 and an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 956. NO:956 differs from one or more residues at a residue position selected from the group consisting of: 3, 21 / 104 / 160 / 178 / 179, 21 / 104 / 160 / 255 / 258, 21 / 104 / 178 / 179 / 255, 21 / 104 / 178 / 255 / 258, 21 / 104 / 255, 21 / 104 / 255 / 258, 21 / 104 / 255, 21 / 104 / 255 / 258, 21 / 104 / 255 / 258 9 / 255 / 260, 21 / 255 / 260, 31, 32, 40, 40 / 104 / 178 / 179, 40 / 160 / 178, 46 / 82 / 259, 103, 104 / 149 / 255 / 258 / 260, 104 / 149 / 258, 104 / 160, 104 / 160 / 178, 104 / 160 / 178 / 179 / 255, 1 04 / 160 / 178 / 179 / 260、104 / 160 / 178 / 255 / 260、104 / 160 / 179、104 / 178 / 179 / 255 / 258、104 / 178 / 255 / 258 / 260、104 / 178 / 255 / 260、104 / 179、104 / 255 / 260、104 / 258 / 260、111 / 149 / 255 / 258, 111 / 149 / 255 / 260, 111 / 160 / 178 / 179 / 255 / 258 / 260, 111 / 160 / 255 / 260, 154, 156, 160, 160 / 178 / 179, 174, 194, 197, 205, 205 / 229, 222, 253, 257, 258 / 260 and 260.

[0204] In some embodiments, the polynucleotide capable of hybridizing under high stringency conditions encodes an engineered glucose dehydrogenase polypeptide having imine reductase activity with improved properties, the engineered glucose dehydrogenase polypeptide comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 1062 and an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 1062. NO:1062 differs from one or more residues at a residue position selected from the group consisting of: 3 / 31 / 154 / 205 / 257, 3 / 31 / 205, 3 / 154 / 205, 31 / 55 / 154 / 205, 31 / 55 / 205, 31 / 154 / 205, 31 / 154 / 205 / 257, 31 / 205, 31 / 205 / 222, 40 / 55 / 205, 40 / 154 / 205, 40 / 154 / 205 / 257, 40 / 174 / 205 / 229, 40 / 205 / 229, 54 / 205 / 229. 5 / 253, 63, 63 / 149 / 154 / 174 / 178 / 253, 63 / 149 / 154 / 174 / 205 / 253, 63 / 149 / 156 / 178 / 205 / 253, 63 / 149 / 205, 63 / 149 / 205 / 229 / 253, 63 / 149 / 205 / 253, 63 / 154, 63 / 154 / 178 / 205 / 253, 63 / 154 / 205, 63 / 154 / 205 / 229, 63 / 154 / 205 / 229 / 253, 63 / 154 / 2 05 / 253、63 / 156、63 / 156 / 205、63 / 205、63 / 205 / 253、63 / 253、67、98、149 / 154、149 / 154 / 205、149 / 156 / 253、149 / 205、149 / 205 / 253、154、154 / 160、154 / 160 / 205、154 / 160 / 205 / 222 / 257、154 / 160 / 253、154 / 174、154 / 174 / 178 / 205、154 / 174 / 205 / 22 9, 154 / 205, 154 / 205 / 222, 154 / 205 / 222 / 257, 154 / 205 / 229, 154 / 205 / 253, 154 / 205 / 257, 154 / 229, 154 / 253, 156 / 205, 156 / 205 / 253, 156 / 229 / 253, 160, 160 / 205, 160 / 205 / 229, 194, 205, 205 / 222, 205 / 229, 205 / 229 / 253, 205 / 253, 208, 226 and 253.

[0205] In some embodiments, the polynucleotide capable of hybridizing under high stringency conditions encodes an engineered glucose dehydrogenase polypeptide having imine reductase activity with improved properties, the engineered glucose dehydrogenase polypeptide comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 4, 14, 34, 112, 736, 908, 956 and / or 1062. In some embodiments, the polynucleotide encodes a polypeptide described herein, but has at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference polynucleotide encoding an engineered glucose dehydrogenase at the nucleotide level. In some embodiments, the reference polynucleotide sequence is selected from SEQ ID NOs: 5-1383.

[0206] In some embodiments, the polynucleotide capable of hybridizing under high stringency conditions encodes an engineered glucose dehydrogenase polypeptide with improved properties having imine reductase activity, the engineered glucose dehydrogenase polypeptide comprising an amino acid sequence with SEQ ID NO: 4 having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity. In some embodiments, the polynucleotide encodes a polypeptide described herein, but has at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity or more at the nucleotide level with a reference polynucleotide encoding an engineered glucose dehydrogenase. In some embodiments, the reference polynucleotide sequence is selected from SEQ ID NO: 5-29.

[0207] In some embodiments, the polynucleotide capable of hybridizing under high stringency conditions encodes an engineered glucose dehydrogenase polypeptide having imine reductase activity with improved properties, the engineered glucose dehydrogenase polypeptide comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 14. In some embodiments, the polynucleotide encodes a polypeptide described herein, but has at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity or more with a reference polynucleotide encoding an engineered glucose dehydrogenase at the nucleotide level. In some embodiments, the reference polynucleotide sequence is selected from SEQ ID NO: 31-103.

[0208] In some embodiments, the polynucleotide capable of hybridizing under high stringency conditions encodes an engineered glucose dehydrogenase polypeptide with improved properties having imine reductase activity, the engineered glucose dehydrogenase polypeptide comprising an amino acid sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 34. In some embodiments, the polynucleotide encodes a polypeptide described herein, but has at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity or more at the nucleotide level to a reference polynucleotide encoding an engineered glucose dehydrogenase. In some embodiments, the reference polynucleotide sequence is selected from SEQ ID NO: 105-701.

[0209] In some embodiments, the polynucleotide capable of hybridizing under high stringency conditions encodes an engineered glucose dehydrogenase polypeptide with improved properties having imine reductase activity, the engineered glucose dehydrogenase polypeptide comprising an amino acid sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 112. In some embodiments, the polynucleotide encodes a polypeptide described herein, but has at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity or more at the nucleotide level to a reference polynucleotide encoding an engineered glucose dehydrogenase. In some embodiments, the reference polynucleotide sequence is selected from SEQ ID NO: 703-875.

[0210] In some embodiments, the polynucleotide capable of hybridizing under high stringency conditions encodes an engineered glucose dehydrogenase polypeptide having imine reductase activity with improved properties, the engineered glucose dehydrogenase polypeptide comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 736. In some embodiments, the polynucleotide encodes a polypeptide described herein, but has at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity or more with a reference polynucleotide encoding an engineered glucose dehydrogenase at the nucleotide level. In some embodiments, the reference polynucleotide sequence is selected from SEQ ID NO: 877-953.

[0211] In some embodiments, the polynucleotide capable of hybridizing under high stringency conditions encodes an engineered glucose dehydrogenase polypeptide having imine reductase activity with improved properties, the engineered glucose dehydrogenase polypeptide comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with SEQ ID NO: 908. In some embodiments, the polynucleotide encodes a polypeptide described herein, but has at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a reference polynucleotide encoding an engineered glucose dehydrogenase at the nucleotide level. In some embodiments, the reference polynucleotide sequence is selected from SEQ ID NO: 955-1059.

[0212] In some embodiments, the polynucleotide capable of hybridizing under high stringency conditions encodes an engineered glucose dehydrogenase polypeptide with improved properties having imine reductase activity, the engineered glucose dehydrogenase polypeptide comprising an amino acid sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 956. In some embodiments, the polynucleotide encodes a polypeptide described herein, but has at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity or more with a reference polynucleotide encoding an engineered glucose dehydrogenase at the nucleotide level. In some embodiments, the reference polynucleotide sequence is selected from SEQ ID NO: 1061-1169.

[0213] In some embodiments, the polynucleotide capable of hybridizing under high stringency conditions encodes an engineered glucose dehydrogenase polypeptide with improved properties having imine reductase activity, the engineered glucose dehydrogenase polypeptide comprising an amino acid sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 1062. In some embodiments, the polynucleotide encodes a polypeptide described herein, but has at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity or more at the nucleotide level to a reference polynucleotide encoding an engineered glucose dehydrogenase. In some embodiments, the reference polynucleotide sequence is selected from SEQ ID NO: 1171-1383.

[0214] In some embodiments, the polynucleotide of any separation in the engineering glucose dehydrogenase polypeptide provided by this paper is operated in a variety of ways to provide expression of polypeptide.In some embodiments, the polynucleotide of encoding polypeptide is provided as expression vector, wherein there are one or more control sequences to regulate the expression of polynucleotide and / or polypeptide.Depend on expression vector, the operation of the polynucleotide of separation before it is inserted into the vector can be desired or necessary.The technology for utilizing recombinant DNA method to modify polynucleotide and nucleotide sequence is well known in the art.

[0215] In some embodiments, the control sequences include, among other sequences, a promoter, a leader, a polyadenylation sequence, a propeptide sequence, a signal peptide sequence, and a transcription terminator. As known in the art, a suitable promoter can be selected based on the host cell used. For bacterial host cells, suitable promoters for directing transcription of the nucleic acid constructs of the present application include, but are not limited to, promoters obtained from the Escherichia coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis α-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens α-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, and prokaryotic β-lactamase genes (see, e.g., Villa-Kamaroff et al., Proc. Natl Acad. Sci. USA 75:3727-3731).

