Engineered galactose oxidase variant enzymes

Abstract

The present invention provides engineered galactose oxidase (GO enzyme), polypeptides having GO enzyme activity and polynucleotides encoding these enzymes, and vectors and host cells comprising these polynucleotides and polypeptides. Also provided are methods for producing GO enzymes. The present invention also provides compositions comprising GO enzymes, and methods for using engineered GO enzymes. The present invention is particularly useful for the production of pharmaceutical compounds and other compounds.

Description

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 62 / 695,570 filed on July 9, 2018 and U.S. Provisional Patent Application Serial No. 62 / 822,286 filed on March 22, 2019, both of which are incorporated herein by reference in their entirety for all purposes. Field of the Invention

[0002] The present invention provides engineered galactose oxidase (GO enzyme), polypeptides having GO enzyme activity and polynucleotides encoding these enzymes, and vectors and host cells comprising these polynucleotides and polypeptides. Also provided are methods for producing GO enzymes. The present invention also provides compositions comprising GO enzymes, and methods for using engineered GO enzymes. The present invention is particularly useful for the production of pharmaceutical compounds and other compounds.

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

[0004] An official copy of the sequence listing is submitted with the specification as an ASCII formatted text file via EFS-Web, with the file name "CX2-176WO2_ST25.txt", the creation date of July 2, 2019, and the size of 11,509 kilobytes. The sequence listing submitted via EFS-Web is part of the specification and is incorporated herein by reference in its entirety. Background of the Invention

[0006] The oxidation of alcohols to aldehydes is a key transformation required in organic synthetic chemistry. There are several chemical reagents capable of performing this type of reaction, but the use of these methods brings several disadvantages. Chemical oxidation approaches are non-chemically selective methods that require the protection of non-target reactive groups when used. These oxidation methods are difficult to control in terms of oxidation state because some chemical reagents can over-oxidize the target alcohol. In addition, the operation of the reaction under oxidizing conditions presents a dangerous situation, which may lead to explosions and serious physical damage to personnel and property. Oxidizing agents and their by-products are reactive substances that are harmful to the environment. Therefore, there is still a need in the art to produce controlled reagents capable of performing selective oxidation chemistry while reducing or eliminating these serious disadvantages. SUMMARY OF THE INVENTION

[0008] The present invention provides engineered galactose oxidase (GO enzyme), polypeptides having mild oxidation activity for primary alcohols and producing corresponding aldehydes in an enantioselective manner, and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. Methods for producing GO enzymes are also provided. The present invention also provides compositions comprising GO enzymes, and methods for using engineered GO enzymes. The present invention is particularly useful for the production of pharmaceutical compounds and other compounds.

[0009] The present invention provides an engineered galactose oxidase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 2, 4, 166, 272, 928, 932, 1264, 1416, 1598, 1866, 1912, 2080, 2300 and / or 2424, or a functional fragment thereof. In some embodiments, the engineered galactose oxidase comprises at least one substitution or set of substitutions in the polypeptide sequence, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 2, 4, 166, 272, 928, 932, 1264, 1416, 1598, 1866, 1912, 2080, 2300, and / or 2424. In some additional embodiments of the engineered galactose oxidase, the polypeptide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 2. In some embodiments, the engineered galactose oxidase comprises at least one substitution or set of substitutions at one or more positions selected from 331 / 406 / 407 / 465 and 331 / 406 / 465 in the polypeptide sequence, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 2. In some embodiments, the engineered galactose oxidase comprises at least one substitution or set of substitutions selected from 331R / 406Y / 407Q / 465A, 331R / 406Y / 465A, and 331R / 406Y / 465Q, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 2. In some additional embodiments, the engineered galactose oxidase comprises at least one substitution or set of substitutions selected from K331R / F406Y / E407Q / F465A, K331R / F406Y / F465A, and K331R / F406Y / F465Q, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:2.

[0010] In some additional embodiments, the engineered galactose oxidase comprises NO:4 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity, and wherein the engineered galactose oxidase comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 170, 171, 173, 191, 192, 193, 194, 197, 198, 199, 202, 204, 205, 220, 227, 243, 247, 248, 252, 269, 294, 296, 324, 332, 407, 463, 465, 466, 493, 515, 517, 520, 521 and 522, wherein the amino acid positions of the polypeptide sequence are referenced to SEQ ID NO: 4. In some embodiments, the engineered galactose oxidase comprises at least one substitution or set of substitutions selected from the group consisting of 170L, 171A, 171C, 171L, 173C, 173S, 191A, 192I, 192M, 192Q, 193T, 194V, 197K, 197S, 198A, 198G, 198T, 199G, 199R, 199T, 202C, 202T, 204Q, 204S, 204V, 205A, 220E, 220P, 220R, 227L, 243C, 243V, 247G, 248E , 248T, 252T, 269Q, 269Y, 294K, 294N, 294S, 296A, 296L, 296S, 296W, 324G, 324S, 332R, 407V, 463K, 463R, 463V, 465G, 466R, 493G, 515T, 517D, 517E, 517L, 517M, 517S, 520G, 520L, 520P, 520S, 520V, 521G, 521P, 521S, 521V and 522S, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:4.In some embodiments, the engineered galactose oxidase comprises at least one substitution or set of substitutions selected from I170L, V171A, V171C, V171L, A173C, A173S, R191A, N192I, N192M, N192Q, D193T, A194V, G197K, G197S, S198A, S198G, S198T, P199G, P199R, P199T, I202C, I202T, L204Q, L204S, L204V, T205A, V220E, V220P, V220R, M227L, T243C, T243V, D247G, A248E , A248T, S252T, V269Q, V269Y, G294K, G294N, G294S, F296A, F296L, F296S, F296W, A324G, A324S, S332R, E407V, I463K, I463R, I463V, A465G, E466R, V493G, L515T, G517D, G517E, G517L, G517M, G517S, T520G, T520L, T520P, T520S, T520V, T521G, T521P, T521S, T521V and N522S, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:4.

[0011] In some additional embodiments, the engineered galactose oxidase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:4, and wherein the engineered galactose oxidase comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 171, 173, 188, 192, 197, 199, 203, 220, 223, 243, 252, 294, 295, 296, 332, 407, 465, 466, 493, 515, 517, 520, and 521, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:4. In some embodiments, the engineered galactose oxidase comprises at least one substitution or set of substitutions selected from the group consisting of: 171A, 173S, 188T, 192Q, 197K, 197S, 197T, 199A, 199N, 199S, 203V, 220C, 220E, 220M, 220P, 220S, 223H, 223L, 223M, 223N, 243A, 243S, 252M, 252R, 252T, 252V, 294E, 294Q, 294S, 295E, 295G, 295N, 295R, 295S, 296S, 332Q, 407F, 407I, 407M, 465G, 465M, 465T, 466G, 466R, 493T, 515V, 517D, 517K, 517S, 520A, 520S, 521A, 521G, 521Q, 521V and 521Y, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:4.In some embodiments, the engineered galactose oxidase comprises at least one substitution or set of substitutions selected from the group consisting of V171A, A173S, S188T, N192Q, G197K, G197S, G197T, P199A, P199N, P199S, T203V, V220C, V220E, V220M, V220P, V220S, T223H, T223L, T223M, T223N, T243A, T243S, S252M, S252R, S252T, S252V, G2 94E, G294Q, G294S, V295E, V295G, V295N, V295R, V295S, F296S, S332Q, E407F, E407I, E407M, A465G, A465M, A465T, E466G, E466R, V493T, L515V, G517D, G517K, G517S, T520A, T520S, T521A, T521G, T521Q, T521V and T521Y, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:4.