[1978] ), and the tac promoter (See, e.g., DeBoer et al., Proc. Natl Acad. Sci. USA 80:21-25

[1983] ). Exemplary promoters for filamentous fungal host cells include those obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral α-amylase, Aspergillus niger acid-stable α-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (See, e.g., WO 99 / 11665). 96 / 00787), and the NA2-tpi promoter (a hybrid of the promoters from the Aspergillus niger neutral α-amylase gene and the Aspergillus oryzae triose phosphate isomerase gene), and mutant promoters, truncated promoters, and hybrid promoters thereof. Exemplary yeast cell promoters can be derived from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are known in the art (see, e.g., Romanos et al., Yeast 8:423-488

[1992] ). In some embodiments, the control sequence is a suitable transcription terminator sequence, and the transcription terminator sequence is a sequence recognized by the host cell to terminate transcription. The terminator sequence is operably connected to the 3' end of the nucleic acid sequence of the coded polypeptide. Any terminator having a function in the host cell of selection can be used in the present invention. For example, the exemplary transcription terminator for filamentous fungal host cells can be obtained from the following genes: Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger α-glucosidase and Fusarium oxysporum trypsin-like protease. The exemplary terminator for yeast host cells can be obtained from the following genes: Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1) and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are known in the art (see, for example, Romanos etc., above). In some embodiments, the control sequence is a suitable leader sequence, which is a non-translated region of an mRNA that is important for translation by a host cell. The leader sequence is operably connected to the 5' end of the nucleic acid sequence encoding the polypeptide. Any leader sequence that works in the selected host cell can be used. The exemplary leader sequence for filamentous fungal host cells is obtained from the following genes: Aspergillus oryzae TAKA amylase and Aspergillus nidulans triosephosphate isomerase. Suitable leader sequences for yeast host cells include, but are not limited to, those obtained from the following genes: Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae α-factor and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP). The control sequence can also be a polyadenylation sequence, which is a sequence that is operably connected to the 3' end of the nucleic acid sequence, and when transcribed, it is recognized by the host cell as a signal that polyadenosine residues are added to the transcribed mRNA. Any polyadenylation sequence that works in the selected host cell can be used in the present invention. Exemplary polyadenylation sequences for filamentous fungal host cells include, but are not limited to, those from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger alpha-glucosidase. Useful polyadenylation sequences for yeast host cells are also known in the art (see, e.g., Guo and Sherman, Mol. Cell. Bio., 15:5983-5990

[1995] ). In some embodiments, the control sequence is a signal peptide coding region, which encodes an amino acid sequence connected to the amino terminus of the polypeptide and directs the encoded polypeptide to the secretory pathway of the cell. The 5' end of the coding sequence of the nucleic acid sequence may inherently contain a signal peptide coding region, which is naturally connected to the segment of the coding region encoding the secretory polypeptide in accordance with the translation reading frame. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding region that is exogenous to the coding sequence. Any signal peptide coding region that directs the expressed polypeptide to the secretory pathway of the selected host cell can be used for the expression of the engineered glucose dehydrogenase polypeptide provided herein. The effective signal peptide coding region for bacterial host cells includes but is not limited to the signal peptide coding region obtained from the following genes: Bacillus NClB 11837 maltogenic amylase, Bacillus stearothermophilus α-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis β-lactamase, Bacillus stearothermophilus neutral protease (nprT, nprS, nprM) and Bacillus subtilis prsA. Additional signal peptides are known in the art (see, e.g., Simonen and Palva, Microbiol. Rev., 57:109-137

[1993] ). Effective signal peptide coding regions for filamentous fungal host cells include, but are not limited to, those obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase. Useful signal peptides for yeast host cells include, but are not limited to, those from the genes for Saccharomyces cerevisiae alpha factor and Saccharomyces cerevisiae invertase. In some embodiments, the control sequence is a propeptide coding region encoding an amino acid sequence positioned at the amino terminus of a polypeptide. The polypeptide produced is referred to as a "proenzyme", "propolypeptide" or in some cases a "zymogen". The propolypeptide can be converted into a mature active polypeptide by catalyzing or autocatalyzing the cleavage of the propeptide from the propolypeptide. The propeptide coding region includes, but is not limited to, the following genes: Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae α-factor, Rhizomucor miehei aspartic protease, and Myceliophthora thermophila lactase (see, e.g., WO95 / 33836). When both the signal peptide and propeptide regions are present at the amino terminus of a polypeptide, the propeptide region is positioned adjacent to the amino terminus of the polypeptide and the signal peptide region is positioned adjacent to the amino terminus of the propeptide region. In some embodiments, regulatory sequences are also utilized. These sequences promote regulation of polypeptide expression relative to host cell growth. Examples of regulatory systems are those that cause gene expression to be turned on or off in response to chemical or physical stimuli (including the presence of regulatory compounds). In prokaryotic host cells, suitable regulatory sequences include but are not limited to lac, tac and trp operator systems. In yeast host cells, suitable regulatory systems include but are not limited to ADH2 systems or GAL1 systems. In filamentous fungi, suitable regulatory sequences include but are not limited to TAKA alpha-amylase promoters, Aspergillus niger glucoamylase promoters and Aspergillus oryzae glucoamylase promoters. The present invention also provides recombinant expression vector, described recombinant expression vector comprises the polynucleotide of coding engineering glucose dehydrogenase polypeptide and depends on one or more expression regulation regions of the type of the host that they will be introduced, such as promoter and terminator, replication origin etc.In some embodiments, various nucleic acids described above and control sequence can be combined together to produce recombinant expression vector, and this recombinant expression vector comprises one or more convenient restriction sites, to allow to insert or replace the nucleotide sequence of coding variant glucose dehydrogenase polypeptide in such site.Alternatively, polynucleotide sequence of the present invention is expressed by inserting polynucleotide sequence or the nucleic acid construct comprising this polynucleotide into suitable expression vector.When producing expression vector, encoding sequence is located in carrier, makes encoding sequence operably connected with the suitable control sequence for expressing. The recombinant expression vector can be any vector (e.g., plasmid or virus) that can be easily subjected to recombinant DNA procedures and can result in the expression of variant glucose dehydrogenase polynucleotide sequences. The selection of the vector will generally depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector can be a linear plasmid or a closed circular plasmid. In some embodiments, the expression vector is an autonomously replicating vector (i.e., a vector that exists as an extrachromosomal entity, whose replication is independent of chromosomal replication, such as a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome). The vector may include any means for ensuring self-replication. In some alternative embodiments, the vector may be a vector that, when introduced into a host cell, is integrated into the genome and replicates with the chromosome into which it is integrated. In addition, a single vector or plasmid or two or more vectors or plasmids or transposons that together contain the total DNA to be introduced into the host cell genome may be used. In some embodiments, the expression vector preferably comprises one or more selection markers that allow for easy selection of transformed cells. A "selection marker" is a gene whose product provides biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophic types, etc. Examples of bacterial selection markers include, but are not limited to, dal genes from Bacillus subtilis or Bacillus licheniformis, or markers that confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracycline resistance. Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1 and URA3. Selectable markers for use in filamentous fungal host cells include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenylyltransferase), and trpC (anthranilate synthase), and their equivalents. In another aspect, the present invention provides a host cell comprising a polynucleotide encoding at least one engineered glucose dehydrogenase polypeptide of the present invention, the polynucleotide being operably linked to one or more control sequences for expressing the engineered glucose dehydrogenase in the host cell. Host cells for use in expressing polypeptides encoded by expression vectors of the present invention are well known in the art and include, but are not limited to, bacterial cells such as Escherichia coli, Vibrio fluvialis, Streptomyces, and Salmonella typhimurium cells; fungal cells such as yeast cells (e.g., Saccharomyces cerevisiae and Pichia pastoris [ATCC Accession No. 201178]); insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, BHK, 293, and Bowes melanoma cells; and plant cells. Exemplary host cells are Escherichia coli strains (such as W3110 (ΔfhuA) and BL21). Therefore, in another aspect, the invention provides a method for producing an engineered glucose dehydrogenase polypeptide, wherein the method comprises culturing a host cell capable of expressing a polynucleotide encoding an engineered glucose dehydrogenase polypeptide under conditions suitable for expression of the polypeptide. In some embodiments, the method further comprises the step of separating and / or purifying the glucose dehydrogenase polypeptide as described herein. Suitable culture media and growth conditions for the host cells described above are well known in the art. Polynucleotides for expressing the glucose dehydrogenase polypeptide can be introduced into cells by various methods known in the art. Techniques include, among others, electroporation, bioparticle bombardment, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion. Engineered glucose dehydrogenases with properties disclosed herein can be obtained by subjecting polynucleotides encoding naturally occurring or engineered glucose dehydrogenase polypeptides to mutagenesis and / or directed evolution methods known in the art and as described herein. Exemplary directed evolution techniques are mutagenesis and / or DNA shuffling (see, e.g., Stemmer, Proc. Natl. Acad. Sci. USA 91: 10747-10751

[1994] ; WO 95 / 22625; WO 97 / 0078; WO 97 / 35966; WO 98 / 27230; WO 00 / 42651; WO 01 / 75767 and U.S. Patent No. 6,537,746). Other directed evolution procedures that can be used include, among others, the staggered extension process (StEP), in vitro recombination (see, e.g., Zhao et al., Nat. Biotechnol., 16:258-261

[1998] ), mutagenic PCR (see, e.g., Caldwell et al., PCR Methods Appl., 3:S136-S140

[1994] ), and cassette mutagenesis (see, e.g., Black et al., Proc. Natl. Acad. Sci. USA 93:3525-3529

[1996] ). For example, mutagenesis and directed evolution methods can be readily applied to polynucleotides to generate libraries of variants that can be expressed, screened, and assayed. Mutagenesis and directed evolution methods are well known in the art (see, e.g., U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, 5,837,458, 5,928,905, 6,096,548, 6,117,679, 6,132,970, 6,165,793, 6,180,406, 6,251,674, 6,265,201, 6,277,638, 6,287,861, 6,287,862, 6,291,242, 6,297,053, 6,298,061, 6,299,071, 6,304,330,331, 6,313,333,334,336, 6,326,337,338, 6,338,339, 6,340,351,333, 6,352,361,373, 6,362,373,373, 6,373,333, 6,373,334,336, 6,373,335, 6,373,336, 6,373,337,338, 6,373,339, 6,384,339, 6,385,339, 6,386,339, 6,387,339, 6,388,339, 6,389,339, 6,394,339, 6,4 3, No. 6,303,344, No. 6,309,883, No. 6,319,713, No. 6,319,714, No. 6,323,030, No. 6,326,204, No. 6,335,160, No. 6,335,198, No. 6,344,356, No. 6,352,859, No. 6,355,484, No. 6,358,740, No. 6,358,742, No. 6,365,377, No. 6,365,408, No. 6,368,861, No. 6,372,497, No. 6,337,186, No. 6,376,246, No. 6,379,9 64, 6,387,702, 6,391,552, 6,391,640, 6,395,547, 6,406,855, 6,406,910, 6,413,745, 6,413,774, 6,420,175, 6,423,542, 6,426,224, 6,436,675, 6,444,468, 6,455,253, 6,479,652, 6,482,647, 6,483,011, 6,484,105, 6,489,146, 6,500, 617, 6,500,639, 6,506,602, 6,506,603, 6,518,065, 6,519,065, 6,521,453, 6,528,311, 6,537,746, 6,573,098, 6,576,467, 6,579,678, 6,586,182, 6,602,986, 6,605,430, 6,613,514, 6,653,072, 6,686,515, 6,703,240, 6,716,631, 6,825,001, No. 6,902,922, No. 6,917,882, No. 6,946,296, No. 6,961,664, No. 6,995,017, No. 7,024,312, No. 7,058,515, No. 7,105,297, No. 7,148,054, No. 7,220,566, No. 7,288,375, No. 7,384,387, No. 7,421,347, No. 7,4 No. 30,477, No. 7,462,469, No. 7,534,564, No. 7,620,500, No. 7,620,502, No. 7,629,170, No. 7,702,464, No. 7,747,391, No. 7,747,393, No. 7,751,986, No. 7,776,598, No. 7,783,428, No. 7,795,030, No. 7,853,410, No. No. 7,868,138, No. 7,783,428, No. 7,873,477, No. 7,873,499, No. 7,904,249, No. 7,957,912, No. 7,981,614, No. 8,014,961, No. 8,029,988, No. 8,048,674, No. 8,058,001, No. 8,076,138, No. 8,108,150, No. 8,170,806 No. 8,224,580, No. 8,377,681, No. 8,383,346, No. 8,457,903, No. 8,504,498, No. 8,589,085, No. 8,762,066, No. 8,768,871, No. 9,593,326, and all related U.S. and PCT and non-U.S. counterparts; Ling et al., Anal. Biochem., 254:157-78,

[1997] ;Dale et al., Meth. Mol. Biol., 57:369-74

[1996] ;Smith, Ann. Rev. Genet., 19:423-462

[1985] ;Botstein et al., Science, 229:1193-1201

[1985] ;Carter, Biochem. J., 237:1-7

[1986] ;Kramer et al., Cell, 38:879-887

[1984] ;Wells et al., Gene, 34:315-323

[1985] ;Minshull et al., Curr. Op. Chem. Biol., 3:284-290

[1999] ;Christians et al., Nat. Biotech nol., 17:259-264

[1999] ; Crameri et al., Nature, 391:288-291

[1998] ; Crameri, et al., Nat. Biotechnol., 15:436-438

[1997] ; Zhang et al., Proc. Nat. Acad. Sci. USA, 94:4504-4509

[1997] ; Crameri et al., Nat. Biotechnol., 14:315-319

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[1994] ; Stemmer, Proc. Nat. Acad. Sci. USA, 91:10747-10751

[1994] ; WO 95 / 22625; WO 97 / 0078; WO 97 / 35966; WO 98 / 27230; WO 00 / 42651; WO 01 / 75767 and WO 2009 / 152336, all of which are incorporated herein by reference). In some embodiments, the enzyme clones obtained after mutagenesis are screened by subjecting the enzyme to a specified temperature (or other assay conditions, such as testing the activity of the enzyme to a wide range of substrates), and measuring the amount of enzyme activity remaining after heat treatment or other assay conditions. The clones of the polynucleotides encoding the glucose dehydrogenase polypeptide are then sequenced to identify changes in the nucleotide sequence, if any, and are used to express the enzyme in a host cell. Enzyme activity from an expression library can be measured using any suitable method known in the art (e.g., standard biochemical techniques, such as HPLC analysis). In some embodiments, clones obtained after mutagenesis can be screened for engineered glucose dehydrogenases with one or more desired improved enzyme properties (e.g., improved regioselectivity). Enzyme activity from expression libraries can be measured using standard biochemical techniques such as HPLC analysis, and / or product derivatization (before or after separation), such as using dansyl chloride or OPA (see, e.g., Yaegaki et al., J Chromatogr. 356 (1): 163-70

[1986] ). When the sequence of the engineered polypeptide is known, the polynucleotide encoding the enzyme can be prepared by a standard solid phase method according to a known synthesis method. In some embodiments, up to about 100 base fragments can be synthesized individually, then connected (for example, by an enzymatic ligation method or a chemical ligation method or a polymerase-mediated method) to form any desired continuous sequence. For example, the polynucleotides and oligonucleotides encoding the part of glucose dehydrogenase can be prepared by chemical synthesis as known in the art (for example, Beaucage et al., Tet. Lett. 22: 1859-69

[1981] classical phosphoramidite method or by Matthes et al., EMBO J. 3: 801-05

[1984] described method), because it is typically implemented with an automatic synthesis method. According to the phosphoramidite method, oligonucleotides are synthesized (for example, in an automatic DNA synthesizer), purified, annealed, connected and cloned into a suitable vector. In addition, any nucleic acid can be obtained from any of various commercial sources. In some embodiments, additional changes can be made by synthesizing oligonucleotides containing deletions, insertions, and / or substitutions and combining the oligonucleotides in various permutations to generate engineered glucose dehydrogenases with improved properties. Thus, in some embodiments, a method for preparing an engineered glucose dehydrogenase polypeptide comprises: (a) synthesizing a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to an even-numbered sequence selected from SEQ ID NOs: 6-1384, and having one or more residue differences compared to SEQ ID NOs: 4, 14, 34, 112, 736, 908, 956, and / or 1062; and (b) expressing the glucose dehydrogenase polypeptide encoded by the polynucleotide. In some embodiments of the method, the polynucleotide encoding optionally has one or several (e.g., up to 3, 4, 5 or up to 10) amino acid residue deletions, insertions and / or substitutions of engineered glucose dehydrogenases. In some embodiments, the amino acid sequence optionally has 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1-45 or 1-50 amino acid residue deletions, insertions and / or substitutions. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45 or 50 amino acid residue deletions, insertions and / or substitutions. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24 or 25 amino acid residue deletions, insertions and / or substitutions. In some embodiments, the substitutions may be conservative substitutions or non-conservative substitutions. In some embodiments, any one of the engineered glucose dehydrogenases expressed in host cells can be recovered from cells and / or culture medium using any one or more of the well-known techniques for protein purification, including, among others, lysozyme treatment, ultrasonic treatment, filtration, salting out, ultracentrifugation, and chromatography. Suitable solutions for lysis and efficient protein extraction from bacteria such as Escherichia coli are commercially available (e.g., CelLytic B TM , Sigma-Aldrich, St. Louis MO). Chromatographic techniques for separating glucose dehydrogenase polypeptides include, among others, reverse phase chromatography, high performance liquid chromatography, ion exchange chromatography, gel electrophoresis and affinity chromatography. The conditions used to purify a particular enzyme will depend in part on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, and will be apparent to those skilled in the art. In some embodiments, affinity techniques can be used to separate improved glucose dehydrogenase. For affinity chromatography purification, any antibody that specifically binds to a glucose dehydrogenase polypeptide can be used. In order to produce antibodies, various host animals can be immunized by injecting a glucose dehydrogenase polypeptide or a fragment thereof, including but not limited to rabbits, mice, rats, etc. The glucose dehydrogenase polypeptide or fragment can be attached to a suitable carrier such as BSA by way of a side chain functional group or a linker attached to a side chain functional group. In some embodiments, affinity purification can use a specific ligand or dye affinity column that binds to glucose dehydrogenase (see, e.g., EP0641862; Stellwagen, "Dye Affinity Chromatography," in Current Protocols in Protein Science ,Unit 9.2-9.2.16