[0012] In yet other embodiments, the engineered galactose oxidase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 166, and wherein the engineered galactose oxidase comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 171 / 220 / 243 / 407 / 465, 171 / 220 / 295 / 296 / 407, 171 / 220 / 296 / 407 / 520, 171 / 220 / 332 / 407, 171 / 220 / 407 / 520, 171 / 220 / 465 / 520, 171 / 220 / 407 / 465 / 520. 3 / 296 / 332、171 / 295 / 296 / 332 / 465、171 / 295 / 332、171 / 296 / 407、171 / 296 / 407 / 465、171 / 332、171 / 407、171 / 407 / 520、173、173 / 192 / 243 / 465、173 / 465、192 / 220 / 295 / 296 / 332 / 521、192 / 220 / 295 / 520 / 521、192 / 220 / 296 / 332 / 520、192 / 220 / 407、192 / 294 / 465 / 515、192 / 295 / 296、192 / 295 / 296 / 332、192 / 295 / 296 / 520 / 521、192 / 296、198 / 294 / 296、198 / 295、198 / 295 / 296、204、204 / 243 / 465 / 517 / 521、220、220 / 243、220 / 243 / 295 / 296 / 332 / 407 / 521、220 / 243 / 295 / 296 / 407、220 / 243 / 407、220 / 243 / 407 / 465、220 / 243 / 407 / 520 / 521、220 / 252 / 332 / 407、220 / 295 / 296、220 / 295 / 296 / 332 、220 / 295 / 296 / 332 / 407 / 465 / 521、220 / 295 / 296 / 332 / 407 / 520、220 / 295 / 296 / 332 / 407 / 521、220 / 295 / 296 / 407、220 / 295 / 296 / 465 / 520、220 / 295 / 332、220 / 295 / 332 / 465 / 520 / 521、220 / 295 / 407 / 465、220 / 295 / 407 / 520 / 521、220 / 295 / 407 / 521、220 / 295 / 465 / 520、220 / 295 / 465 / 521、220 / 296、220 / 296 / 332 / 407, 220 / 296 / 332 / 407 / 520 / 521, 220 / 296 / 407, 220 / 296 / 465, 220 / 296 / 465 / 521, 220 / 332 / 407 / 465 / 521, 220 / 407, 220 / 407 / 520, 220 / 465, 220 / 465 / 517, 220 / 465 / 518 0 / 465 / 520、221 / 227 / 243 / 465、227、243 / 295、243 / 295 / 407、243 / 515 / 517、277 / 296 / 407 / 520 / 521、284 / 295 / 296、294 / 296 / 407、294 / 465、294 / 465 / 515、294 / 521、295 / 296、 295 / 296 / 332 / 407、295 / 296 / 407、295 / 296 / 407 / 521、295 / 296 / 521、295 / 332 / 407 / 465、295 / 332 / 407 / 521、295 / 332 / 520 / 521、295 / 407、296、296 / 332 / 407 / 520、296 / 332 / 40 7 / 521, 296 / 407, 296 / 520 / 521, 332 / 407, 332 / 407 / 520 / 521, 332 / 407 / 521, 407, 407 / 465, 407 / 520, 465, 465 / 515, 465 / 515 / 517 / 521, 465 / 517 / 521 and 517, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:166. In some embodiments, the engineered galactose oxidase comprises at least one substitution or set of substitutions selected from the group consisting of: 171C / 220E / 295E / 296V / 407Q, 171C / 220E / 407V, 171C / 220M / 296V / 407V / 520A, 171C / 220S / 243V / 407Q / 465T, 171C / 220S / 332Q / 407V, 171C / 243V / 296V / 332Q, 171C / 295E / 332Q, 171C / 296V / 407V / 465T, 171C / 220S / 332Q / 407V 1C / 332Q, 171C / 407V, 171C / 407V / 520A, 171L / 220E / 465T / 520A, 171L / 295E / 296V / 332Q / 465T, 171L / 296V / 407I, 173S, 173 S / 192Q / 243A / 465G, 173S / 465G, 192Q / 294S / 465G / 515V, 192T / 220E / 295E / 296V / 332Q / 521G, 192T / 220E / 295E / 520A / 521G,192T / 220E / 407V、192T / 220M / 296V / 332Q / 520A、192T / 295E / 296V、192T / 295G / 296V / 332Q、192T / 295G / 296V / 520A / 521G、192T / 296V、198A / 294E / 296S、198R / 295N、198R / 295S / 296S、204A / 243A / 465G / 517R / 521Q、204S、220C / 465G、220C / 465G / 517R、220E、220E / 243V / 295E / 296V / 332Q / 407V / 521G、220E / 243V / 295G / 296V / 407V、220E / 243V / 407I、220E / 243V / 407V / 465T、220E / 252V / 332Q / 407I、220E / 295E / 296V / 332Q / 407V / 520A、220E / 295E / 296V / 465T / 520A、220E / 295E / 465T / 521G、220E / 295G / 407V / 465T、220E / 296V / 407V、220E / 332Q / 407Q / 465T / 521G、220E / 407I、220E / 407V、220E / 407V / 520A、220E / 465T / 520A、220M / 243V / 407V / 520A / 521G、220M / 295E / 332Q、220M / 295E / 465T / 520A、220S / 243V、220S / 243V / 407Q / 520A / 521G、220S / 295E / 296V、220S / 295E / 296V / 332Q / 407Q / 521G、220S / 295E / 332Q / 465T / 520A / 521G、220S / 295E / 407I / 521G、220S / 295G / 296V / 332Q、220S / 295G / 296V / 332Q / 407I / 465T / 521G、220S / 295G / 296V / 407V、220S / 295G / 407I / 520A / 521G、220S / 296V、220S / 296V / 332Q / 407V、220S / 296V / 332Q / 407V / 520A / 521G、220S / 296V / 465T、220S / 296V / 465T / 521G、220S / 407I、221I / 227N / 243A / 465G、227N、243A / 515V / 517R、243V / 295E、243V / 295E / 407I、277T / 296V / 407I / 520A / 521G、284I / 295E / 296V, 294E / 296S / 407M, 294S / 465G, 294S / 465G / 515V, 294S / 521Q, 295E / 296V, 295E / 296V / 332Q / 407I, 295E / 296V / 407Q, 295 E / 296V / 521G, 295E / 332Q / 407V / 465T, 295E / 332Q / 407V / 521G, 295E / 332Q / 520A / 521G, 295E / 407I, 295G / 296V / 407I / 521G, 296S / 407M, 2 521G, 465G / 515V, 465G / 515V / 517R / 521Q, 465G / 517H / 521Q, 465M and 517N, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 166. In some embodiments, the engineered galactose oxidase comprises at least one substitution or set of substitutions selected from the group consisting of: V171C / V220E / R295E / F296V / E407Q, V171C / V220E / E407V, V171C / V220M / F296V / E407V / T520A, V171C / V220S / T243V / E407Q / A465T, V171C / V220S / S332Q / E407V, V171C / T243V / F296V / S332Q, V171C / R295E / S332Q, V171C / F296V / E407V / A465T, V171C / S332Q, V171C / E407V, V171C / E407V / T520A, V171L / V220E / A465T / T520A, V171L / R295E / F296V / S332Q / A465T, V171L / F296V / E407I, A173S, A173S / N192Q / T243A / A465G, A173S / A465G, N192Q / G294S / A465G / L515V, N192T / V220E / R295E / F296V / S332Q / T521G, N192T / V220E / R295E / T520A / T521G, N192T / V220E / E407V, N192T / V220M / F296V / S332Q / T520A,N192T / R295E / F296V、N192T / R295G / F296V / S332Q、N192T / R295G / F296V / T520A / T521G、N192T / F296V、S198A / G294E / F296S、S198R / R295N、S198R / R295S / F296S、L204A / T243A / A465G / G517R / T521Q、L204S、V220C / A465G、V220C / A465G / G517R、V220E、V220E / T243V / R295E / F296V / S332Q / E407V / T521G、V220E / T243V / R295G / F296V / E407V、V220E / T243V / E407I、V220E / T243V / E407V / A465T、V220E / S252V / S332Q / E407I、V220E / R295E / F296V / S332Q / E407V / T520A、V220E / R295E / F296V / A465T / T520A、V220E / R295E / A465T / T521G、V220E / R295G / E407V / A465T、V220E / F296V / E407V、V220E / S332Q / E407Q / A465T / T521G、V220E / E407I、V220E / E407V、V220E / E407V / T520A、V220E / A465T / T520A、V220M / T243V / E407V / T520A / T521G、V220M / R295E / S332Q、V220M / R295E / A465T / T520A、V220S / T243V、V220S / T243V / E407Q / T520A / T521G、V220S / R295E / F296V、V220S / R295E / F296V / S332Q / E407Q / T521G、V220S / R295E / S332Q / A465T / T520A / T521G、V220S / R295E / E407I / T521G、V220S / R295G / F296V / S332Q、V220S / R295G / F296V / S332Q / E407I / A465T / T521G、V220S / R295G / F296V / E407V、V220S / R295G / E407I / T520A / T521G、V220S / F296V、V220S / F296V / S332Q / E407V、V220S / F296V / S332Q / E407V / T520A / T521G、V220S / F296V / A465T, V220S / F296V / A465T / T521G, V220S / E407I, T221I / M227N / T243A / A465G, M227N, T24 3A / L515V / G517R, T243V / R295E, T243V / R295E / E407I, A277T / F296V / E407I / T520A / T521G, V284I / R295E / F 296V, G294E / F296S / E407M, G294S / A465G, G294S / A465G / L515V, G294S / T521Q, R295E / F296V, R295E / F296 V / S332Q / E407I, R295E / F296V / E407Q, R295E / F296V / T521G, R295E / S332Q / E407V / A465T, R295E / S332Q / E4 07V / T521G, R295E / S332Q / T520A / T521G, R295E / E407I, R295G / F296V / E407I / T521G, F296S / E407M, F296V , F296V / S332Q / E407V / T520A, F296V / S332Q / E407V / T521G, F296V / T520A / T521G, S332Q / E407I, S332Q / E40 7V, S332Q / E407V / T520A / T521G, S332Q / E407V / T521G, E407I, E407I / T520A, E407Q / A465T, E407V, E407V / T520A, A465G / L515V, A465G / L515V / G517R / T521Q, A465G / G517H / T521Q, A465M and G517N, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 166. 、