[2001] ). Methods of using engineered glucose dehydrogenases In some embodiments, the glucose dehydrogenases described herein can be used in methods of converting one or more suitable substrates into a product. In another aspect, the engineered glucose dehydrogenase polypeptide disclosed herein can be used in a method for converting a substrate compound (2) or a structural analog thereof and a substrate compound (3) or a structural analog thereof into a product of compound (1) or a corresponding structural analog thereof. Generally, the structural analog of compound (1) is included in structural formula (IV). In some embodiments, the present disclosure provides methods for preparing compounds of structural formula (IV): in R1 is selected from the group consisting of a hydrogen atom, or an optionally substituted alkyl, alkenyl, alkynyl, alkoxy, arylalkoxy, hydroxyalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl and heteroarylalkyl; and R2 is independently selected from alkyl, alkenyl, alkynyl, hydroxyalkyl, carboxyl, aminocarbonyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, alkylamino, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl and heteroarylalkyl; and R3 is independently selected from methyl, d3-methyl and ethyl; The method comprises the steps of contacting a ketone substrate of structural formula (V) and an amine substrate of formula (VI) with an engineered polypeptide disclosed herein under suitable reaction conditions: The product of formula (IV) is thereby prepared. In some embodiments, the present disclosure provides a method for preparing compound (1); The method comprises reacting a substrate of compound (2) under suitable reaction conditions. and a substrate of compound (3) The step of contacting with the engineered polypeptide disclosed herein, thereby producing a product of compound (1). In the embodiments provided herein and illustrated in the Examples, various ranges of suitable reaction conditions that can be used in the method include, but are not limited to, substrate loading, co-substrate loading, pH, temperature, buffer, solvent system, polypeptide loading, and reaction time. Additional suitable reaction conditions for biocatalytic conversion of substrate compounds to product compounds using the engineered glucose dehydrogenase described herein can be easily optimized by routine experimentation according to the guidance provided herein, including but not limited to contacting the engineered glucose dehydrogenase polypeptide and one or more substrate compounds under experimental reaction conditions of concentration, pH, temperature, and solvent conditions, and detecting product compounds. In some embodiments, suitable reaction conditions include for each of one or more substrates, at least about 0.5g / L to about 25g / L, 1g / L to about 25g / L, 5g / L to about 25g / L, about 10g / L to about 25g / L or 20g / L to about 25g / L substrate compound loading. In some embodiments, suitable reaction conditions include for each of one or more substrates, at least about 0.5g / L, at least about 1g / L, at least about 5g / L, at least about 10g / L, at least about 15g / L, at least about 20g / L or at least about 30g / L or even larger substrate compound loading. When carrying out the glucose dehydrogenase-mediated method described herein, the engineered polypeptide can be added to the reaction mixture in the form of a purified enzyme, a partially purified enzyme, a complete cell transformed with a gene encoding the enzyme, an extract and / or lysate of such a cell, and / or an enzyme fixed on a solid support. The complete cell or cell extract transformed with a gene encoding the engineered glucose dehydrogenase, its lysate and the separated enzyme can be used in various forms, including solids (e.g., freeze-dried, spray-dried, etc.) or semisolids (e.g., crude pastes). Cell extracts or cell lysates can be partially purified by precipitation (ammonium sulfate, polyethyleneimine, heat treatment, etc.), and then desalting procedures (e.g., ultrafiltration, dialysis, etc.) are performed before freeze-drying. Any enzyme preparation (including complete cell preparations) can be cross-linked or fixed to a solid phase (e.g., Eupergit C, etc.) using a known cross-linking agent (such as, for example, glutaraldehyde) to stabilize. The gene encoding the engineered glucose dehydrogenase polypeptide can be transformed into the host cell individually or together into the same host cell. For example, in some embodiments, one group of host cells can be transformed with a gene encoding a kind of engineered glucose dehydrogenase polypeptide, and another group of host cells can be transformed with a gene encoding another kind of engineered glucose dehydrogenase polypeptide. The two groups of transformed cells can be used together in the reaction mixture in the form of intact cells or in the form of a lysate or extract derived therefrom. In other embodiments, the host cell can be transformed with a gene encoding more than one engineered glucose dehydrogenase polypeptide. In some embodiments, the engineered polypeptide can be expressed in the form of a secreted polypeptide, and the culture medium containing the secreted polypeptide can be used for the glucose dehydrogenase reaction. In some embodiments, the improved activity and / or regioselectivity and / or stereoselectivity of the engineered glucose dehydrogenase polypeptide disclosed herein provides a method in which a higher conversion percentage can be achieved with a lower concentration of the engineered polypeptide. In some embodiments of the method, suitable reaction conditions include about 1% (w / w), 2% (w / w), 5% (w / w), 10% (w / w), 20% (w / w), 30% (w / w), 40% (w / w), 50% (w / w), 75% (w / w), 100% (w / w) or more of the amount of the engineered polypeptide of the substrate compound load. In some embodiments, the engineered polypeptide is present at about 0.01 g / L to about 50 g / L; about 0.05 g / L to about 50 g / L; about 0.1 g / L to about 40 g / L; about 1 g / L to about 40 g / L; about 2 g / L to about 40 g / L; about 5 g / L to about 40 g / L; about 5 g / L to about 30 g / L; about 0.1 g / L to about 10 g / L; about 0.5 g / L to about 10 g / L; about 1 g / L to about 10 g / L; about 0.1 g / L to about 5 g / L; about 0.5 g / L to about 5 g / L or about 0.1 g / L to about 2 g / L. In some embodiments, the glucose dehydrogenase polypeptide is present at about 0.01 g / L, 0.05 g / L, 0.1 g / L, 0.2 g / L, 0.5 g / L, 1 g / L, 2 g / L, 5 g / L, 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, or 50 g / L. During the reaction process, the pH of the reaction mixture can change. The pH of the reaction mixture can be maintained at a desired pH or within a desired pH range. This can be carried out by adding acid or alkali before and / or during the reaction process. Alternatively, pH can be controlled by using a buffer. Therefore, in some embodiments, reaction conditions include a buffer. Suitable buffers for maintaining the desired pH range are known in the art, and include, for example, but not limited to borate, phosphate, 2-(N-morpholino) ethanesulfonic acid (MES), 3-(N-morpholino) propanesulfonic acid (MOPS), acetate, triethanolamine and 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris) etc. In some embodiments, reaction conditions include water as a suitable solvent, without the presence of a buffer. In the embodiment of the method, reaction conditions include suitable pH. The desired pH or desired pH range can be maintained by using an acid or base, a suitable buffer or a combination of buffering and acid or base addition. The pH of the reaction mixture can be controlled before and / or during the reaction process. In some embodiments, suitable reaction conditions include a solution of about 4 to about 10 pH, about 5 to about 10 pH, about 5 to about 9 pH, about 6 to about 9 pH, about 6 to about 8 pH. In some embodiments, reaction conditions include a solution of about 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 pH. In the embodiments of the method herein, suitable temperature is used for reaction conditions, for example, considering the increase of reaction rate at higher temperatures and the activity of enzyme during the reaction time period. Therefore, in some embodiments, suitable reaction conditions include a temperature of about 10 ℃ to about 60 ℃, about 10 ℃ to about 55 ℃, about 15 ℃ to about 60 ℃, about 20 ℃ to about 60 ℃, about 20 ℃ to about 55 ℃, about 25 ℃ to about 55 ℃ or about 30 ℃ to about 50 ℃. In some embodiments, suitable reaction conditions include a temperature of about 10 ℃, 15 ℃, 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃ or 60 ℃. In some embodiments, the temperature during the enzymatic reaction can be maintained at a specific temperature during the entire reaction process. In some embodiments, the temperature during the enzymatic reaction can be adjusted with the change of the temperature curve during the reaction process. In some embodiments, the method of the present invention is carried out in a solvent. Suitable solvents include water, aqueous buffer solutions, organic solvents, polymer solvents and / or co-solvent systems, which generally include aqueous solvents, organic solvents and / or polymer solvents. Aqueous solvents (water or aqueous co-solvent systems) can be pH buffered or non-buffered. In some embodiments, the method using engineered glucose dehydrogenase polypeptides can be carried out in an aqueous co-solvent system comprising: organic solvents (e.g., ethanol, isopropanol (IPA), dimethyl sulfoxide (DMSO), dimethyl formamide (DMF), ethyl acetate, butyl acetate, 1-octanol, heptane, octane, methyl tert-butyl ether (MTBE), toluene, etc.), ions or polar solvents (e.g., 1-ethyl 4-methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium tetrafluoroborate, 1-butyl 3-methylimidazolium hexafluorophosphate, glycerol, polyethylene glycol, etc.). In some embodiments, co-solvents can be polar solvents, such as polyols, dimethyl sulfoxide (DMSO) or lower alcohols. The non-aqueous co-solvent component of aqueous co-solvent system can be miscible with aqueous component, thereby single liquid phase is provided, or can be partially miscible or immiscible with aqueous component, thereby two liquid phases are provided. Exemplary aqueous co-solvent system can comprise water and one or more co-solvents selected from organic solvent, polar solvent and polyol solvent.Usually, the co-solvent component of aqueous co-solvent system is selected so that it can not unfavorably inactivate glucose dehydrogenase under reaction conditions.Suitable co-solvent system can be easily identified by utilizing enzyme activity assay (such as those described herein) to measure specific engineered glucose dehydrogenase and the interested substrate of restriction in candidate solvent system. In some embodiments of the method, suitable reaction conditions include aqueous co-solvents, wherein the co-solvent includes about 1% to about 50% (v / v), about 1% to about 40% (v / v), about 2% to about 40% (v / v), about 5% to about 30% (v / v), about 10% to about 30% (v / v) or about 10% to about 20% (v / v) of DMSO. In some embodiments of the method, suitable reaction conditions may include an aqueous co-solvent comprising about 1% (v / v), about 5% (v / v), about 10% (v / v), about 15% (v / v), about 20% (v / v), about 25% (v / v), about 30% (v / v), about 35% (v / v), about 40% (v / v), about 45% (v / v) or about 50% (v / v) of ethanol. In some embodiments, the reaction conditions include a surfactant for stabilizing or enhancing the reaction. The surfactant may include a nonionic surfactant, a cationic surfactant, an anionic surfactant, and / or an amphiphilic surfactant. Exemplary surfactants include, for example, but are not limited to, nonylphenoxypolyethoxyethanol (NP40), TRITON TM X-100 polyethylene glycol tert-octylphenyl ether, polyoxyethylene-stearamide, hexadecyl trimethyl ammonium bromide, oleyl amido sodium sulfate, polyoxyethylene sorbitan monostearate, hexadecyl dimethylamine etc. Any surfactant that can stabilize or enhance the reaction can be used. The concentration of the surfactant used in the reaction can be usually 0.1mg / ml to 50mg / ml, especially 1mg / ml to 20mg / ml. In some embodiments, the reaction conditions include a defoamer, which helps reduce or prevent the formation of foam in the reaction solution, such as when the reaction solution is mixed or sprayed. Defoamers include non-polar oils (e.g., mineral oils, silicones, etc.), polar oils (e.g., fatty acids, alkylamines, alkylamides, alkyl sulfates, etc.), and hydrophobic substances (e.g., treated silica, polypropylene, etc.), some of which also function as surfactants. Exemplary defoamers include (Dow Corning), polyglycol copolymers, oxy / ethoxylated alcohols, and polydimethylsiloxanes. In some embodiments, the defoamer may be present at about 0.001% (v / v) to about 5% (v / v), about 0.01% (v / v) to about 5% (v / v), about 0.1% (v / v) to about 5% (v / v), or about 0.1% (v / v) to about 2% (v / v). In some embodiments, the defoamer may be present at about 0.001% (v / v), about 0.01% (v / v), about 0.1% (v / v), about 0.5% (v / v), about 1% (v / v), about 2% (v / v), about 3% (v / v), about 4% (v / v), or about 5% (v / v) or more, as desired to promote the reaction. The amount of reactants used in the glucose dehydrogenase reaction will generally vary depending on the amount of desired product and the amount of glucose dehydrogenase substrate used concomitantly. One of ordinary skill in the art will readily understand how to vary these amounts to tailor them to the level of desired productivity and production scale. In some embodiments, the order in which the reactants are added is not critical. The reactants can be added together simultaneously to a solvent (e.g., a single-phase solvent, a two-phase aqueous co-solvent system, etc.), or alternatively, some reactants can be added separately, while some reactants can be added together at different time points. For example, cofactors, cosubstrates, and substrates can be added to the solvent first. Solid reactants (e.g., enzymes, salts, etc.) can be provided to the reaction in a variety of different forms, including powders (e.g., lyophilized powders, spray-dried powders, etc.), solutions, emulsions, suspensions, etc. The reactants can be readily lyophilized or spray-dried using methods and equipment known to those of ordinary skill in the art. For example, a protein solution can be frozen at -80°C in small aliquots and then added to a pre-cooled lyophilization chamber, followed by application of a vacuum. When using an aqueous co-solvent system, to improve mixing efficiency, the glucose dehydrogenase and co-substrate can be added and mixed into the aqueous phase first. The glucose dehydrogenase substrate can be added and mixed in, followed by the organic phase, or the substrate can be dissolved in the organic phase and mixed in. Alternatively, the glucose dehydrogenase substrate can be premixed in the organic phase and then added to the aqueous phase. The method of the present invention is generally allowed to proceed until further conversion of substrate to product does not vary significantly with reaction time (e.g., less than 10% of substrate is converted or less than 5% of substrate is converted). In some embodiments, the reaction is allowed to proceed until the substrate is completely or nearly completely converted to product. The conversion of substrate to product can be monitored using known methods by detecting substrate and / or product (with or without derivatization). Suitable analytical methods include gas chromatography, HPLC, MS, etc. In some embodiments of the method, suitable reaction conditions include a substrate loading of at least about 5 g / L, 10 g / L, 20 g / L, or more for each of the one or more substrates, and wherein the method produces at least about 50%, 60%, 70%, 80%, 90%, 95% or more conversion of the substrate compound to the product compound in about 24 h or less, in about 12 h or less, in about 6 h or less, or in about 4 h or less. When used in a method under appropriate reaction conditions, the engineered glucose dehydrogenase polypeptides of the invention produce an excess of desired product over undesired products of at least 90%, 95%, 96%, 97%, 98%, 99% or more diastereomeric excess. In some other embodiments of the method for converting one or more compounds into product compounds using engineered glucose dehydrogenase polypeptides, suitable reaction conditions can be included in the reaction solution for each of one or more substrates, and then contacted with polypeptide.Then the reaction solution is further supplemented with other substrate compounds added continuously or in batches, for each of one or more substrates, at least about 1g / L / h, at least about 2g / L / h, at least about 4g / L / h, at least about 6g / L / h or higher speed over time.Therefore, according to these suitable reaction conditions, for each of one or more substrates, polypeptide is added to the solution with at least about 1g / L, 5g / L or 10g / L initial substrate loading. After this addition of the polypeptide, additional substrate is then continuously added to the solution at a rate of about 2 g / L / h, 4 g / L / h, or 6 g / L / h for each of the one or more substrates until a much higher final substrate loading of at least about 30 g / L or more is reached for each of the one or more substrates. Thus, in some embodiments of the method, suitable reaction conditions include adding the polypeptide to a solution having an initial substrate loading of at least about 1 g / L, 5 g / L, or 10 g / L, and then adding additional substrate to the solution at a rate of about 2 g / L / h, 4 g / L / h, or 6 g / L / h until a final substrate loading of at least about 30 g / L or more is reached for each of the one or more substrates. The substrate supplementation reaction conditions allow for a higher substrate loading to be achieved while maintaining a high conversion rate of at least about 5%, 25%, 50%, 75%, 90% or more of substrate conversion to product for each or both of the one or more substrates. Any method disclosed herein for preparing a compound of formula (IV) or compound (1) using an engineered polypeptide can be performed under a suitable range of reaction conditions (including but not limited to an amine substrate range, a ketone substrate range, temperature, pH, solvent system, substrate loading, polypeptide loading, cofactor loading, and reaction time). In one example, in some embodiments, the preparation of a compound of formula (IV) or compound (1) can be performed, wherein suitable reaction conditions include: (a) an amine substrate loading of about 1 g / L to 200 g / L substrate compound; (b) a ketone substrate loading of about 1 g / L to 200 g / L substrate compound; (c) about 0.5 g / L to 25 g / L of the engineered polypeptide; (d) 0-20% DMSO; (e) 1 g / L of GDH cofactor cycle enzyme, 0.1 g / L of NAD+ cofactor, and up to 120 g / L of glucose; (f) at a pH of 5.5-8; and (g) a temperature of about 30°C to 60°C. In some embodiments, suitable reaction conditions include: (a) about 20 g / L of an amine substrate compound; (b) about 20 g / L of a ketone substrate compound; (c) about 5 g / L of an engineered polypeptide; (d) 10% DMSO; (e) 1 g / L GDH cofactor cycle enzyme, 0.1 g / L NAD+ cofactor, and 20 g / L glucose; (f) at a steady-state pH of 6.5; and (g) about 30°C. In some embodiments, additional reaction components or additional techniques are performed to supplement the reaction conditions. These may include taking measures to stabilize the enzyme or prevent enzyme inactivation, reduce product inhibition, and shift the reaction equilibrium toward the formation of the desired product. In further embodiments, any of the above-described methods for converting one or more substrate compounds into product compounds may also include one or more steps selected from the group consisting of extraction, separation, purification and crystallization of the product compounds. Methods, techniques and protocols for extracting, separating, purifying and / or crystallizing products from biocatalytic reaction mixtures produced by the methods disclosed above are known to those of ordinary skill and / or are learned by routine experimentation. In addition, illustrative methods are provided in the Examples below. Various features and embodiments of the invention are illustrated in the following representative examples, which are intended to be illustrative rather than limiting. experiment The following examples, including the experiments and results obtained, are provided for illustrative purposes only and should not be construed as limiting the present invention. In the examples hereinafter, the following abbreviations apply: ppm (parts per million); M (mole / liter); mM (millimol / liter), uM and μM (micromol / liter); nM (nanomoles / liter); mol (mole); gm and g (gram); mg (milligram); ug and μg (microgram); L and l (liter); ml and mL (milliliter); cm (centimeter); mm (millimeter); um and μm (micrometer); sec. (second); min(s) (minute); h(s) and hr(s) (hour); U (unit); MW (molecular weight); rpm (revolutions per minute). number); psi and PSI (pounds per square inch); °C (degrees Celsius); RT and rt (room temperature); CAM and cam (chloramphenicol); DMSO (dimethyl sulfoxide); PMBS (polymyxin B sulfate); IPTG (isopropyl β-D-1-thiogalactopyranoside); LB (Luria-Bertani broth); TB (Terrific broth; 12 g / L Bactotryptone, 24 g / L yeast extract, 4 mL / L glycerol, 65 mM potassium phosphate, pH 7.0, 1 mM MgSO4); HEPES (HEPES zwitterionic buffer; 4-(2-hydroxyethyl)-piperazineethanesulfonic acid); SFP (shake flask powder); CDS (coding sequence); DNA (deoxyribonucleic acid); RNA (ribonucleic acid); Escherichia coli W3110 (a commonly used laboratory strain of E. coli, available from the Coli Genetic Stock Center [CGSC], New Haven, CT); HTP (high throughput); HPLC (high pressure liquid chromatography); FIOPC (fold increase over positive control); Microfluidics (Microfluidics, Corp., Westwood, MA); Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO); Difco (Difco Laboratories, BD Diagnostic Systems, Detroit, MI); Agilent (Agilent Technologies, Inc., Santa Clara, CA); Corning (Corning, Inc., Palo Alto, CA); Alto, CA); Dow Corning (Dow Corning, Corp., Midland, MI) and Gene Oracle (Gene Oracle, Inc., Mountain View, CA). Example 1 E. coli expression host containing recombinant GDH gene The initial GDH enzyme for producing variants of the present invention is obtained from U.S. Patent No. 7,816,111 and No. 7,939,309. The GDH coding gene is cloned into expression vector pCK110900 (see Fig. 3 of U.S. Patent Application Publication No. 2006 / 0195947) and is operably connected to the lac promoter under the control of the lac1 repressor. The expression vector also comprises a P15a origin of replication and a chloramphenicol resistance gene. The resulting plasmid is transformed into Escherichia coli W3110 using standard methods known in the art. As known in the art, transformants are isolated by subjecting cells to chloramphenicol selection (see, for example, U.S. Patent No. 8,383,346 and WO2010 / 144103). Example 2 High-throughput preparation of wet cell pellets containing GDH The E. coli cells containing recombinant GDH encoding genes from monoclonal colonies are inoculated into 180 μl LB containing 1% glucose and 30 μg / mL chloramphenicol in the holes of 96-well shallow-well microtiter plates. The plate is sealed with a seal that can be permeated with O2, and the culture is grown overnight at 30°C, 200rpm and 85% humidity. Then, each cell culture of 10 μl is transferred to the hole of a 96-well deep-well plate containing 390mL TB and 30 μg / mL CAM. The deep-well plate is sealed with a seal that can be permeated with O2, and cultivated at 30°C, 250rpm and 85% humidity until OD600 0.6-0.8 is reached. Then the cell culture is induced with the IPTG reaching a final concentration of 1mM, and cultured overnight under the same conditions as initially used. Then the cells are centrifuged at 4000rpm for 10 minutes for precipitation. In some cases, overnight cultures are merged from two plates, then centrifuged, and duplicate precipitates are obtained. The supernatant was discarded and the pellet was frozen at -80°C before lysis. Example 3 High-throughput preparation of cell lysate containing GDH First, 200 μl lysis buffer containing 200 mM triethanolamine (TEoA) buffer pH 8.5, 1 mg / mL lysozyme and 0.5 mg / mL PMBS was added to the cell paste in each well produced as described in Example 2. Cells were lysed at room temperature for 2 hours with shaking on a bench top shaker. Plates were then centrifuged at 4000 rpm and 4°C for 15 min. Clarified supernatants were used for biocatalytic reactions to determine their activity levels. Example 4 Preparation of lyophilized lysates from shake flask (SF) cultures Selected HTP cultures grown as described above were plated onto LB agar plates containing 1% glucose and 30 μg / ml CAM and grown overnight at 37° C. A single colony from each culture was transferred to 6 ml LB containing 1% glucose and 30 μg / ml CAM. The cultures were grown at 30° C., 250 rpm for 18 h and subcultured approximately 1:50 into 250 ml TB containing 30 μg / ml CAM to a final OD of 0.05. 600 Grow the culture at 30°C, 250 rpm for approximately 195 minutes to an OD between 0.6-0.8. 600 , and induced with 1mM IPTG.Then the culture was grown at 30°C, 250rpm for 20h. The culture was centrifuged at 4000rpm for 20min. The supernatant was discarded and the precipitate was resuspended in 30ml of 200mM TEoA buffer, pH 8.5. The cells were precipitated (4000rpm for 20min) and frozen at -80°C for 120 minutes. The frozen precipitate was resuspended in 30ml of 200mM TEoA buffer, pH 8.5, and cracked using a Microfluidizer system (Microfluidics) at 18,000psi. The lysate was precipitated (10,000rpm for 60min) and the supernatant was frozen and lyophilized to produce a shake flask (SF) enzyme. Example 5 Improved activity of GDH relative to SEQ ID NO: 4 in high throughput screening SEQ ID NO:4 was selected as the parent enzyme, which has evolved towards improved glucose dehydrogenase activity and stability and is disclosed in U.S. Patent Nos. 7,816,111 and 7,939,309. Compared with the wild-type enzyme of SEQ ID NO:2, SEQ ID NO:4 comprises the following mutations: E170K, Q252L, I165M, P194T. A library of engineered genes was generated using recognized techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial mutations). The polypeptide encoded by each gene was produced with HTP as described in Example 2, duplicate precipitates were obtained, and soluble lysates were produced as described in Example 3. To lyse the cells, 200 μl of lysis buffer containing 200 mM TEoA buffer pH 8.5, 1 mg / mL lysozyme and 0.5 mg / mL PMBS was added to the cell mass. The cells were incubated at room temperature for 2 hours with shaking on a benchtop shaker. The plate was then centrifuged at 4000 rpm and 4°C for 15 minutes, and the clarified supernatant was used for subsequent biocatalytic reactions. HTP reactions were performed in 96-well deep well plates containing 200 μL 0.2M TEoA pH 8.5, 1.25 g / L chlorobiphenylpyruvic acid, 3.6 g / L L-alanine-3,3,3-d3 ester (~5x molar equivalents), 5% DMSO, 25 μL of the above HTP supernatant, 0.5 g / L PDH 101, 25 mM phosphite, 1.5 g / L NAD+, or only 20 g / L glucose and 6 g / L NAD+. The HTP plates were incubated at 30°C, 400 rpm for 12 hours in a Thermotron (3 mm swing, model #AJ185, Infors). The reaction was quenched with 0.1% formic acid in methanol at a ratio of 1:10, the supernatant was further diluted with water at a ratio of 1:25, and then loaded into RapidFire for analysis. The activity relative to SEQ ID NO: 4 was calculated as the fold increase over the positive control (FIOPC). It was determined by dividing the mass spectrometric signal of the product in each sample under the specified reaction conditions by the mass spectrometric signal of D3-chlorobiphenylpyruvate present in the parental variant (positive control) in the same plate and is shown in Table 5.1. Example 6 Improved activity of GDH relative to SEQ ID NO: 14 in high throughput screening After screening variants as described in Example 5, SEQ ID NO: 14 was selected as the parent enzyme. Libraries of engineered genes were generated using recognized techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were generated as described in Example 2 with HTP, duplicate precipitates were obtained, and soluble lysates were generated as described in Example 3. To lyse the cells, 200 μl of lysis buffer containing 200 mM TEoA buffer pH 8.5, 1 mg / mL lysozyme and 0.5 mg / mL PMBS was added to the cell mass. The cells were incubated at room temperature for 2 hours with shaking on a benchtop shaker. The plate was then centrifuged at 4000 rpm and 4°C for 15 minutes, and the clarified supernatant was used for subsequent biocatalytic reactions. HTP reactions were performed in 96-well deep well plates containing 200 μL 0.2M TEoA pH 8.5, 1.25 g / L chlorobiphenylpyruvic acid, 3.5 g / L L-alanine ethyl ester (~5x molar equivalents), 5% DMSO, 25 μL of the above HTP supernatant, and 0.5 g / L PDH 101, 25 mM phosphite, 1.5 g / L NAD+. The HTP plates were incubated at 30°C, 400 rpm for 12 