[0013] In some embodiments, the engineered galactose oxidase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 272, and wherein the engineered galactose oxidase comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 4, 8, 8 / 29 / 92 / 196 / 258 / 426, 8 / 46 / 92 / 279 / 296 / 426 / 549 / 553 / 597, 8 / 56 / 192 / 194 / 460 / 571 / 598, 8 / 56 / 243 / 560 / 598, 8 / 92 / 258 / 36 3 / 549 / 553 / 567、8 / 192 / 194 / 243 / 460 / 553 / 560、8 / 192 / 460 / 560 / 598、8 / 258 / 363 / 426 / 553 / 597、16、16 / 24 / 43 / 56 / 103 / 220 / 295 / 296 / 499 / 549、16 / 43 / 56 / 103 / 148 / 220 / 295 / 296 / 499 / 549、16 / 43 / 63 / 103 / 295 / 304 / 499 / 549、16 / 43 / 63 / 148 / 295 / 499、16 / 43 / 103 / 148 / 220 / 295 / 426 / 549、16 / 43 / 148 / 220 / 295 / 499、16 / 43 / 295 / 296 / 549、16 / 43 / 426 / 549、16 / 56 / 63 / 148 / 295 / 296 / 304 / 426、16 / 56 / 296、16 / 56 / 426 / 499、16 / 63 / 103 / 220 / 295 / 426 / 549、16 / 103 / 220 / 296 / 465 / 549、16 / 103 / 220 / 465、16 / 148 / 220 / 295 / 296 / 304 / 499、16 / 148 / 220 / 295 / 426 / 499 / 549、16 / 148 / 220 / 296 / 54 9、16 / 148 / 295 / 426 / 549、16 / 220 / 499、16 / 295 / 296 / 426 / 499 / 549、16 / 295 / 426 / 465 / 499 / 549、24、24 / 36、24 / 36 / 43 / 148 / 319 / 560 / 637、24 / 36 / 92 / 148、24 / 36 / 92 / 222 / 560 / 637、24 / 36 / 92 / 279 / 319 / 363 / 637、24 / 36 / 148 / 222 / 279 / 560 / 637、24 / 36 / 363 / 465 / 637、24 / 43 / 92 / 279 / 363 / 560 / 637、24 / 43 / 92 / 279 / 499、24 / 43 / 148 / 295 / 560、24 / 43 / 148 / 363、24 / 43 / 148 / 560、24 / 43 / 222、24 / 92 / 148 / 279 / 363、24 / 92 / 279 / 363 / 465 / 499 / 560 / 637、24 / 148 / 319 / 465 / 637、24 / 148 / 637、24 / 637、29、29 / 46 / 92 / 196 / 258 / 279 / 363 / 426 / 481 / 567 / 597、36、 36 / 43 / 92 / 148 / 222 / 279 / 295 / 499 / 560 / 637、36 / 92 / 148 / 279 / 319 / 363 / 560 / 637、36 / 92 / 148 / 499 / 637、36 / 92 / 319 / 363 / 637、36 / 92 / 560、36 / 148 / 222 / 319 / 465 / 499、36 / 222 / 279 / 319 / 363 / 560、43、43 / 56 / 220 / 296 / 426 / 499 / 549、43 / 56 / 220 / 426 / 549 、43 / 92 / 148 / 222 / 279 / 499 / 560、43 / 92 / 222 / 465 / 499 / 637、43 / 148 / 279 / 295 / 560、43 / 148 / 549、43 / 222 / 279、43 / 295 / 499、46、46 / 196 / 228 / 279 / 296 / 465 / 553、56、63、92、92 / 148 / 295 / 319 / 465 / 637、92 / 279 / 465 / 637、103、134、148、148 / 295 / 465、192 / 243, 194, 196, 196 / 258 / 363 / 426 / 465 / 549 / 597, 220, 220 / 295 / 520, 220 / 295 / 521, 220 / 296 / 304 / 426 / 549, 222, 257, 258, 279, 279 / 560 / 637, 295, 296, 304, 319, 363, 363 / 426 / 481 / 553, 426, 465, 499, 549, 553, 560, 567, 597 and 637, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:272. In some embodiments, the engineered galactose oxidase comprises at least one substitution or set of substitutions selected from the group consisting of: 4Q, 8I / 29T / 92V / 196D / 258L / 426P, 8I / 46V / 92V / 279T / 296T / 426P / 549G / 553S / 597D, 8I / 92V / 258L / 363E / 549G / 553S / 567M, 8I / 258L / 363E / 426P / 553S / 597D,8V、8V / 56I / 192Q / 194G / 460Q / 571A / 598E、8V / 56I / 243S / 560T / 598E、8V / 192Q / 194E / 243S / 460Q / 553S / 560T、8V / 192Q / 460A / 560T / 598E、16E、16E / 43E / 426L / 549W、16E / 56F / 426L / 499V、16E / 103I / 220E / 296E / 465F / 549W、16E / 103I / 220E / 465F、16E / 148R / 220E / 295T / 426L / 499V / 549W、16E / 220E / 499V、16E / 295Q / 426L / 465F / 499V / 549W、16S、16S / 24A / 43E / 56F / 103I / 220E / 295T / 296L / 499V / 549W、16S / 43E / 56F / 103I / 148R / 220E / 295S / 296E / 499V / 549W、16S / 43E / 63V / 103I / 295Q / 304C / 499V / 549W、16S / 43E / 63V / 148R / 295Q / 499V、16S / 43E / 103I / 148R / 220E / 295Q / 426L / 549W、16S / 43E / 148R / 220E / 295S / 499V、16S / 43E / 295T / 296E / 549W、16S / 56F / 63V / 148R / 295T / 296E / 304C / 426L、16S / 56F / 296E、16S / 63V / 103I / 220E / 295Q / 426L / 549W、16S / 148R / 220E / 295Q / 296E / 304C / 499V、16S / 148R / 220E / 296E / 549W、16S / 148R / 295Q / 426L / 549W、16S / 295Q / 296E / 426L / 499V / 549W、24A、24E、24P、24P / 36P、24P / 36P / 43A / 148A / 319R / 560W / 637L、24P / 36P / 92D / 148A、24P / 36P / 92D / 222D / 560W / 637L、24P / 36P / 92D / 279L / 319R / 363L / 637L、24P / 36P / 148A / 222D / 279L / 560W / 637L、24P / 36P / 363L / 465F / 637L、24P / 43A / 92D / 279L / 499F、24P / 43A / 148A / 295E / 560W、24P / 43A / 148A / 363L、24P / 43A / 148A / 560W、24P / 43A / 222D、<h2 style=";text-align:left;direction:ltr">24P / 43Q / 92D / 279L / 363L / 560W / 637L, 24P / 92D / 148A / 279L / 363L, 24P / 92D / 279L / 363L / 465F / 499F / 560W / 637L, 24P / 148A / 319S / 465F / 637L, 24P / 148A / 637L, 24P, 637L, 24Q, 29T, 46V, 92V, 196D, 258L, 279T, 363E, 426P, 481D, 567M, 597D, 29Y, 36P, 36P, 43Q, 92D, 148A, 222D, 279L, 295D, 499F, 560W, 637L 36P / 92D / 148A / 279L / 319S / 363L / 560W / 637L,36P / 92D / 148A / 499F / 637L,36P / 92D / 319R / 363L / 637L,36P / 92D / 560W,36P / 148A / 222D / 319S / 465F / 499 F, 36P / 222D / 279L / 319S / 363L / 560W, 43A, 43A / 92D / 148A / 222D / 279L / 499F / 560W, 43A / 148A / 279L / 295D / 560W, 43A / 222D / 279L, 43E / 56F / 220E / 296E / 4 26L / 499V / 549W、43E / 56F / 220E / 426L / 549W、43E / 148R / 549W、43E / 295Q / 49 9V、43Q、43Q / 92D / 222Y / 465F / 499F / 637L、46V、46V / 196D / 228W / 279T / 296T / 465F / 553S, 56I, 63V, 92D / 148A / 295E / 319S, 465F / 637L, 92D / 279L, 465F / 637L, 92V, 103I, 134H, 148A, 148R, 148R / 295Q / 465F, 192Q / 243S, 194E, 196D, 196D / 258L / 363E / 426P / 465F / 549G / 597D、220E、220E / 295E / 520A、220E / 295E / 521G、220E / 296E / 304C / 426L / 549W、222D、222T、222Y、257D、258L、279L / 560W / 637L, 279T, 295E, 295Q, 295S, 295T, 296E, 296T, 304C, 319S, 363E, 363E / 426P / 481D / 553S, 426A, 426L, 465G, 499F, 499V, 549G, 549Q, 553S, 560W,567M, 597D and 637L, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:272. In some embodiments, the engineered galactose oxidase comprises at least one substitution or set of substitutions selected from the group consisting of A4Q, S8I / N29T / S92V / E196D / S258L / S426P, S8I / A46V / S92V / M279T / V296T / S426P / R549G / Q553S / N597D, S8I / S92V / S258L / S363E / R549G / Q553S / S567M, S8I / S258L / S363E / S426P / Q553S / N597D, S8V, S8V / Y56I / N192Q / A194G / R460Q / K571A / N5 98E, S8V / Y56I / T243S / R560T / N598E, S8V / N192Q / A194E / T243S / R460Q / Q553S / R560T, S8V / N192Q / R460A / R560T / N598E, A16E, A16E / F43E / S42 6L / R549W, A16E / Y56F / S426L / I499V, A16E / S103I / S220E / V296E / T465F / R549W、A16E / S103I / S220E / T465F、A16E / Q148R / S220E / R295T / S426L / I499V / R549W, A16E / S220E / I499V, A16E / R295Q / S426L / T465F / I499V / R549W, A16S, A16S / S24A / F43E / Y56F / S103I / S220E / R295T / V296L / I499 V / R549W、A16S / F43E / Y56F / S103I / Q148R / S220E / R295S / V296E / I499V / R549W、A16S / F43E / T63V / S103I / R295Q / S304C / I499V / R549W、A16S / F4 3E / T63V / Q148R / R295Q / I499V, A16S / F43E / S103I / Q148R / S220E / R295 Q / S426L / R549W, A16S / F43E / Q148R / S220E / R295S / I499V, A16S / F43E / R 295T / V296E / R549W, A16S / Y56F / T63V / Q148R / R295T / V296E / S304C / S42 6L, A16S / Y56F / V296E, A16S / T63V / S103I / S220E / R295Q / S426L / R549W,<h2 style=";text-align:left;direction:ltr">A16S / Q148R / S220E / R295Q / V296E / S304C / I499V、A16S / Q148R / S220E / V2 96E / R549W、A16S / Q148R / R295Q / S426L / R549W、A16S / R295Q / V296E / S426L / I499V / R549W、S24A、S24E、S24P、S24P / K36P、S24P / K36P / F43A / Q148A / N3 19R / R560W / R637L、S24P / K36P / S92D / Q148A、S24P / K36P / S92D / V222D / R56 0W / R637L、S24P / K36P / S92D / M279L / N319R / S363L / R637L、S24P / K36P / Q148A / V222D / M279L / R560W / R637L、S24P / K36P / S363L / T465F / R637L、S24P / F43A / S92D / M279L / I499F、S24P / F43A / Q148A / R295E / R560W、S24P / F43A / Q 148A / S363L、S24P / F43A / Q148A / R560W、S24P / F43A / V222D、S24P / F43Q / S9 2D / M279L / S363L / R560W / R637L、S24P / S92D / Q148A / M279L / S363L、S24P / S92D / M279L / S363L / T465F / I499F / R560W / R637L、S24P / Q148A / N319S / T46 5F / R637L、S24P / Q148A / R637L、S24P / R637L、S24Q、N29T / A46V / S92V / E196 D / S258L / M279T / S363E / S426P / Q481D / S567M / N597D、N29Y、K36P、K36P / F4 3Q / S92D / Q148A / V222D / M279L / R295D / I499F / R560W / R637L、K36P / S92D / Q148A / M279L / N319S / S363L / R560W / R637L、K36P / S92D / Q148A / I499F / R63 7L, K36P / S92D / N319R / S363L / R637L, K36P / S92D / R560W, K36P / Q148A / V222D / N319S / T465F / I499F, K36P / V222D / M279L / N319S / S363L / R560W, F43A,F43A / S92D / Q148A / V222D / M279L / I499F / R560W, F43A / Q148A / M279L / R295D / R560W, F43A / V222D / M279L, F43E / Y5 6F / S220E / V296E / S426L / I499V / R549W, F43E / Y56F / S220E / S426L / R549W, F43E / Q148R / R549W, F43E / R295Q / I499 V, F43Q, F43Q / S92D / V222Y / T465F / I499F / R637L, A46V, A46V / E196D / F228W / M279T / V296T / T465F / Q553S, Y56I, T 63V, S92D / Q148A / R295E / N319S / T465F / R637L, S92D / M279L / T465F / R637L, S92V, S103I, N134H, Q148A, Q148R, Q14 8R / R295Q / T465F, N192Q / T243S, A194E, E196D, E196D / S258L / S363E / S426P / T465F / R549G / N597D, S220E, S220E / R295E / T520A, S220E / R295E / T521G, S220E / V296E / S304C / S426L / R549W, V222D, V222T, V222Y, S257D, S258L, M279 L / R560W / R637L, M279T, R295E, R295Q, R295S, R295T, V296E, V296T, S304C, N319S, S363E, S363E / S426P / Q481D / Q553S, S426A, S426L, T465G, I499F, I499V, R549G, R549Q, Q553S, R560W, S567M, N597D and R637L, wherein the amino acid positions of the polypeptide sequences are numbered with reference to SEQ ID NO: 272. 、

[0014] In some additional embodiments, the engineered galactose oxidase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 272, and wherein the engineered galactose oxidase comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 4 / 8 / 56 / 598, 4 / 56 / 192 / 194 / 257 / 571 / 598, 4 / 56 / 194 / 329 / 598, 4 / 194 / 243 / 549 / 598, 8 / 29 / 46 / 196 / 363 / 481 / 549 / 553, 8 / 56 / 192 / 194 / 257 / 571 / 598. / 194 / 243 / 329、8 / 56 / 192 / 194 / 243 / 329 / 460 / 560、8 / 56 / 192 / 194 / 243 / 460 / 560 / 598、8 / 56 / 192 / 243 / 460、8 / 56 / 192 / 243 / 598、8 / 56 / 194 / 257 / 460 / 549 / 560 / 598、8 / 92 / 196 / 258 / 426 / 597、8 / 92 / 196 / 481 / 597、8 / 192 / 194 / 243 / 329 / 460 / 560、8 / 196 / 258 / 279 / 481 / 549 / 553、8 / 243 / 460 / 560 / 571 / 598、8 / 257 / 460 / 560 / 598、8 / 258 / 363 / 426 / 549、8 / 279 / 363 / 426 / 481 / 549 / 553、16 / 43 / 63 / 103 / 295 / 296 / 499、16 / 43 / 103 / 148 / 295 / 426 / 499 / 549、16 / 43 / 103 / 304 / 499 / 549、16 / 43 / 148 / 295 / 296 / 304 / 499 / 549、16 / 43 / 148 / 296 / 426 / 499、16 / 43 / 295 / 296 / 499、16 / 56 / 103 / 220 / 295、16 / 56 / 103 / 2 20 / 295 / 296、16 / 56 / 103 / 295 / 549、16 / 56 / 148 / 295 / 296 / 304 / 426 / 549、16 / 56 / 220 / 295、16 / 56 / 220 / 499 / 549、16 / 56 / 295、16 / 56 / 295 / 296 / 549、16 / 56 / 295 / 499、16 / 56 / 499 / 549、16 / 63 / 103 / 148 / 426 / 499 / 549、16 / 63 / 103 / 426 / 499 / 549、16 / 63 / 148 / 220 / 295 / 296 / 426 / 499、16 / 63 / 148 / 499 / 549、16 / 103、16 / 103 / 148、16 / 103 / 148 / 220 / 499、16 / 103 / 148 / 295 / 426 / 499 / 549、16 / 103 / 220 / 295、16 / 148 / 220 / 295 / 426 / 499、16 / 148 / 295 / 426、16 / 148 / 295 / 549、16 / 148 / 426 / 549、16 / 220 / 295 / 296、16 / 304 / 426 / 499、16 / 304 / 499 / 549、24 / 36 / 92 / 279 / 295 / 363 / 499、24 / 43 / 92 / 148 / 279 / 295 / 319 / 637、24 / 43 / 92 / 222 / 279、24 / 43 / 222 / 319、24 / 43 / 363 / 637、24 / 92、24 / 92 / 148 / 279、24 / 92 / 222 / 279 / 319 / 637、24 / 222 / 637、24 / 279 / 319、29 / 46 / 92 / 196 / 426 / 481 / 549 / 597、29 / 46 / 481 / 549 / 553 / 597、29 / 426 / 549、29 / 549 / 553、36 / 43 / 222 / 279 / 363 / 560、36 / 92 / 148 / 222 / 279 / 319 / 363 / 499 / 560 / 637、36 / 92 / 222 / 637、36 / 148 / 279 / 319 / 499、43 / 148 / 222 / 279 / 560 / 637、43 / 220 / 295 / 549、56 / 148 / 220 / 295 / 499、56 / 194 / 243 / 257 / 329 / 460、56 / 243、63 / 103 / 148 / 220 / 295 / 549、63 / 220 / 295 / 304 / 426 / 499 / 549、63 / 220 / 295 / 304 / 549、92、92 / 222 / 279 / 499 / 560 / 637、92 / 258 / 363 / 426 / 481 / 549 / 597、103 / 295 / 499 / 549、148 / 220 / 304、148 / 222、148 / 222 / 560 / 637、148 / 279 / 319 / 499、194 / 243 / 329 / 460、194 / 243 / 329 / 560 / 571 / 598、220 / 295 / 549、279 / 296 / 481 / 549 / 553 / 567 / 597、279 / 560 / 637、295 / 296 / 426 / 549、295 / 296 / 549、295 / 499 / 560 / 637、296 / 363 / 426 / 481 / 549、319 / 560、319 / 637 and 363 / 560, wherein the amino acid positions of the polypeptide sequences are numbered with reference to SEQ ID NO:272. In some embodiments, the engineered galactose oxidase comprises at least one substitution or set of substitutions selected from the group consisting of 4Q / 8V / 56I / 598L, 4Q / 56I / 192Q / 194G / 257N / 571A / 598E, 4Q / 56I / 194E / 329A / 598E, 4Q / 194G / 243S / 549G / 598E, 8I / 29Y / 46V / 196D / 363E / 481D / 549G / 553S, 8I / 92V / 196D / 258L / 426P / 597D, 8I / 92V / 196D / 481D / 597D, 8I / 196D / 258L / 279T / 481 D / 549G / 553S, 8I / 258L / 363E / 426P / 549G, 8I / 279T / 363E / 426P / 481D / 549G / 553S, 8V / 56I, 8V / 56I / 192Q / 194E / 243S / 329A / 460A / 560T, 8V / 56 I / 192Q / 194E / 243S / 460A / 560T / 598E, 8V / 56I / 192Q / 194G / 243S / 329A, 8V / 56I / 192Q / 243S / 460Q, 8V / 56I / 192Q / 243S / 598E, 8V / 56I / 194G / 257 D / 460A / 549G / 560T / 598L, 8V / 192Q / 194G / 243S / 329A / 460Q / 560T, 8V / 243S / 460Q / 560T / 571A / 598L, 8V / 257D / 460Q / 560T / 598E, 16E / 43E / 103 I / 148R / 295Q / 426L / 499V / 549W, 16E / 43E / 103I / 304C / 499V / 549W, 16E / 43E / 295T / 296E / 499V, 16E / 56F / 103I / 220E / 295T / 296E, 16E / 56F / 103 I / 295T / 549W, 16E / 56F / 148R / 295Q / 296E / 304C / 426L / 549W, 16E / 56F / 295Q / 296E / 549W, 16E / 63V / 148R / 220E / 295Q / 296E / 426L / 499V, 16E / 10 3I / 220E / 295Q, 16E / 148R / 220E / 295S / 426L / 499V, 16E / 148R / 295Q / 426 L, 16E / 148R / 295Q / 549W, 16E / 148R / 426L / 549W, 16E / 220E / 295Q / 296E,16E / 304C / 499V / 549W、16S / 43E / 63V / 103I / 295Q / 296E / 499V、16S / 43E / 148R / 295Q / 296E / 304C / 499V / 549W、16S / 43E / 148R / 296E / 426L / 499V、16S / 56F / 103I / 220E / 295S、16S / 56F / 220E / 295S、16S / 56F / 220E / 499V / 549W、16S / 56F / 295Q、16S / 56F / 295T / 499V、16S / 56F / 499V / 549W、16S / 63V / 103I / 148R / 426L / 499V / 549W、16S / 63V / 103I / 426L / 499V / 549W、16S / 63V / 148R / 499V / 549W、16S / 103I、16S / 103I / 148R、16S / 103I / 148R / 220E / 499V、16S / 103I / 148R / 295S / 426L / 499V / 549W、16S / 304C / 426L / 499V、24P / 36P / 92D / 279L / 295E / 363L / 499F、24P / 43A / 92D / 148A / 279L / 295D / 319S / 637L、24P / 43A / 92D / 222Y / 279L、24P / 43A / 222Y / 319R、24P / 43A / 363L / 637L、24P / 92D、24P / 92D / 148A / 279L、24P / 92D / 222D / 279L / 319S / 637L、24P / 222D / 637L、24P / 279L / 319R、29T / 46V / 92V / 196D / 426P / 481D / 549G / 597D、29T / 46V / 481D / 549G / 553S / 597D、29T / 549G / 553S、29Y / 426P / 549G、36P / 43Q / 222Y / 279L / 363L / 560W、36P / 92D / 148A / 222D / 279L / 319S / 363L / 499F / 560W / 637L、36P / 92D / 222D / 637L、36P / 148A / 279L / 319S / 499F、43E / 220E / 295S / 549W、43Q / 148A / 222D / 279L / 560W / 637L、56F / 148R / 220E / 295Q / 499V、56I / 194G / 243S / 257N / 329A / 460A、56I / 243S、63V / 103I / 148R / 220E / 295S / 549W、63V / 220E / 295T / 304C / 426L / 499V / 549W, 63V / 220E / 295T / 304C / 549W, 92D, 92D / 222D / 279L / 499F / 560W / 637L, 92V / 258L / 363E / 426P / 481 D / 549G / 597D, 103I / 295T / 499V / 549W, 148A / 222D, 148A / 222D / 560W / 637L, 148A / 279L / 319S / 499F, 148R / 220E / 304C, 194E / 243S / 329A / 5 60T / 571A / 598E, 194G / 243S / 329A / 460Q, 220E / 295T / 549W, 279L / 560W / 637L, 279T / 296T / 481D / 549G / 553S / 567M / 597D, 295D / 499F / 560W / 637L, 295Q / 296E / 426L / 549W, 295S / 296E / 549W, 296T / 363E / 426P / 481D / 549G, 319R / 637L, 319S / 560W and 363L / 560W, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:272. In some embodiments, the engineered galactose oxidase comprises at least one substitution or set of substitutions selected from the group consisting of A4Q / S8V / Y56I / N598L, A4Q / Y56I / N192Q / A194G / S257N / K571A / N598E, A4Q / Y56I / A194E / L329A / N598E, A4Q / A194G / T243S / R549G / N598E, S8I / N29Y / A46V / E196D / S363E / Q481D / R549G / Q553S, S8I / S92V / E196D / S258L / S426P / N597D, S8I / S92V / E196D / Q481D / N597D. 6D / S258L / M279T / Q481D / R549G / Q553S, S8I / S258L / S363E / S426P / R549G, S8I / M279T / S363E / S426P / Q481D / R549G / Q553S, S8V / Y56I, S8V / Y56I / N192Q / A194E / T243S / L329A / R460A / R560T, S8V / Y56I / N192Q / A194E / T243S / R460A / R560T / N 598E、S8V / Y56I / N192Q / A194G / T243S / L329A、S8V / Y56I / N192Q / T243S / R460Q、S8V / Y56I / N192Q / T243S / N598E、S8V / Y56I / A194G / S257D / R460A / R549G / R560T / N598L、S8V / N192Q / A194G / T243S / L329A / R460Q / R560T、S8V / T243S / R460Q / R560T / K571A / N598L、S8V / S257D / R460Q / R560T / N598E、A16E / F43E / S103I / Q148R / R295Q / S426L / I499V / R549W、A16E / F43E / S103I / S304C / I499V / R549W、A16E / F43E / R295T / V296E / I499V、A16E / Y56F / S103I / S220E / R295T / V296E、A16E / Y56F / S103I / R295T / R549W、A16E / Y56F / Q148R / R295Q / V296E / S304C / S426L / R549W、A16E / Y56F / R295Q / V296E / R549W、A16E / T63V / Q148R / S220E / R295Q / V296E / S426L / I499V、A16E / S103I / S220E / R295Q、A16E / Q148R / S220E / R295S / S426L / I499V、A16E / Q148R / R295Q / S426L、A16E / Q148R / R295Q / R549W、A16E / Q148R / S426L / R549W、A16E / S220E / R295Q / V296E、A16E / S304C / I499V / R549W、A16S / F43E / T63V / S103I / R295Q / V296E / I499V、A16S / F43E / Q148R / R295Q / V296E / S304C / I499V / R549W、A16S / F43E / Q148R / V296E / S426L / I499V、A16S / Y56F / S103I / S220E / R295S、A16S / Y56F / S220E / R295S、A16S / Y56F / S220E / I499V / R549W、A16S / Y56F / R295Q、A16S / Y56F / R295T / I499V、A16S / Y56F / I499V / R549W、A16S / T63V / S103I / Q148R / S426L / I499V / R549W、A16S / T63V / S103I / S426L / I499V / R549W、A16S / T63V / Q148R / I499V / R549W、<h2 style=";text-align:left;direction:ltr">A16S / S103I, A16S / S103I / Q148R, A16S / S103I / Q148R, S220E / I499V, A16S / S103I / Q148R, R295S, S426L, I499V, R549W, A16S / S304C, S426L, I499V, S2 4P / K36P / S92D / M279L / R295E / S363L / I499F、S24P / F43A / S92D / Q148A / M279L / R295D / N319S / R637L、S24P / F43A / S92D / V222Y / M279L、S24P / F43A / V222 Y / N319R, S24P / F43A / S363L / R637L, S24P / S92D, S24P / S92D / Q148A / M279L, S24P / S92D / V222D / M279L, N319S / R637L, S24P / V222D / R637L, S24P / M279L / N319R、N29T / A46V / S92V / E196D / S426P / Q481D / R549G / N597D、N29T / A46V / Q481D / R549G / Q553S / N597D、N29T / R549G / Q553S、N29Y / S426P / R549G、K36P / F43Q / V222Y / M279L / S363L / R560W、K36P / S92D / Q148A / V222D / M279L / N319S / S363L / I499F / R560W / R637L、K36P / S92D / V222D / R637L、K36P / Q148A / M2 79L / N319S / I499F、F43E / S220E / R295S / R549W、F43Q / Q148A / V222D / M279L / R560W / R637L、Y56F / Q148R / S220E / R295Q / I499V、Y56I / A194G / T243S / S257 N / L329A / R460A、Y56I / T243S、T63V / S103I / Q148R / S220E / R295S / R549W、T 63V / S220E / R295T / S304C / S426L / I499V / R549W、T63V / S220E / R295T / S304C / R549W、S92D、S92D / V222D / M279L / I499F / R560W / R637L、S92V / S258L / S363E / S426P / Q481D / R549G / N597D、S103I / R295T / I499V / R549W、Q148A / V222D、Q148A / V222D / R560W / R637L, Q148A / M279L / N319S / I499F, Q148R / S220E / S304C, A194E / T243S / L329A / R560 T / K571A / N598E, A194G / T243S / L329A / R460Q, S220E / R295T / R549W, M279L / R560W / R637L, M279T / V296T / Q4 81D / R549G / Q553S / S567M / N597D, R295D / I499F / R560W / R637L, R295Q / V296E / S426L / R549W, R295S / V296E / R549W, V296T / S363E / S426P / Q481D / R549G, N319R / R637L, N319S / R560W and S363L / R560W, wherein the amino acid positions of the polypeptide sequences are numbered with reference to SEQ ID NO: 272. 、