hours in a Thermotron (3 mm swing, model # AJ185, Infors). The reaction was quenched with 0.1% formic acid in methanol at a ratio of 1:20, the supernatant was further diluted with water at a ratio of 1:20, and then loaded into RapidFire for analysis. Activity relative to SEQ ID NO: 14 was calculated as the fold increase over the positive control (FIOPC) determined by dividing the mass spectrometry signal of the product in each sample under the specified reaction conditions by the mass spectrometry signal of the chlorobiphenylpyruvate product in the parental variant (positive control) present in the same plate. Example 7 Improved activity of GDH relative to SEQ ID NO: 34 After screening variants as described in Example 6, SEQ ID NO: 34 was selected as the parent enzyme. Libraries of engineered genes were generated using recognized techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were generated as described in Example 2 with HTP, duplicate precipitates were obtained, and soluble lysates were generated as described in Example 3. To lyse the cells, 200 μl of lysis buffer containing 200 mM TEoA buffer pH 8.5, 1 mg / mL lysozyme and 0.5 mg / mL PMBS was added to the cell mass. The cells were incubated at room temperature for 2 hours with shaking on a benchtop shaker. The plate was then centrifuged at 4000 rpm and 4°C for 15 minutes, and the clarified supernatant was used for subsequent biocatalytic reactions. HTP reaction was carried out in a 96-well deep well plate containing 200 μL 0.2M TEoA pH 8.5, 10 g / L chlorobiphenylpyruvic acid, 28 g / L L-alanine ethyl ester (~5x molar equivalent), 5% DMSO, 50 μL of the above HTP supernatant, and 1 g / L GDH 105, 10 g / L (~55 mM) glucose, 0.1 g / L NAD+. The HTP plate was incubated at 30°C, 400 rpm for 12 hours in a Thermotron (3 mm swing, model # AJ185, Infors). The reaction was quenched with 0.1% formic acid in methanol at a ratio of 1:20, the supernatant was further diluted with water at a ratio of 1:400, and then loaded into RapidFire for analysis. Activity relative to SEQ ID NO: 34 was calculated as the fold increase over the positive control (FIOPC) determined by dividing the mass spectrometry signal of the product in each sample under the specified reaction conditions by the mass spectrometry signal of the chlorobiphenylpyruvate product in the parental variant (positive control) present in the same plate. Example 8 Improved activity of GDH relative to SEQ ID NO: 112 After screening variants as described in Example 7, SEQ ID NO: 112 was selected as the parent enzyme. Libraries of engineered genes were generated using recognized techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were generated as described in Example 2 with HTP, a single precipitate was obtained, and soluble lysates were generated as described in Example 3. To lyse the cells, 200 μl of lysis buffer containing 200 mM TEoA buffer pH 8.5, 1 mg / mL lysozyme and 0.5 mg / mL PMBS was added to the cell mass. The cells were incubated at room temperature for 2 hours with shaking on a benchtop shaker. The plate was then centrifuged at 4000 rpm and 4°C for 15 minutes, and the clarified supernatant was used for subsequent biocatalytic reactions. HTP reaction was carried out in a 96-well deep well plate containing 200 μL 0.2M TEoA, pH 9, 20 g / L chlorobiphenylpyruvic acid, 56 g / L L-alanine ethyl ester (~5x molar excess), 10% DMSO, 20 μL of the above HTP supernatant, and 1 g / L GDH 105, 20 g / L (~110 mM) glucose, 0.1 g / L NAD+. The HTP plate was incubated at 30°C, 400 rpm for 12 hours in a Thermotron (3 mm swing, model # AJ185, Infors). The reaction was quenched with 0.1% formic acid in methanol at a ratio of 1:20, the supernatant was further diluted with water at a ratio of 1:400, and then loaded into RapidFire for analysis. Activity relative to SEQ ID NO: 112 was calculated as the fold increase over the positive control (FIOPC) determined by dividing the mass spectrometry signal of the product in each sample under the specified reaction conditions by the mass spectrometry signal of the chlorobiphenylpyruvate product in the parental variant (positive control) present in the same plate. Example 9 Improved activity of GDH relative to SEQ ID NO: 736 After screening variants as described in Example 8, SEQ ID NO: 736 was selected as the parent enzyme. Libraries of engineered genes were generated using recognized techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were generated as described in Example 2 with HTP, a single precipitate was obtained, and soluble lysates were generated as described in Example 3. To lyse the cells, 200 μl of lysis buffer containing 200 mM TEoA buffer pH 8.5, 1 mg / mL lysozyme and 0.5 mg / mL PMBS was added to the cell mass. The cells were incubated at room temperature for 2 hours with shaking on a benchtop shaker. The plate was then centrifuged at 4000 rpm and 4°C for 15 minutes, and the clarified supernatant was used for subsequent biocatalytic reactions. HTP reaction is carried out in 96-well deep well plates containing 200 μ L 0.2M TEOA, pH 9, 20 g / L chlorobiphenylpyruvic acid, 56 g / L L-alanine ethyl ester (~5x molar excess), 10% DMSO, 4 μ L of the above HTP supernatant, and 1 g / L GDH 105, 20 g / L (~110 mM) glucose, 0.1 g / L NAD+. HTP plate is incubated at 30° C., 400 rpm for 12 hours in Thermotron (3 mm swing, model # AJ185, Infors). The reaction is quenched with 1:1.5 ratio of acetonitrile, then 1:20 ratio of 0.1% formic acid in methanol, the supernatant is further diluted with water at a ratio of 1:400, and then loaded into RapidFire for analysis. Activity relative to SEQ ID NO: 736 was calculated as the fold increase over the positive control (FIOPC) determined by dividing the mass spectrometry signal of the product in each sample under the specified reaction conditions by the mass spectrometry signal of the chlorobiphenylpyruvate product in the parental variant (positive control) present in the same plate. Example 10 Improved activity of GDH relative to SEQ ID NO: 908 After screening variants as described in Example 9, SEQ ID NO: 908 was selected as the parent enzyme. Libraries of engineered genes were generated using recognized techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were generated as described in Example 2 with HTP, a single precipitate was obtained, and soluble lysates were generated as described in Example 3. To lyse the cells, 200 μl of lysis buffer containing 200 mM TEoA buffer pH 8.5, 1 mg / mL lysozyme and 0.5 mg / mL PMBS was added to the cell mass. The cells were incubated at room temperature for 2 hours with shaking on a benchtop shaker. The plate was then centrifuged at 4000 rpm and 4°C for 15 minutes, and the clarified supernatant was used for subsequent biocatalytic reactions. HTP reaction was carried out in a 96-well deep well plate containing 200 μL 0.2M TEoA, pH 9, 20 g / L chlorobiphenylpyruvic acid, 22 g / L L-alanine ethyl ester (~2x molar excess), 10% DMSO, 2 μL of the above HTP supernatant, and 1 g / L GDH 105, 20 g / L (~110 mM) glucose, 0.1 g / L NAD+. The HTP plate was incubated at 30°C, 400 rpm for 12 hours in a Thermotron (3 mm swing, model # AJ185, Infors). The reaction was quenched with 1:2 ratio of acetonitrile, then 1:20 ratio of 0.1% formic acid in methanol, the supernatant was further diluted with water at a ratio of 1:100, and then loaded into RapidFire for analysis. Activity relative to SEQ ID NO: 908 was calculated as the fold increase over the positive control (FIOPC) determined by dividing the mass spectrometry signal of the product in each sample under the specified reaction conditions by the mass spectrometry signal of the chlorobiphenylpyruvate product in the parental variant (positive control) present in the same plate. Embodiment 11 Improved activity of GDH relative to SEQ ID NO: 956 After screening variants as described in Example 10, SEQ ID NO: 956 was selected as the parent enzyme. Libraries of engineered genes were generated using recognized techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial mutations). Each gene-encoded polypeptide was generated as described in Example 2 with HTP, a single precipitate was obtained, and a soluble lysate was generated as described in Example 3. To lyse the cells, 200 μl of lysis buffer containing 200 mM TEoA buffer pH 8.5, 1 mg / mL lysozyme and 0.5 mg / mL PMBS was added to the cell mass. The cells were incubated at room temperature for 2 hours with shaking on a benchtop shaker. The plate was then centrifuged at 4000 rpm and 4°C for 15 minutes, and the clarified supernatant was used for subsequent biocatalytic reactions. HTP reaction was carried out in a 96-well deep well plate containing 200 μL 0.2M TEoA, pH 9, 20 g / L chlorobiphenylpyruvic acid, 22 g / L L-alanine ethyl ester (~2x molar excess), 10% DMSO, 2 μL of the above HTP supernatant, and 1 g / L GDH 105, 20 g / L (~110 mM) glucose, 0.1 g / L NAD+. The HTP plate was incubated at 30°C, 400 rpm for 12 hours in a Thermotron (3 mm swing, model # AJ185, Infors). The reaction was quenched with 1:2 ratio of acetonitrile, then 1:20 ratio of 0.1% formic acid in methanol, the supernatant was further diluted with water at a ratio of 1:100, and then loaded into RapidFire for analysis. Activity relative to SEQ ID NO: 956 was calculated as the fold increase over the positive control (FIOPC). It was determined by dividing the mass spectrometry signal of the product in each sample under the specified reaction conditions by the mass spectrometry signal of the chlorobiphenylpyruvate product in the parental variant (positive control) present in the same plate. Example 12 Improved activity of GDH relative to SEQ ID NO: 1062 After screening variants as described in Example 11, SEQ ID NO: 1062 was selected as the parent enzyme. Libraries of engineered genes were generated using recognized techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial mutations). Each gene-encoded polypeptide was generated as described in Example 2 with HTP, a single precipitate was obtained, and a soluble lysate was generated as described in Example 3. To lyse the cells, 200 μl of lysis buffer containing 200 mM TEoA buffer pH 8.5, 1 mg / mL lysozyme and 0.5 mg / mL PMBS was added to the cell mass. The cells were incubated at room temperature for 2 hours with shaking on a benchtop shaker. The plate was then centrifuged at 4000 rpm and 4°C for 15 minutes, and the clarified supernatant was used for subsequent biocatalytic reactions. HTP reactions were performed in 96-well deep well plates containing 200 μL 0.2M TEoA, pH 9, 20 g / L chlorobiphenylpyruvic acid, 22 g / L L-alanine ethyl ester (~2x molar excess), 10% DMSO, 2 μL (for 12 hr reaction at 30°C) or 10 uL (for 1.5 hr reaction at 45°C) of the above HTP supernatant, and 1 g / L GDH 105, 20 g / L (~110 mM) glucose, 0.1 g / L NAD+. The HTP plates were incubated at 30°C or 45°C, 400 rpm for 1.5 hours or 12 hours in a Thermotron (3 mm swing, model #AJ185, Infors). The reaction was quenched with acetonitrile at a ratio of 1:2, followed by 0.1% formic acid in methanol at a ratio of 1:20, and the supernatant was further diluted with water at a ratio of 1:100 and then loaded into RapidFire for analysis. Activity relative to SEQ ID NO: 1062 was calculated as the fold increase over the positive control (FIOPC). It was determined by dividing the mass spectrometry signal of the product in each sample under the specified reaction conditions by the mass spectrometry signal of the chlorobiphenylpyruvate product in the parental variant (positive control) present in the same plate. Example 13 Improvement of GDH activity in pH-controlled reactions Improvements of the five variants listed in Table 13.1 were also evaluated at a manufacturing scale as follows. Shake flask powders of these variants were produced as described in Example 4. 10.5 mL of 200 mM TEoA buffer, pH 9, containing 79.88 g / L or 199.7 g / L alanine ethyl ester (2 molar equivalents or 5 molar equivalents) was added to a 25 mL three-necked vessel equipped with a PTFE-coated magnetic stirring bar and a pH electrode connected to an automatic titrator for pH-controlled addition of base to the vessel on demand through a feed tube. Subsequently, 500 g / L chlorobiphenylpyruvic acid in 1.5 mL DMSO, 10 g / L GDH (SEQ ID NO: 4) in 1.5 mL 200 mM TEoA buffer pH 9, 350 g / L glucose, 1 g / L NAD+, and 29 mg, 50 mg, or 67 mg GDH variant in 1.5 mL 200 mM TEoA buffer pH 9 were added. The automatic titrator maintained the pH at 6.5 by adding 1 N NaOH, which was continuously recorded. The reaction was carried out at 30° C. or 45° C. for 20 hrs. Samples were collected at 20 hrs and quenched with 20% triethylamine in acetonitrile at a ratio of 1:2. After centrifugation at 4000 rpm for 10 minutes at 20°C, the supernatant was diluted 250x with 50% acetonitrile and then analyzed by LC-UV described in Example 15. The conversion of substrate to the desired product isomer was calculated based on LC-UV-MS of the product standard curve. The productivity of these five GDH variants was calculated using the substrate conversion normalized by the enzyme powder weight percentage of each substrate loading. The improvements are listed in Table 13.1. Sequences of other variants found throughout multiple rounds of evolution to have similar activity to the reference sequence, SEQ ID NOs: 1385-1388, are included in the sequence listing. Embodiment 14 RapidFire Assay Detection of GDH Reaction Products The formation of chlorobiphenylalaninate product in the HTP assay mixture prepared in Examples 5-12 was analyzed by RapidFire SPE-MS / MS using the instrumentation and parameters described in Table 14.1 below. Embodiment 15 LC-UV-MS analysis and detection of GDH reaction products Using the instrument parameters and conditions shown in Table 15.1 and Table 15.2, a portion of the samples in Examples 5-12 and all of the samples in Example 13 were analyzed using the LC-UV-MS method. They were prepared as dilutions in water and acetonitrile as described in Example 13. The dilution level depends on the starting conditions of the assay. The mass of the product is used to determine the peak of the product isomer, and the byproduct and UV signals are used to quantify the desired product isomer and compared with the product standard to calculate the conversion. All publications, patents, patent applications, and other documents cited in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document was individually indicated to be incorporated by reference for all purposes. While various specific embodiments have been illustrated and described, it will be appreciated that various changes can be made without departing from the spirit and scope of the invention.