[0015] In some embodiments, the engineered galactose oxidase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 928, and wherein the engineered galactose oxidase comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of 4 / 43 / 46 / 56 / 63 / 279 / 295 / 319 / 567 / 598, 4 / 43 / 46 / 295 / 319 / 549 / 560, 4 / 43 / 46 / 426 / 549 / 560, 4 / 43 / 148 / 196 / 279 / 319 / 363 / 564 / 598. 6 / 24 / 29、16 / 24 / 29 / 92 / 220 / 279 / 597 / 598、16 / 24 / 29 / 279 / 549、16 / 24 / 29 / 279 / 637、16 / 24 / 43 / 92 / 549、16 / 24 / 43 / 192 / 220 / 279 / 549、16 / 29 / 36 / 192 / 319 / 549 / 597 / 598 / 637、16 / 29 / 36 / 279 / 549、16 / 29 / 43 / 92 / 192 / 319、16 / 29 / 43 / 192 / 222 / 319 / 549 / 637、16 / 29 / 43 / 222 / 279 / 549、16 / 29 / 92 / 19 2 / 549 / 637、16 / 29 / 92 / 220 / 222 / 319 / 549 / 598、16 / 29 / 92 / 279 / 549、16 / 29 / 92 / 319 / 549、16 / 29 / 92 / 549 / 637、16 / 29 / 192 / 220 / 549、16 / 29 / 192 / 220 / 549 / 597、16 / 29 / 192 / 222 / 279 / 549、16 / 29 / 192 / 222 / 637、16 / 29 / 192 / 549、16 / 29 / 220 / 222 / 279 / 549 / 637、16 / 29 / 220 / 222 / 597 / 598、16 / 29 / 222 / 549 / 598 / 637、16 / 29 / 549 / 637、16 / 36 / 43 / 192 / 597 / 637、16 / 36 / 92 / 220 / 222 / 279 / 549、16 / 36 / 192 / 549 / 597 / 598 / 637、16 / 36 / 319 / 549 / 597 / 598 / 637、16 / 43 / 56 / 192 / 549 / 597 / 598 / 637、16 / 43 / 92 / 222 / 597 / 598、16 / 43 / 192 / 549、16 / 43 / 220 / 549 / 637、16 / 43 / 279 / 319 / 597、16 / 43 / 279 / 549 / 597、16 / 43 / 319 / 549 / 598、16 / 43 / 597、16 / 92 / 192 / 279 / 319 / 549 / 637、16 / 92 / 192 / 279 / 637、16 / 92 / 220 / 549、16 / 92 / 319 / 597 / 637、16 / 192 / 319 / 549 / 637、16 / 192 / 549、16 / 220 / 222 / 279 / 549 / 598 / 637、16 / 220 / 279 / 549、16 / 220 / 319 / 549 / 597 / 598、16 / 222 / 319 / 597 / 598、16 / 222 / 637、16 / 279 / 319 / 549 / 597 / 637、16 / 279 / 549 / 597 / 598 / 637、16 / 279 / 597、16 / 319 / 597、16 / 319 / 597 / 598、16 / 549、16 / 549 / 598、16 / 597、29 / 63 / 134 / 520 / 597 / 598、29 / 63 / 520 / 537 / 538 / 598、29 / 134 / 237 / 537 / 538 / 567 / 571、29 / 237 / 520、29 / 237 / 520 / 538、29 / 237 / 567 / 598、29 / 237 / 597、29 / 597 / 598、36 / 92 / 549、36 / 134 / 237 / 520 / 537 / 538 / 571、36 / 134 / 237 / 567 / 571 / 597 / 598、36 / 520 / 537 / 538 / 597、43 / 46 / 56 / 148 / 258 / 279 / 363 / 549 / 571、43 / 46 / 63 / 258 / 295 / 426 / 560 / 567 / 571、43 / 46 / 196 / 319 / 549 / 560 / 567、43 / 279 / 549 / 560 / 567、46 / 295 / 319 / 426、46 / 560、95、134 / 237 / 520 / 597、134 / 520 / 597 / 598、220 / 222 / 597 / 637、220 / 296 / 407 / 465、224、237 / 520、237 / 520 / 537 / 538 / 598、237 / 520 / 537 / 598、237 / 520 / 538 / 597、237 / 520 / 567 / 571 / 597、237 / 520 / 597 / 598、237 / 538 / 597 / 598、237 / 571、237 / 597 / 598、279 / 319 / 560、294、295 / 549 / 560、343、433、465、483、486、520 / 571 / 598、520 / 597 / 598、549 / 598、556、564、567 / 571 / 597、568 and 609, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:928. In some embodiments, the engineered galactose oxidase comprises at least one substitution or set of substitutions selected from the group consisting of 4Q / 43Q / 46V / 56F / 63V / 279L / 295T / 319S / 567M / 598E, 4Q / 43Q / 46V / 295Q / 319S / 549G / 560W, 4Q / 43Q / 46V / 426L / 549G / 560W, 4Q / 43Q / 148A / 196D / 279L / 319S / 363L / 560W, 16E, 16E / 24P / 29T / 92D / 220E / 279T / 597D / 598L, 16E / 24P / 29T / 279T / 549W, 16 E / 24P / 29T / 279T / 637L, 16E / 24P / 29Y, 16E / 24P / 43E / 92D / 549W, 16E / 24 P / 43E / 192N / 220E / 279T / 549W, 16E / 29T / 36P / 192N / 319R / 549W / 597D / 59 8L / 637L, 16E / 29T / 36P / 279T / 549W, 16E / 29T / 43A / 92D / 192N / 319R, 16E / 29T / 43E / 222D / 279T / 549W, 16E / 29T / 92D / 220E / 222D / 319R / 549W / 598L, 16E / 29T / 92D / 279T / 549W, 16E / 29T / 192N / 220E / 549W, 16E / 29T / 192N / 2 20E / 549W / 597D, 16E / 29T / 192N / 222D / 637L, 16E / 29T / 192N / 549W, 16E / 2 9T / 220E / 222D / 279T / 549W / 637L, 16E / 29T / 222D / 549W / 598L / 637L, 16E / 29Y / 43E / 192N / 222D / 319R / 549W / 637L, 16E / 29Y / 92D / 319R / 549W, 16E / 2 9Y / 192N / 222D / 279T / 549W, 16E / 29Y / 192N / 549W, 16E / 29Y / 220E / 222D / 597D / 598L, 16E / 29Y / 549W / 637L, 16E / 36P / 43A / 192N / 597D / 637L, 16E / 3 6P / 92D / 220E / 222D / 279T / 549W, 16E / 36P / 192N / 549W / 597D / 598L / 637L, 16E / 36P / 319R / 549W / 597D / 598L / 637L, 16E / 43A / 92D / 222D / 597D / 598L,<h2 style=";text-align:left;direction:ltr">16E / 43A / 220E / 549W / 637L、16E / 43A / 279T / 549W / 597D、16E / 43E / 56V / 192N / 549W / 597D / 598L / 637L、16E / 43E / 192N / 549W、16E / 43E / 279T / 319R / 597D、 16E / 43E / 597D、16E / 92D / 192N / 279T / 319R / 549W / 637L、16E / 92D / 220E / 549 W、16E / 192N / 319R / 549W / 637L、16E / 192N / 549W、16E / 220E / 222D / 279T / 549 W / 598L / 637L、16E / 220E / 279T / 549W、16E / 220E / 319R / 549W / 597D / 598L、16 E / 222D / 319R / 597D / 598L、16E / 222D / 637L、16E / 279T / 319R / 549W / 597D / 63 7L, 16E / 279T / 597D, 16E / 319R / 597D, 16E / 319R / 597D / 598L, 16E / 549W, 16E / 549W / 598L, 16E / 597D, 16S / 29T / 92D / 549W / 637L, 16S / 29Y / 92D / 192N / 549 W / 637L、16S / 43E / 319R / 549W / 598L、16S / 92D / 192N / 279T / 637L、16S / 92D / 319R / 597D / 637L、16S / 279T / 549W / 597D / 598L / 637L、29H / 63V / 134A / 520A / 5 97D / 598E, 29H / 63V / 520A / 537G / 538D / 598E, 29H / 134A / 237D / 537G / 538D / 567M / 571A, 29H / 237D / 520A, 29H / 237D / 520A / 538D, 29H / 237D / 567M / 598E, 2 9H / 237D / 597D, 29H / 597D / 598E, 36P / 92D / 549W, 36V / 134A / 237D / 520A / 537G / 538D / 571A, 36V / 134A / 237D / 567M / 571A / 597D / 598E, 36V / 520A / 537G / 53 8D / 597D, 43Q / 46V / 56F / 148A / 258L / 279L / 363L / 549G / 571A, 43Q / 46V / 63V / 258L / 295Q / 426L / 560W / 567M / 571A, 43Q / 46V / 196D / 319S / 549G / 560W / 567M43Q / 279L / 549G / 560W / 567M, 46V / 295T / 319S / 426L, 46V / 560W, 95E, 134A / 237D / 520A / 597D, 134A / 520A / 597D / 598E, 220E / 222D / 597D / 637L, 220E / 2 96S / 407I / 465G, 224D, 237D / 520A, 237D / 520A / 537G / 538D / 598E, 237D / 52 0A / 537G / 598E, 237D / 520A / 538D / 597D, 237D / 520A / 567M / 571A / 597D, 237 98E, 237D / 538D / 597D / 598E, 237D / 571A, 237D / 597D / 598E, 279L / 319S / 560W, 294E, 295Q / 549G / 560W, 343G, 433G, 465G, 483R, 486P, 520A / 571A / 598E, 520A / 597D / 598E, 549G / 598E, 556A, 556S, 556V, 564D, 564E, 564T, 564W, 567M / 571A / 597D, 568E, 568P and 609D, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:928. In some embodiments, the engineered galactose oxidase comprises at least one substitution or set of substitutions selected from the group consisting of A4Q / F43Q / A46V / I56F / T63V / M279L / R295T / N319S / S567M / N598E, A4Q / F43Q / A46V / R295Q / N319S / R549G / R560W, A4Q / F43Q / A46V / S426L / R549G / R560W, A4Q / F43Q / Q148A / E196D / M279L / N319S / S363L / R560W, A16E, A16E / S24P / N29T / S92D / S220E / M279T / N597D / N 598L, A16E / S24P / N29T / M279T / R549W, A16E / S24P / N29T / M279T / R637L, A16 E / S24P / N29Y, A16E / S24P / F43E / S92D / R549W, A16E / S24P / F43E / Q192N / S22 0E / M279T / R549W, A16E / N29T / K36P / Q192N / N319R / R549W / N597D / N598L / R6 37L、A16E / N29T / K36P / M279T / R549W、A16E / N29T / F43A / S92D / Q192N / N319R、<h2 style=";text-align:left;direction:ltr">A16E / N29T / F43E / V222D / M279T / R549W、A16E / N29T / S92D / S220E / V222D / N3 19R / R549W / N598L、A16E / N29T / S92D / M279T / R549W、A16E / N29T / Q192N / S22 0E / R549W、A16E / N29T / Q192N / S220E / R549W / N597D、A16E / N29T / Q192N / V22 2D / R637L、A16E / N29T / Q192N / R549W、A16E / N29T / S220E / V222D / M279T / R549 W / R637L、A16E / N29T / V222D / R549W / N598L / R637L、A16E / N29Y / F43E / Q192N / V222D / N319R / R549W / R637L、A16E / N29Y / S92D / N319R / R549W、A16E / N29Y / Q 192N / V222D / M279T / R549W, A16E / N29Y / Q192N / R549W, A16E / N29Y / S220E / V222D / N597D / N598L, A16E / N29Y / R549W / R637L, A16E / K36P / F43A / Q192N / N59 7D / R637L, A16E / K36P / S92D / S220E / V222D / M279T / R549W, A16E / K36P / Q192N / R549W / N597D / N598L / R637L, A16E / K36P / N319R / R549W / N597D / N598L / R63 7L, A16E / F43A / S92D / V222D / N597D / N598L, A16E / F43A / S220E / R549W / R637L, A16E / F43A / M279T / R549W / N597D, A16E / F43E / I56V / Q192N / R549W / N597D / N598L / R637L、A16E / F43E / Q192N / R549W、A16E / F43E / M279T / N319R / N597D、 A16E / F43E / N597D、A16E / S92D / Q192N / M279T / N319R / R549W / R637L、A16E / S9 2D / S220E / R549W, A16E / Q192N / N319R / R549W / R637L, A16E / Q192N / R549W, A16E / S220E / V222D / M279T / R549W / N598L / R637L, A16E / S220E / M279T / R549W<h2 style=";text-align:left;direction:ltr">A16E / S220E / N319R / R549W / N597D / N598L、A16E / V222D / N319R / N597D / N59 8L、A16E / V222D / R637L、A16E / M279T / N319R / R549W / N597D / R637L、A16E / M 279T / N597D, A16E / N319R / N597D, A16E / N319R / N597D / N598L, A16E / R549W, A16E / R549W / N598L, A16E / N597D, A16S / N29T / S92D / R549W / R637L, A16S / N 29Y / S92D / Q192N / R549W / R637L, A16S / F43E / N319R / R549W / N598L, A16S / S92D / Q192N / M279T / R637L, A16S / S92D / N319R / N597D / R637L, A16S / M279T / R 549W / N597D / N598L / R637L、N29H / T63V / N134A / T520A / N597D / N598E、N29H / T63V / T520A / S537G / N538D / N598E、N29H / N134A / N237D / S537G / N538D / S56 7M / K571A、N29H / N237D / T520A、N29H / N237D / T520A / N538D、N29H / N237D / S 567M / N598E、N29H / N237D / N597D、N29H / N597D / N598E、K36P / S92D / R549W、 K36V / N134A / N237D / T520A / S537G / N538D / K571A, K36V / N134A / N237D / S567M / K571A / N597D / N598E, K36V / T520A / S537G / N538D / N597D, F43Q / A46V / I5 6F / Q148A / S258L / M279L / S363L / R549G / K571A、F43Q / A46V / T63V / S258L / R 295Q / S426L / R560W / S567M / K571A、F43Q / A46V / E196D / N319S / R549G / R560W / S567M、F43Q / M279L / R549G / R560W / S567M、A46V / R295T / N319S / S426L、A46V / R560W、T95E、N134A / N237D / T520A / N597D、N134A / T520A / N597D / N598E、S220E / V222D / N597D / R637L, S220E / V296S / E407I / T465G, K224D, N237D / T520A, N237D / T520A / S537G / N538D / N598E, N237D / T520A / S537G / N5 98E、N237D / T520A / N538D / N597D、N237D / T520A / S567M / K571A / N597D、 N237D / T520A / N597D / N598E, N237D / N538D / N597D / N598E, N237D / K571A , N237D / N597D / N598E, M279L / N319S / R560W, G294E, R295Q / R549G / R560W, K343G, S433G, T465G, T483R, K486P, T520A / K571A / N598E, T520A / N597D / N598E, R549G / N598E, K556A, K556S, K556V, S564D, S564E, S564T, S564W, S567M / K571A / N597D, S568E, S568P and S609D, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:928. ,

[0016] In some additional embodiments, the engineered galactose oxidase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:928, and wherein the engineered galactose oxidase comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 4 / 43 / 46 / 426 / 549 / 560, 36 / 63 / 520, 95, 394, 483, 520 / 597, 556, 562, 568 and 598, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:928. In some embodiments, the engineered galactose oxidase comprises at least one substitution or set of substitutions selected from the group consisting of 4Q / 43Q / 46V / 426L / 549G / 560W, 36V / 63V / 520A, 95V, 394A, 483R, 520A / 597D, 556V, 562D, 568D, 568P, and 598E, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:928. In some embodiments, the engineered galactose oxidase comprises at least one substitution or set of substitutions selected from the group consisting of A4Q / F43Q / A46V / S426L / R549G / R560W, K36V / T63V / T520A, T95V, K394A, T483R, T520A / N597D, K556V, T562D, S568D, S568P, and N598E, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:928.

[0017] In some additional embodiments, the engineered galactose oxidase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:928, and wherein the engineered galactose oxidase comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 63 / 196, 173, 189, 194, 196, 197, 198, 198 / 447, 220 / 294 / 296 / 332, 220 / 294 / 465, 290, 292, 294 / 407 / 465, 327, 407 / 465 and 638, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:928. In some embodiments, the engineered galactose oxidase comprises at least one substitution or set of substitutions selected from the group consisting of 63A / 196L, 173S, 173V, 189A, 194R, 194V, 194W, 196A, 196G, 196I, 196L, 196Q, 196R, 196V, 197A, 197E, 197P, 197Q, 197R, 198G, 198T, 198T / 447I, 220E / 294E / 296S / 332Q, 220M / 294E / 465G, 290A, 290G, 292G, 294E / 407M / 465G, 327R, 407V / 465G, and 638A, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:928. In some embodiments, the engineered galactose oxidase comprises at least one substitution or set of substitutions selected from the group consisting of T63A / E196L, A173S, A173V, S189A, A194R, A194V, A194W, E196A, E196G, E196I, E196L, E196Q, E196R, E196V, G197A, G197E, G197P, G197Q, G197R, S198G, S198T, S198T / V447I, S220E / G294E / V296S / S332Q, S220M / G294E / T465G, S290A, S290G, S292G, G294E / E407M / T465G, Q327R, E407V / T465G and V638A, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:928.

[0018] In some embodiments, the engineered galactose oxidase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 932, and wherein the engineered galactose oxidase comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 16 / 43 / 220 / 258 / 538 / 637, 16 / 258 / 426 / 465 / 538 / 549 / 637, 18, 24 / 222 / 237 / 520 / 538, 24 / 222 / 520, 43 / 222 / 237 / 258 / 426 / 597, 43 / 258 / 407 / 426 / 465 / 538 / 549 / 637. / 538 / 549 / 637, 63, 63 / 95 / 173 / 343 / 564 / 568 / 609, 95 / 173 / 258 / 426 / 556 / 564, 95 / 173 / 556 / 609, 173 / 556, 194, 220 / 294 / 295 / 319 / 407 / 426 / 465 / 538 / 549 / 637, 222 / 237, 222 / 520 / 597, 237 / 258 / 549 / 597, 237 / 265 / 279, 258 / 267, 258 / 319 / 426 / 465 / 549 / 637, 258 / 426 and 258 / 538 / 549 / 637, wherein the amino acid positions of the polypeptide sequences refer to SEQ ID NO:932.In some embodiments, the engineered galactose oxidase comprises at least one substitution or set of substitutions selected from the group consisting of: 16E / 43A / 220M / 258S / 538D / 637L, 16E / 258S / 426S / 465G / 538D / 549W / 637L, 18K, 24P / 222D / 237D / 520A / 538D, 24P / 222D / 520A, 43A / 258S / 407I / 426S / 465G / 538D / 549W / 637L, 43E / 222D / 237D / 258S / 426S / 597D, 63T, 63T / 95E / 173S / 343G / 564D / 568P / 609D, 95E / 173 S / 556V / 609D, 95V / 173S / 258S / 426S / 556V / 564W, 173S / 556V, 194R, 220M / 294E / 295S / 319S / 407I / 426S / 465G / 538D / 549W / 637L, 222D / 237D, 222D / 520A / 597D, 237D / 258S / 549G / 597D, 237D / 265S / 279L, 258S / 267T, 258S / 319S / 426S / 465G / 549W / 637L, 258S / 426S and 258S / 538D / 549W / 637L, wherein the amino acid positions of the polypeptide sequences refer to SEQ ID NO:932.In some embodiments, the engineered galactose oxidase comprises at least one substitution or set of substitutions selected from the group consisting of A16E / Q43A / S220M / L258S / N538D / R637L, A16E / L258S / L426S / T465G / N538D / R549W / R637L, T18K, S24P / V222D / N237D / T520A / N538D, S24P / V222D / T520A, Q43A / L258S / E407I / L426S / T465G / N538D / R549W / R637L, Q43E / V222D / N237 D / L258S / L426S / N597D, V63T, V63T / T95E / A173S / K343G / S564D / S568P / S609D, T95E / A173S / K556V / S609D, T95V / A173S / L258S / L426S / K556V / S564W, A173S / K556V, A194R, S220M / G2 94E / Q295S / N319S / E407I / L426S / T465G / N538D / R549W / R637L, V222D / N237D, V222D / T520A / N597D, N237D / L258S / R549G / N597D, N237D / P265S / M279L, L258S / M267T, L258S / N319S / L426S / T465G / R549W / R637L, L258S / L426S and L258S / N538D / R549W / R637L, wherein the amino acid positions of the polypeptide sequences are numbered with reference to SEQ ID NO:932.

[0019] In some embodiments, the engineered galactose oxidase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1264, and wherein the engineered galactose oxidase comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 18 / 95 / 327 / 548, 28, 36, 43, 43 / 46 / 56 / 63 / 191, 43 / 237 / 279 / 538 / 597 / 598, 43 / 237 / 294 / 538, 43 / 237 / 520, 43 / 237 / 520 / 549 / 598, 43 / 237 / 5 20 / 597, 43 / 279 / 294, 43 / 538, 43 / 549 / 597, 51 / 55 / 111 / 150 / 367 / 564, 55, 61, 95 / 327 / 548, 99, 183, 198, 224, 229, 237 / 520 / 538 / 597, 243, 252, 258, 291, 295, 312, 335, 342, 343, 367 / 371 / 564 / 594, 371, 384, 468, 485, 520, 544, 549, 564 / 604, 567, 568, 570, 594, 596, 604, 635 and 637, wherein the amino acid positions of the polypeptide sequences refer to SEQ ID NO:1264.In some embodiments, the engineered galactose oxidase comprises at least one substitution or set of substitutions selected from the group consisting of: 18K / 95E / 327R / 548M, 28P, 28S, 36N, 43E, 43E / 237D / 279L / 538D / 597D / 598E, 43E / 237D / 294E / 538D, 43E / 237D / 520A, 43E / 237D / 520A / 549G / 598E, 43E / 237D / 520A / 597D, 43E / 279L / 294E, 43E / 538D, 43E / 549G / 597D, 43F / 46A / 56Y / 63T / 191V, 51P / 55W / 111Q / 150P / 367I / 564D, 55M , 55R, 61E, 95E / 327R / 548M, 99H, 183D, 198R, 224G, 229S, 237D / 520A / 538D / 597D , 243K, 243L, 252G, 258H, 291V, 295T, 312T, 335R, 342R, 342S, 343S, 367I / 371D / 564D / 594Q, 371A, 371P, 384G, 468N, 485L, 520E, 520N, 544P, 549E, 549G, 564K / 604G, 567G, 568A, 570K, 594C, 596G, 604M, 635K, 637N and 637W, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:1264.In some embodiments, the engineered galactose oxidase comprises at least one substitution or set of substitutions selected from the group consisting of T18K / V95E / Q327R / T548M, C28P, C28S, K36N, Q43E, Q43E / N237D / M279L / N538D / N597D / N598E, Q43E / N237D / G294E / N538D, Q43E / N237D / T520A, Q43E / N237D / T520A / R549G / N598E, Q43E / N237D / T520A / N597D, Q43E / M279L / G294E, Q43E / N538D, Q4 3E / R549G / N597D, Q43F / V46A / I56Y / V63T / R191V, K51P / T55W / T111Q / S150P / K367I / W564D, T55M , T55R, K61E, V95E / Q327R / T548M, S99H, R183D, S198R, K224G, C229S, N237D / T520A / N538D / N59 7D, S243K, S243L, S252G, S258H, F291V, Q295T, S312T, H335R, K342R, K342S, K343S, K367I / K371 D / W564D / T594Q, K371A, K371P, C384G, S468N, Y485L, T520E, T520N, R544P, R549E, R549G, W564K / S604G, M567G, S568A, S570K, T594C, T596G, S604M, T635K, R637N and R637W, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:1264.

[0020] In some additional embodiments, the engineered galactose oxidase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1264, and wherein the engineered galactose oxidase comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 28, 28 / 99 / 520 / 637, 55 / 295, 55 / 342, 55 / 568, 55 / 568 / 594, 55 / 568 / 637, 61 / 224 / 343 / 520 / 637, 99 / 343 / 637, 99 / 520 / 637, 99 / 637, 224 / 520 / 637, 295 / 342, 295 / 342 / 568, 342 / 568, 342 / 594, 343 / 520 / 637, 403 / 520 / 637, 520 / 637, 568 / 637, 594 and 637, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:1264. In some embodiments, the engineered galactose oxidase comprises at least one substitution or set of substitutions selected from the group consisting of 28P, 28P / 99H / 520E / 637N, 55R / 295T, 55R / 342S, 55R / 568A, 55R / 568A / 594C, 55R / 568A / 637W, 61E / 224G / 343S / 520E / 637N, 99H / 343S / 637N, 99H / 520E / 637N, 99H / 637N, 224G / 520E / 637N, 295T / 342S, 295T / 342S / 568A, 342S / 568A, 342S / 594C, 343S / 520E / 637N, 403P / 520E / 637N, 520E / 637N, 568A / 637W, 594C and 637W, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:1264.In some embodiments, the engineered galactose oxidase comprises at least one substitution or set of substitutions selected from the group consisting of C28P, C28P / S99H / T520E / R637N, T55R / Q295T, T55R / K342S, T55R / S568A, T55R / S568A / T594C, T55R / S568A / R637W, K61E / K224G / K343S / T520E / R637N, S99H / K343S / R637N, S99H / T 520E / R637N, S99H / R637N, K224G / T520E / R637N, Q295T / K342S, Q295T / K342S / S568A, K342S / S568A, K342S / T594C, K343S / T520E / R637N, S403P / T520E / R637N, T520E / R637N, S568A / R637W, T594C and R637W, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:1264.