Claims

1. An engineered glucose dehydrogenase polypeptide, wherein the amino acid difference between the engineered glucose dehydrogenase polypeptide and SEQ ID NO:4 is H147A / S / I and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:

4.

2. An engineered glucose dehydrogenase polypeptide, wherein the amino acid difference between the engineered glucose dehydrogenase polypeptide and SEQ ID NO: 14 is 1-6 amino acid substitutions selected from the following: S17T, L95V, E96A / C / S, T118M, I147Q / T, F155A / N / Q / R, V156T / Q / A / L, A159C / T, I195G, F200W and Y253H / G / S, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 14, and wherein the engineered glucose dehydrogenase polypeptide consists of a polypeptide sequence that exhibits at least one improved property compared to the engineered glucose dehydrogenase polypeptide of SEQ ID NO:

4.

3. An engineered glucose dehydrogenase polypeptide, wherein the amino acid difference between the engineered glucose dehydrogenase polypeptide and SEQ ID NO: 14 is F155A / N / Q / R, or a combination of F155A / N and 1-4 amino acid substitutions selected from the group consisting of L95V, E96A / C / S, I147Q, V156T / Q / A, A159C / T, I195G, F200W, and Y253H, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 14, and wherein the engineered glucose dehydrogenase polypeptide consists of a polypeptide sequence that exhibits at least one improved property compared to the engineered glucose dehydrogenase polypeptide of SEQ ID NO:

4.

4. An engineered glucose dehydrogenase polypeptide, wherein the amino acid difference between the engineered glucose dehydrogenase polypeptide and SEQ ID NO: 14 is E96A / C / S, or a combination of E96A / C and 1-5 amino acid substitutions selected from the group consisting of L95V, T118M, I147Q, F155A / N, V156T / Q / A / L, A159C / T, I195G, F200W, and Y253H, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 14, and wherein the engineered glucose dehydrogenase polypeptide consists of a polypeptide sequence that exhibits at least one improved property compared to the engineered glucose dehydrogenase polypeptide of SEQ ID NO:

4.

5. An engineered glucose dehydrogenase polypeptide, wherein the amino acid difference between the engineered glucose dehydrogenase polypeptide and SEQ ID NO: 34 is 1-10 amino acid substitutions selected from the group consisting of S17T, K21R, K42T, D44N, N46W, E47A / L / S, K49A, E51L / A, E96N / S / T / A / C / K / G, G114A, V132L, I147T, V149D / E, L154E, N155R / Q, H157N, L167G / Q, K170Q, A177T, P178S / A / G / K, I195N / G / R / S, N196G / H, E198H, K199R, P214G, I218P, T251A / L, L252A / T, Y253L / G / R / S, F256I, Q257E / W, G259D / E and R260G / E, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:34, and wherein the engineered glucose dehydrogenase polypeptide consists of a polypeptide sequence that exhibits at least one improved property compared to the engineered glucose dehydrogenase polypeptide of SEQ ID NO:

4.

6. An engineered glucose dehydrogenase polypeptide, wherein the amino acid difference between the engineered glucose dehydrogenase polypeptide and SEQ ID NO:34 is E96T / A / C / S / N / K / G, or a combination of E96T / A / C / S and 1-9 amino acid substitutions selected from the group consisting of S17T, K21R, K42T, D44N, N46W, E47A / L / S, K49A, E51A / L, G114A, I147T, N155R / Q, A177T, P178S, I195G / N, K199R, Y253G / L / R / S and R260G, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:34, and wherein the engineered glucose dehydrogenase polypeptide consists of a polypeptide sequence that exhibits at least one improved property compared to the engineered glucose dehydrogenase polypeptide of SEQ ID NO:

4.

7. An engineered glucose dehydrogenase polypeptide, wherein the amino acid difference between the engineered glucose dehydrogenase polypeptide and SEQ ID NO:34 is I195G / N / R / S, or a combination of I195G / N and 1-9 amino acid substitutions selected from the group consisting of S17T, K21R, K42T, D44N, N46W, E47A / L / S, K49A, E51A / L, E96A / C / S / T, G114A, I147T, N155R / Q, P178S, K199R, Y253G / L / S / R and R260G, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:34, and wherein the engineered glucose dehydrogenase polypeptide consists of a polypeptide sequence that exhibits at least one improved property compared to the engineered glucose dehydrogenase polypeptide of SEQ ID NO:

4.

8. An engineered glucose dehydrogenase polypeptide, wherein the amino acid difference between the engineered glucose dehydrogenase polypeptide and SEQ ID NO:34 is S17T, or a combination of S17T and 1-9 amino acid substitutions selected from the group consisting of K21R, K42T, D44N, N46W, E47A / L / S, K49A, E51A / L, E96A / T / C / S, G114A, I147T, N155R / Q, P178S, I195G / N, K199R, Y253L / G / S / R and R260G, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:34, and wherein the engineered glucose dehydrogenase polypeptide consists of a polypeptide sequence that exhibits at least one improved property compared to the engineered glucose dehydrogenase polypeptide of SEQ ID NO:

4.

9. An engineered glucose dehydrogenase polypeptide, wherein the amino acid difference between the engineered glucose dehydrogenase polypeptide and SEQ ID NO: 112 is 1-8 amino acid substitutions selected from the group consisting of K21R, K42T, G94A, E96C / G / S / K / A, P98A, E103L, A114G, I147T / A / R, V149D / E, R155N / C, L167Q, P178S, K179N, G195N / R, N196G / A, E198H, A201P, Q205E, P214G, I218P, T251A / L, L252A / T, S253G / R, F256I, and Q257E / W, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 112, and wherein the engineered glucose dehydrogenase polypeptide is replaced by the amino acid positions of SEQ ID NO: The polypeptide sequence composition exhibits at least one improved property compared to the engineered glucose dehydrogenase polypeptide of NO:

4.

10. An engineered glucose dehydrogenase polypeptide, wherein the amino acid difference between the engineered glucose dehydrogenase polypeptide and SEQ ID NO: 112 is E96C / S, or a combination of E96C / S / A / G / K and 1-7 amino acid substitutions selected from the group consisting of K21R, K42T, A114G, I147T, V149D / E, R155N / C, L167Q, P178S, K179N, G195N / R, N196G, E198H, A201P, P214G, I218P, T251A, L252A / T, S253G / R, F256I and Q257E / W, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 112, and wherein the engineered glucose dehydrogenase polypeptide consists of a polypeptide sequence that exhibits at least one improved property compared to the engineered glucose dehydrogenase polypeptide of SEQ ID NO:

4.

11. An engineered glucose dehydrogenase polypeptide, wherein the amino acid difference between the engineered glucose dehydrogenase polypeptide and SEQ ID NO:736 is 1-6 amino acid substitutions selected from the group consisting of K21R, I25L, K42T, L47A, E96A / T, A114G, V149E, R155N, P178S, K179N, G195N, E198L / H, V209L, T251A / I / L, L252T, S253G, F256I and Q257E / W, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:736, and wherein the engineered glucose dehydrogenase polypeptide consists of a polypeptide sequence that exhibits at least one improved property compared to the engineered glucose dehydrogenase polypeptide of SEQ ID NO:

4.

12. An engineered glucose dehydrogenase polypeptide, wherein the amino acid difference between the engineered glucose dehydrogenase polypeptide and SEQ ID NO: 736 is P178S and a combination of 1-5 amino acid substitutions selected from the group consisting of K21R, I25L, K42T, L47A, E96A, A114G, V149E, R155N, K179N, E198H, V209L, T251A, L252T, S253G, F256I, and Q257E / W, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 736, and wherein the engineered glucose dehydrogenase polypeptide consists of a polypeptide sequence that exhibits at least one improved property compared to the engineered glucose dehydrogenase polypeptide of SEQ ID NO:

4.

13. An engineered glucose dehydrogenase polypeptide, wherein the amino acid difference between the engineered glucose dehydrogenase polypeptide and SEQ ID NO: 736 is R155N, or a combination of R155N and 1-5 amino acid substitutions selected from the group consisting of K21R, I25L, K42T, L47A, E96A / T, A114G, V149E, P178S, K179N, G195N, E198H / L, V209L, T251A, L252T, S253G, F256I, and Q257E / W, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 736, and wherein the engineered glucose dehydrogenase polypeptide consists of a polypeptide sequence that exhibits at least one improved property compared to the engineered glucose dehydrogenase polypeptide of SEQ ID NO:

4.