[0021] In some embodiments, the engineered galactose oxidase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1416, and wherein the engineered galactose oxidase comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 13, 13 / 26 / 156 / 274 / 359 / 429, 13 / 156 / 262 / 274 / 315, 13 / 156 / 262 / 315 / 429 / 437, 13 / 156 / 274 / 437 / 568 / 606, 13 / 262 / 274 / 359 / 429. 80、13 / 262 / 274 / 595、13 / 262 / 437 / 488、13 / 274、13 / 274 / 315 / 437、13 / 274 / 373 / 437、13 / 328 / 437、13 / 373、13 / 437、13 / 437 / 541、26 / 262 / 274 / 315 / 437、35、37、37 / 89、37 / 89 / 274、37 / 263 / 274 / 380 / 559 / 561、37 / 380、45、45 / 262 / 274 / 373 / 437、89、89 / 263 / 274 / 380、89 / 263 / 559、89 / 274 / 380、10 5、154、156、156 / 274 / 315、200、217、217 / 274 / 380 / 561、217 / 274 / 478、217 / 354 / 380、217 / 380、224、239、241、253、262、262 / 274、262 / 274 / 315、262 / 274 / 437、262 / 373 / 595、262 / 380、262 / 437、262 / 541、263、263 / 274、263 / 274 / 380、263 / 354 / 380 / 559、263 / 380、263 / 380 / 441、274、274 / 328、274 / 354, 274 / 359, 274 / 373 / 437, 274 / 380, 274 / 380 / 441, 274 / 380 / 559, 274 / 393 / 437, 274 / 437, 274 / 437 / 541, 274 / 437 / 568, 315, 328, 336, 354, 354 / 380, 359, 366, 373, 373 / 595, 375, 380, 380 / 437, 380 / 559 / 561, 393, 429, 437, 438, 439, 441, 478, 478 / 561, 488, 541, 550, 559, 561, 568, 595, 605,627 and 641, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:1416. In some embodiments, the engineered galactose oxidase comprises at least one substitution or set of substitutions selected from the group consisting of: 13A / 26M / 156V / 274G / 359F / 429V, 13A / 156L / 262V / 274G / 315G, 13A / 156L / 274G / 437K / 568K / 606S, 13A / 156V / 262V / 315G / 429V / 437V, 13A / 274G, 13A / 274G / 315G / 437V, 13H, 13K, 13K / 262V, 13K / 262V / 274N / 380H, 13K / 262V / 274N / 595W, 13K / 262V / 437 7L / 488L, 13K / 274N / 373T / 437C, 13K / 328R / 437C, 13K / 373T, 13K / 437L, 1 3K / 437L / 541R, 26M / 262V / 274G / 315G / 437R, 35D, 37I, 37M, 37M / 89R, 37M / 89R / 274N, 37V / 380K, 37Y, 37Y / 263S / 274N / 380K / 559S / 561T, 45V, 45V / 262V / 274N / 373T / 437C, 89R, 89R / 263S / 274N / 380K, 89R / 263S / 274N / 380 R, 89R / 263S / 559S, 89R / 274N / 380R, 105R, 154H, 156L, 156V, 156V / 274G / 315G, 200A, 217P, 217P / 274N / 380K / 561T, 217P / 274N / 478M, 217P / 354T / 380K, 217P / 380L, 217P / 380R, 224W, 239M, 241I, 253V, 262V, 262V / 274G / 315G, 262V / 274G / 437R, 262V / 274N, 262V / 373T / 595W, 262V / 380H, 262V / 437C, 262V / 541R, 263S, 263S / 274N, 263S / 274N / 380L, 263S / 274N / 380R, 263S / 354T / 380K / 559S, 263S / 380K, 263S / 380K / 441I, 274G, 274G / 359F, 274G / 437V / 568K, 274N, 274N / 328L, 274N / 354T, 274N / 373T / 437L, 274N / 380H, 274N / 380K, 274N / 380R / 441I, 274N / 380R / 559S, 274N / 393P / 437L,274N / 437L, 274N / 437L / 541R, 315G, 328K, 328L, 328R, 336P, 354D, 354T, 354T / 380K, 359F, 3 66T, 373T, 373T / 595W, 375L, 380H, 380H / 437C, 380K, 380K / 559S / 561T, 380L, 380R, 393D, 393 595W, 605L, 627R, and 641D, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:1416. In some embodiments, the engineered galactose oxidase comprises at least one substitution or set of substitutions selected from the group consisting of: N13A / N26M / Q156V / Q274G / Y359F / T429V, N13A / Q156L / I262V / Q274G / N315G, N13A / Q156L / Q274G / Y437K / S568K / Q606S, N13A / Q156V / I262V / N315G / T429V / Y43 7V, N13A / Q274G, N13A / Q274G / N315G / Y437V, N13H, N13K, N13K / I262V, N13K / I262V / Q274N / P380H, N1 3K / I262V / Q274N / L595W, N13K / I262V / Y437L / N488L, N13K / Q274N / Q373T / Y437C, N13K / G328R / Y437C, N13K / Q373T, N13K / Y437L, N13K / Y437L / L541R, N26M / I262V / Q274G / N315G / Y437R, N35D, D37I, D37M, D37M / Y89R, D37M / Y89R / Q274N, D37V / P380K, D37Y, D37Y / P263S / Q274N / P380K / G559S / I561T, G45V, G 45V / I262V / Q274N / Q373T / Y437C, Y89R, Y89R / P263S / Q274N / P380K, Y89R / P263S / Q274N / P380R, Y89R / P263S / G559S, Y89R / Q274N / P380R, S105R, A154H, Q156L, Q156V, Q156V / Q274G / N315G, G200A, D217P,D217P / Q274N / P380K / I561T, D217P / Q274N / V478M, D217P / A354T / P380K, D217P / P380L, D217P / P380R, G224W, Q239M, V241I, L253 V, I262V, I262V / Q274G / N315G, I262V / Q274G / Y437R, I262V / Q274N, I262V / Q373T / L595W, I262V / P380H, I262V / Y437C, I262V / L5 41R, P263S, P263S / Q274N, P263S / Q274N / P380L, P263S / Q274N / P380R, P263S / A354T / P380K / G559S, P263S / P380K, P263S / P380K / T441I, Q274G, Q274G / Y359F, Q274G / Y437V / S568K, Q274N, Q274N / G328L, Q274N / A354T, Q274N / Q373T / Y437L, Q274N / P380H, Q274N / P380K、Q274N / P380R / T441I、Q274N / P380R / G559S、Q274N / V393P / Y437L、Q274N / Y437L、Q274N / Y437L / L541R、N315G、G328K、G32 8L, G328R, A336P, A354D, A354T, A354T / P380K, Y359F, V366T, Q373T, Q373T / L595W, N375L, P380H, P380H / Y437C, P380K, P380K / G5 59S / I561T, P380L, P380R, V393D, V393G, V393P, V393T, T429V, Y437C, Y437G, Y437L, Y437R, Y437V, F438S, A439G, T441I, V478L, V478M, V478M / I561T, N488L, N488T, L541R, T550S, G559S, I561S, I561T, S568K, L595W, F605L, A627R and G641D, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 1416. 、

[0022] In some embodiments, the engineered galactose oxidase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1598, and wherein the engineered galactose oxidase comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 13 / 99 / 156 / 262 / 437 / 559 / 641, 13 / 99 / 156 / 380 / 437, 13 / 99 / 156 / 380 / 437 / 559 / 563 / 641, 13 / 99 / 257 / 262 / 263 / 559, 13 / 99 / 262 / 263 / 3 80 / 437 / 563 / 641、13 / 99 / 263、13 / 99 / 263 / 380、13 / 99 / 263 / 380 / 437、13 / 99 / 263 / 380 / 437 / 641、13 / 99 / 380 / 437 / 559、13 / 99 / 437 / 563、13 / 99 / 563 / 641、13 / 156 / 262 / 263 / 437 / 559、13 / 156 / 263 / 437 / 437、13 / 156 / 380、13 / 262、13 / 262 / 263、13 / 262 / 263 / 380 / 437 / 559、13 / 380 / 437、13 / 380 / 437 / 559、13 / 437, 13 / 470 / 559 / 563, 29, 30, 43 / 46 / 56 / 63 / 99 / 156 / 262 / 263 / 403 / 559 / 563, 62, 99, 99 / 156 / 262, 99 / 156 / 262 / 263 / 380 / 437 / 559, 99 / 156 / 262 / 263 / 437, 99 / 156 / 262 / 263 / 559, 99 / 156 / 263 / 559, 99 / 156 / 380, 99 / 156 / 380 / 437, 99 / 156 / 437, 99 / 262 / 263 / 437 / 559 / 641, 99 / 262 / 437 / 559 3 / 437 / 563、99 / 380 / 437 / 559 / 641、99 / 380 / 563、99 / 437、108、149、175、177、184、194、197、208、234、251、254、262、262 / 263、262 / 263 / 437 / 559、262 / 263 / 559 / 563、262 / 437 / 641、263 / 380、263 / 437 / 559 / 563、278、280、287、356、373、380、407、409、463、466、489、559 / 641、565、569、592、596、601、610 and 615, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:1598. In some embodiments, the engineered galactose oxidase comprises at least one substitution or set of substitutions selected from the group consisting of 13K / 99H / 156L / 262V / 437C / 559S / 641D, 13K / 99H / 156L / 380K / 437C, 13K / 99H / 156L / 380K / 437V / 559S / 563A / 641D, 13K / 99H / 257R / 262V / 263S / 559S, 13K / 99H / 262V / 263S / 380K / 437R / 563A / 641D, 13K / 99H / 263S, 13K / 99H / 263S / 380K 380K / 437V, 13K / 99H / 263S / 380K / 437V / 641D, 13K / 99H / 380K / 437R / 559S , 13K / 99H / 437V / 563A, 13K / 99H / 563A / 641D, 13K / 156L / 262V / 263S / 437C / 559S, 13K / 156L / 263S / 437C, 13K / 156L / 380K, 13K / 262V, 13K / 262V / 263 S, 13K / 262V / 263S / 380K / 437V / 559S, 13K / 380K / 437C, 13K / 380K / 437V / 5 59S, 13K / 437R, 13K / 437V, 13K / 470L / 559S / 563A, 29V, 30E, 43F / 46A / 56Y / 63T / 99H / 156L / 262V / 263S / 403P / 559S / 563A, 62D, 62G, 62Q, 99H, 99H / 1 56L / 262V, 99H / 156L / 262V / 263S / 380K / 437C / 559S, 99H / 156L / 262V / 263 S / 380K / 437V / 559S, 99H / 156L / 262V / 263S / 437C, 99H / 156L / 262V / 263S / 559S, 99H / 156L / 263S / 559S, 99H / 156L / 380K, 99H / 156L / 380K / 437V, 99H / 156L / 437V、99H / 262V / 263S / 437V / 559S / 641D、99H / 262V / 437V / 559S、9 9H / 263S / 437R / 563A, 99H / 380K / 437C / 559S / 641D, 99H / 380K / 563A, 99H / 437V, 108F, 149N, 149R, 175G, 177L, 184L, 194Q, 197A, 197P, 208F, 208L,234L, 251V, 254L, 262V, 262V / 263S, 262V / 263S / 437C / 559S, 262V / 263S / 559S / 563A, 262V / 437V / 641D, 263S / 380K, 263S / 437V / 559S / 563A, 278L, 280M, 280N, 2 87L, 356S, 373D, 373K, 380K, 407Q, 409H, 409R, 463V, 466V, 489I, 489L, 559S / 641D, 565S, 569L, 592G, 592K, 596S, 601G, 601L, 610V and 615I, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:1598. In some embodiments, the engineered galactose oxidase comprises at least one substitution or set of substitutions selected from the group consisting of: N13K / S99H / Q156L / I262V / L437C / G559S / G641D, N13K / S99H / Q156L / P380K / L437C, N13K / S99H / Q156L / P380K / L437V / G559S / I563A / G641D, N13K / S99H / S257R / I262V / P263S / G 559S, N13K / S99H / I262V / P263S / P380K / L437R / I563A / G641D, N13K / S99H / P263S, N13K / S99H / P263S / P380K, N13K / S99H / P263S / P380K / L437V, N13K / S99H / P263S / P380K / L437V / G641D, N13K / S99H / P380K / L437R / G559S, N13K / S99H / L437V / I563A, N13K / S99H / I563A / G641D, N13K / Q156L / I262V / P263S / L437C / G559S, N13K / Q156L / P263S / L437C, N13K / Q156L / P380K, N13K / I262V, N13K / I262V / P263S, N13K / I262V / P263S / P380K / L437V / G55 9S, N13K / P380K / L437C, N13K / P380K / L437V / G559S, N13K / L437R, N13K / L437V, N13K / P470L / G559S / I563A, N 29V, K30E, Q43F / V46A / I56Y / V63T / S99H / Q156L / I262V / P263S / S403P / G559S / I563A, T62D, T62G, T62Q, S99H,S99H / Q156L / I262V, S99H / Q156L / I262V / P263S / P380K / L437C / G559S, S99H / Q156L / I262V / P263S / P380K / L437V / G 559S, S99H / Q156L / I262V / P263S / L437C, S99H / Q156L / I262V / P263S / G559S, S99H / Q156L / P263S / G559S, S99H / Q15 6L / P380K, S99H / Q156L / P380K / L437V, S99H / Q156L / L437V, S99H / I262V / P263S / L437V / G559S / G641D, S99H / I262V / L437V / G559S, S99H / P263S / L437R / I563A, S99H / P380K / L437C / G559S / G641D, S99H / P380K / I563A, S99H / L437V, A 108F, A149N, A149R, A175G, I177L, V184L, A194Q, G197A, G197P, W208F, W208L, M234L, T251V, Y254L, I262V, I262V / P263S, I262V / P263S / L437C / G559S, I262V / P263S / G559S / I563A, I262V / L437V / G641D, P263S / P380K, P263S / L43 7V / G559S / I563A, T278L, S280M, S280N, I287L, N356S, Q373D, Q373K, P380K, E407Q, S409H, S409R, I463V, E466V, P489I, P489L, G559S / G641D, T565S, I569L, P592G, P592K, T596S, N601G, N601L, D610V and L615I, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 1598. 、