14. An engineered glucose dehydrogenase polypeptide, wherein the amino acid difference between the engineered glucose dehydrogenase polypeptide and SEQ ID NO:908 is 1-6 amino acid substitutions selected from the group consisting of R21K, S40T, K42T, M104L, K111L, V149E, A160S, S178L, K179R / N, P214A, L244F, S253G / R, S255P, R260Q and G261E, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:908, and wherein the engineered glucose dehydrogenase polypeptide consists of a polypeptide sequence that exhibits at least one improved property compared to the engineered glucose dehydrogenase polypeptide of SEQ ID NO:

4.

15. An engineered glucose dehydrogenase polypeptide, wherein the amino acid difference between the engineered glucose dehydrogenase polypeptide and SEQ ID NO: 908 is a combination of K179R / N and 1-5 amino acid substitutions selected from the group consisting of R21K, K42T, V149E, P214A, L244F, S253G / R, R260Q and G261E, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 908, and wherein the engineered glucose dehydrogenase polypeptide consists of a polypeptide sequence that exhibits at least one improved property compared to the engineered glucose dehydrogenase polypeptide of SEQ ID NO:

4.

16. An engineered glucose dehydrogenase polypeptide, wherein the amino acid difference between the engineered glucose dehydrogenase polypeptide and SEQ ID NO: 908 is S253G, or a combination of S253G / R and 1-5 amino acid substitutions selected from the group consisting of R21K, K42T, V149E, K179N / R, P214A, L244F, R260Q and G261E, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 908, and wherein the engineered glucose dehydrogenase polypeptide consists of a polypeptide sequence that exhibits at least one improved property compared to the engineered glucose dehydrogenase polypeptide of SEQ ID NO:

4.

17. An engineered glucose dehydrogenase polypeptide, wherein the amino acid difference between the engineered glucose dehydrogenase polypeptide and SEQ ID NO: 908 is V149E, or a combination of V149E and 1-5 amino acid substitutions selected from the group consisting of R21K, K42T, K179N / R, P214A, S253G / R, R260Q, and G261E, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 908, and wherein the engineered glucose dehydrogenase polypeptide consists of a polypeptide sequence that exhibits at least one improved property compared to the engineered glucose dehydrogenase polypeptide of SEQ ID NO:

4.

18. An engineered glucose dehydrogenase polypeptide, wherein the amino acid difference between the engineered glucose dehydrogenase polypeptide and SEQ ID NO: 956 is 1-7 amino acid substitutions selected from the group consisting of P3C / T, R21K, Q31H, A32M, S40T / K, N46I, E82Q, E103K, M104L, K111E / L / I, V149E, L154H / K / Q / W, V156A / T, A160S, L174A, S178L / R / K / A, N179R, T194L, A197K, Q205I / L / M / T, E222F, A229S, G253Y, S255A / F, E257R, A258P, G259N, and R260Q, and wherein the amino acid positions of the polypeptide sequence are referenced to SEQ ID NO: NO:956, and wherein the engineered glucose dehydrogenase polypeptide consists of a polypeptide sequence that exhibits at least one improved property compared to the engineered glucose dehydrogenase polypeptide of SEQ ID NO:

4.

19. An engineered glucose dehydrogenase polypeptide, wherein the amino acid difference between the engineered glucose dehydrogenase polypeptide and SEQ ID NO: 956 is a combination of M104L and 1-4 amino acid substitutions selected from the group consisting of R21K, S40T, V149E, A160S, S178A / L / K / R, N179R, S255A / F, A258P and R260Q, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 956, and wherein the engineered glucose dehydrogenase polypeptide consists of a polypeptide sequence that exhibits at least one improved property compared to the engineered glucose dehydrogenase polypeptide of SEQ ID NO:

4.

20. An engineered glucose dehydrogenase polypeptide, wherein the amino acid difference between the engineered glucose dehydrogenase polypeptide and SEQ ID NO: 956 is a combination of S255A / F and 1-6 amino acid substitutions selected from the group consisting of R21K, M104L, K111E / I / L, V149E, A160S, S178A / L / K / R, N179R, A258P and R260Q, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 956, and wherein the engineered glucose dehydrogenase polypeptide consists of a polypeptide sequence that exhibits at least one improved property compared to the engineered glucose dehydrogenase polypeptide of SEQ ID NO:

4.

21. An engineered glucose dehydrogenase polypeptide, wherein the amino acid difference between the engineered glucose dehydrogenase polypeptide and SEQ ID NO: 956 is R260Q, or a combination of R260Q and 1-6 amino acid substitutions selected from the group consisting of R21K, M104L, K111E / I / L, V149E, A160S, S178A / L / K / R, N179R, S255A / F and A258P, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 956, and wherein the engineered glucose dehydrogenase polypeptide consists of a polypeptide sequence that exhibits at least one improved property compared to the engineered glucose dehydrogenase polypeptide of SEQ ID NO:

4.

22. An engineered glucose dehydrogenase polypeptide, wherein the amino acid differences between the engineered glucose dehydrogenase polypeptide and SEQ ID NO: 956 are M104L and A160S, or a combination of M104L, A160S and 1-3 amino acid substitutions selected from the group consisting of R21K, S178A / K / L / R, N179R, S255A, A258P and R260Q, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 956, and wherein the engineered glucose dehydrogenase polypeptide consists of a polypeptide sequence that exhibits at least one improved property compared to the engineered glucose dehydrogenase polypeptide of SEQ ID NO:

4.

23. An engineered glucose dehydrogenase polypeptide, wherein the amino acid difference between the engineered glucose dehydrogenase polypeptide and SEQ ID NO: 1062 is 1-6 amino acid substitutions selected from the group consisting of P3C / L / T, Q31H, S40T / K, K54E, A55W, Q63M, T67S, P98Q, V149E, L154K / W / H / Q / S, V156A / T, S160A, L174A, S178K, T194H, Q205M / L / T / I, D208L / R / T, E222Q / F / G / R / Y, A226C, A229S, G253Y / D, and E257R, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 1062, and wherein the engineered glucose dehydrogenase polypeptide is replaced by the amino acid positions of SEQ ID NO: The polypeptide sequence composition exhibits at least one improved property compared to the engineered glucose dehydrogenase polypeptide of NO:

4.

24. An engineered glucose dehydrogenase polypeptide, wherein the amino acid difference between the engineered glucose dehydrogenase polypeptide and SEQ ID NO: 1062 is Q205M / L / T, or a combination of Q205M / L / T / I and 1-5 amino acid substitutions selected from the group consisting of P3C / L / T, Q31H, S40K / T, K54E, A55W, Q63M, V149E, L154H / K / Q / W / S, V156A / T, S160A, L174A, S178K, E222F / G / R / Y / Q, A229S, G253D / Y, and E257R, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 1062, and wherein the engineered glucose dehydrogenase polypeptide consists of a polypeptide sequence that exhibits at least one improved property compared to the engineered glucose dehydrogenase polypeptide of SEQ ID NO:

4.

25. An engineered glucose dehydrogenase polypeptide, wherein the amino acid difference between the engineered glucose dehydrogenase polypeptide and SEQ ID NO: 1062 is L154K / W / H, or a combination of L154K / W / H / Q / S and 1-5 amino acid substitutions selected from the group consisting of P3C / L, Q31H, S40T, A55W, Q63M, V149E, S160A, L174A, S178K, Q205I / L / M / T, E222F / G / Y / R, A229S, G253Y and E257R, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 1062, and wherein the engineered glucose dehydrogenase polypeptide consists of a polypeptide sequence that exhibits at least one improved property compared to the engineered glucose dehydrogenase polypeptide of SEQ ID NO:

4.

26. An engineered glucose dehydrogenase polypeptide, wherein the engineered glucose dehydrogenase polypeptide consists of a polypeptide sequence listed in an even-numbered sequence of SEQ ID NO: 6, 8, 14, or 32-1384.

27. The engineered glucose dehydrogenase polypeptide of any one of claims 1-26, wherein the engineered glucose dehydrogenase polypeptide consists of a polypeptide sequence that exhibits at least one improved property compared to the engineered glucose dehydrogenase polypeptide of SEQ ID NO:

4.

28. The engineered glucose dehydrogenase polypeptide of any one of claims 2-26, wherein the improved properties include improved production of the compound of formula (IV) in R is selected from the group consisting of a hydrogen atom, or an optionally substituted alkyl, alkenyl, alkynyl, alkoxy, arylalkoxy, hydroxyalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl, and heteroarylalkyl; and wherein R2 is independently selected from alkyl, alkenyl, alkynyl, hydroxyalkyl, carboxyl, aminocarbonyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, alkylamino, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl and heteroarylalkyl; and wherein R3 is independently selected from methyl, d3-methyl and ethyl.

29. The engineered glucose dehydrogenase polypeptide of any one of claims 2-26, wherein the improved properties include improved production of compound (1) 30. The engineered glucose dehydrogenase polypeptide of any one of claims 2-26, wherein the improved properties include improved utilization of compound (2) 31. The engineered glucose dehydrogenase polypeptide of any one of claims 2-26, wherein the improved properties include improved utilization of compound (3) 32. The engineered glucose dehydrogenase polypeptide of any one of claims 2-26, wherein the improved properties include compound (1) From compound (2) and compound (3) The improvement produced.

33. The engineered glucose dehydrogenase polypeptide of any one of claims 2-26, wherein the improved properties comprise improved enantioselectivity.

34. The engineered glucose dehydrogenase polypeptide of any one of claims 2-26, wherein the improved properties comprise improved stability.

35. The engineered glucose dehydrogenase polypeptide of any one of claims 1-26, wherein the engineered polypeptide is purified.

36. A composition comprising at least one glucose dehydrogenase polypeptide provided according to any one of claims 1-35.

37. An engineered polynucleotide encoding at least one engineered glucose dehydrogenase polypeptide according to any one of claims 1-35.

38. An engineered polynucleotide consisting of any one of the odd-numbered sequences listed in SEQ ID NO: 5, 7, 13, and 31 to 1383.

39. A vector comprising the engineered polynucleotide according to any one of claims 37 to 38.

40. The vector of claim 39, further comprising at least one control sequence, wherein the control sequence is selected from the group consisting of a leader sequence, a polyadenylation sequence, a promoter, a signal peptide sequence, and a transcription terminator.

41. A host cell comprising the vector according to claim 39 and / or 40.

42. The host cell of claim 41, wherein the host cell produces at least one engineered glucose dehydrogenase polypeptide of any one of claims 1-35.

43. A method for producing an engineered glucose dehydrogenase polypeptide in a host cell, the method comprising culturing the host cell according to claim 41 and / or 42 in a culture medium under suitable conditions, thereby producing at least one engineered glucose dehydrogenase polypeptide.

44. The method of claim 43, further comprising the step of recovering the engineered glucose dehydrogenase polypeptide.

45. The method of claim 43 or 44, further comprising the step of purifying the at least one engineered glucose dehydrogenase polypeptide.

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