[0023] In some embodiments, the engineered galactose oxidase comprises a NO:1866 has a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity, and wherein the engineered galactose oxidase comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 29 / 149 / 463 / 601, 29 / 177 / 197 / 592, 29 / 177 / 463, 29 / 197 / 592, 29 / 463, 62 / 208 / 417 / 615, 62 / 286 / 615, 62 / 373 / 466, 62 / 466, 62 / 466 / 597, 149 / 208 / 615, 149 / 463、177 / 194 / 197 / 463 / 565、177 / 197 / 463 / 565、177 / 280 / 463 / 594 / 601、177 / 463 / 565、177 / 463 / 592、184、197、197 / 280 / 463、197 / 463 / 592、197 / 466 / 56 9 / 596, 208 / 251 / 259 / 278, 234, 234 / 384, 251, 251 / 399 / 615, 278, 373 / 466, 384 / 569, 399 / 615, 417 / 615, 463 / 565, 466, 546, 569 and 569 / 597, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:1866.In some embodiments, the engineered galactose oxidase comprises at least one substitution or set of substitutions selected from the group consisting of 29V / 149N / 463V / 601L, 29V / 177L / 197P / 592G, 29V / 177L / 463V, 29V / 197P / 592G, 29V / 463V, 62D / 466V, 62D / 466V / 597A, 62 G / 208F / 417L / 615I, 62G / 286C / 615I, 62Q / 373D / 466V, 62Q / 466V, 62Q / 466V / 597A, 149N / 463V, 149R, 149R / 208F / 615I, 177L / 194Q / 197P / 463V / 565S, 177L / 197P / 463V / 565S, 177L / 280N / 463V / 594M / 601L, 177L / 463V / 565S, 177L / 463V / 592G, 184L, 19 7A, 197A / 466V / 569L / 596S, 197P / 280N / 463V, 197P / 463V / 592G, 208F / 251V / 259N / 278L, 234L, 234L / 384N, 251V, 251V / 399V / 615I, 278L, 373D / 466V, 384N / 569L, 399V / 615I, 417L / 615I, 463V / 565S, 466V, 546E, 569L and 569L / 597A, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:1866.In some embodiments, the engineered galactose oxidase comprises at least one substitution or set of substitutions selected from the group consisting of: N29V / A149N / I463V / N601L, N29V / I177L / G197P / P592G, N29V / I177L / I463V, N29V / G197P / P592G, N29V / I463V, T62D / E466V, T62D / E466V / N597A, T62G / W 208F / I417L / L615I, T62G / T286C / L615I, T62Q / Q373D / E466V, T62Q / E466V, T62Q / E466V / N597A, A1 49N / I463V, A149R, A149R / W208F / L615I, I177L / A194Q / G197P / I463V / T565S, I177L / G197P / I463V / T565S, I177L / S280N / I463V / T594M / N601L, I177L / I463V / T565S, I177L / I463V / P592G, V184L, G1 97A, G197A / E466V / I569L / T596S, G197P / S280N / I463V, G197P / I463V / P592G, W208F / T251V / D259N / T278L, M234L, M234L / C384N, T251V, T251V / T399V / L615I, T278L, Q373D / E466V, C384N / I569L, T399V / L615I, I417L / L615I, I463V / T565S, E466V, K546E, I569L and I569L / N597A, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:1866.

[0024] In some embodiments, the engineered galactose oxidase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1912, and wherein the engineered galactose oxidase comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 3, 4, 9, 18, 26, 29, 30, 38, 40, 42, 43, 44, 48, 50, 75, 79, 135, 136, 142, 156, 159, 161, 197, 486, and 601, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 1912. In some embodiments, the engineered galactose oxidase comprises at least one substitution or set of substitutions selected from 3K, 4K, 9L, 18S, 26C, 26H, 26T, 29A, 29M, 29T, 29V, 29Y, 30L, 30N, 30R, 38M, 40P, 42F, 43D, 43G, 43P, 43T, 44H, 48C, 48P, 50D, 50H, 50I, 50T, 50V, 75N, 79A, 79P, 79S, 135D, 136A, 136G, 142C, 142G, 142H, 142S, 142V, 156L, 156T, 159G, 159K, 159S, 161Q, 161V, 197D, 197L, 486A, 486I, 486L, 486P, 486R, 486V and 601L, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:1912. In some embodiments, the engineered galactose oxidase comprises at least one substitution or set of substitutions selected from the group consisting of S3K, A4K, A9L, T18S, N26C, N26H, N26T, N29A, N29M, N29T, N29V, N29Y, K30L, K30N, K30R, T38M, W40P, T42F, Q43D, Q43G, Q43P, Q43T, Y44H, G48C, G48P, P50D, P50H, P50I, P50T, P50V , Q75N, Q79A, Q79P, Q79S, G135D, Q136A, Q136G, A142C, A142G, A142H, A142S, A142V, Q156L, Q156T, L159G, L159K, L159S, R161Q, R161V, P197D, P197L, K486A, K486I, K486L, K486P, K486R, K486V and N601L, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:1912.

[0025] In some embodiments, the engineered galactose oxidase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1912, and wherein the engineered galactose oxidase comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 29, 29 / 30 / 50 / 79 / 136 / 197, 29 / 30 / 50 / 79 / 197 / 407, 29 / 30 / 79 / 136 / 156 / 197, 29 / 30 / 79 / 197 / 407. 197 / 407、29 / 30 / 136 / 197 / 407 / 486、29 / 30 / 136 / 407、29 / 30 / 197、29 / 30 / 197 / 407、29 / 50 / 197 / 407 / 486、29 / 197 / 407、29 / 197 / 407 / 486、30、30 / 50 / 79 / 136 / 156 / 197、43 / 197 / 407、50 / 136 / 197 / 486、65、79、79 / 136 / 197 / 407、79 / 156 / 197 / 407、136、136 / 197 / 407、136 / 197 / 486、156 / 161 / 486、197、197 / 407、197 / 486、279 / 291 / 375 / 420 / 429 / 436 / 453 / 465、279 / 291 / 375 / 465 / 536 / 538、279 / 291 / 465、279 / 291 / 465 / 536、279 / 375 / 420 / 429 / 453 / 465 / 472 / 538、279 / 375 / 420 / 465、279 / 465、291 / 375 / 420 / 430 / 465 / 538、291 / 375 / 436 / 465 / 536 / 538, 486 and 615, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:1912. In some embodiments, the engineered galactose oxidase comprises at least one substitution or set of substitutions selected from: 29A, 29A / 30N / 79E / 136G / 156C / 197D,29A / 30R / 50V / 79S / 136G / 197D、29I、29I / 30N / 79S / 197D、29I / 30N / 79S / 197D / 407D、29I / 30R / 197D / 407D、29S、29S / 30N / 50V / 79S / 197D / 407D、29S / 30R / 79S / 197D、29S / 30R / 79S / 197D / 407D、29S / 30R / 136G / 197D / 407D / 486S、29S / 30R / 136G / 407D、29S / 30R / 197D、29S / 50V / 197D / 407D / 486S、29S / 197D / 407D、29S / 197D / 407D / 486S、29T、30N / 50T / 79S / 136G / 156C / 197D、30R、43D / 197D / 407D、50V / 136G / 197D / 486I、65A、79S、79S / 136G / 197D / 407D、79S / 156C / 197D / 407D、136G、136G / 197D / 407D、136G / 197D / 486I、156M / 161A / 486A、197D、197D / 407D、197D / 486S、279L / 291Y / 375D / 420I / 429V / 436M / 453V / 465G、279L / 291Y / 375D / 465L / 536N / 538D、279L / 291Y / 465L、279L / 291Y / 465L / 536N、279L / 375D / 420I / 429V / 453V / 465L / 472L / 538D、279L / 375D / 420I / 465G、279L / 465G、279L / 465L、291Y / 375D / 420I / 430I / 465G / 538D、291Y / 375D / 436M / 465G / 538D、291Y / 375D / 453V / 465L、291Y / 420I / 465L / 481T / 538D、291Y / 429V / 465L、291Y / 453V / 465L / 536N / 538D、291Y / 465L、291Y / 465L / 538D、375D / 420I / 465G、375D / 420I / 465L、375D / 429V / 453V / 465G、375D / 465G、375D / 465L、420I / 436M / 465L、429V / 465G、453V / 465G、453V / 465G / 478F / 481T、453V / 465L、465G、465G / 536N / 538D、486S and 615I, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:1912. In some embodiments, the engineered galactose oxidase comprises at least one substitution or set of substitutions selected from the group consisting of: N29A, N29A / K30N / Q79E / Q136G / Q156C / P197D, N29A / K30R / P50V / Q79S / Q136G / P197D, N29I, N29I / K30N / Q79S / P197D, N29I / K30N / Q79S / P197D / Q407D, N29I / K30R / P197D / Q407D, N29S, N29S / K30N / P50V / Q79S / P197D / Q407D, N29S / K30R / P50V / Q79S / Q136G / P197D 7D、N29S / K30R / Q79S / P197D / Q407D、N29S / K30R / Q136G / P197D / Q407D / K 486S, N29S / K30R / Q136G / Q407D, N29S / K30R / P197D, N29S / P50V / P197D / Q407D / K486S, N29S / P197D / Q407D, N29S / P197D / Q407D / K486S, N29T, K3 0N / P50T / Q79S / Q136G / Q156C / P197D, K30R, Q43D / P197D / Q407D, P50V / Q1 36G / P197D / K486I, N65A, Q79S, Q79S / Q136G / P197D / Q407D, Q79S / Q156C / P197D / Q407D, Q136G, Q136G / P197D / Q407D, Q136G / P197D / K486I, Q156 M / R161A / K486A, P197D, P197D / Q407D, P197D / K486S, M279L / F291Y / N37 5D / L420I / T429V / L436M / T453V / T465G, M279L / F291Y / N375D / T465L / D53 6N / N538D, M279L / F291Y / T465L, M279L / F291Y / T465L / D536N, M279L / N3 75D / L420I / T429V / T453V / T465L / F472L / N538D, M279L / N375D / L420I / T 465G, M279L / T465G, M279L / T465L, F291Y / N375D / L420I / V430I / T465G / N538D, F291Y / N375D / L436M / T465G / N538D, F291Y / N375D / T453V / T465L,F291Y / L420I / T465L / Q481T / N538D, F291Y / T429V / T465L, F291Y / T453V / T465L / D536N / N538D, F2 91Y / T465L, F291Y / T465L / N538D, N375D / L420I / T465G, N375D / L420I / T465L, N375D / T429V / T453V / T465G, N375D / T465G, N375D / T465L, L420I / L436M / T465L, T429V / T465G, T453V / T465G, T453V / T465G / V478F / Q481T, T453V / T465L, T465G, T465G / D536N / N538D, K486S and L615I, wherein the amino acid positions of the polypeptide sequences are numbered with reference to SEQ ID NO: 1912. 、

[0026] In some embodiments, the engineered galactose oxidase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:2080, and wherein the engineered galactose oxidase comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 24, 47, 63, 78, 95, 119, 121, 197, 207, 214, 219, 220, 249, 294, 324, 365, 408, 414, 437, 480, 485, 520, 556, 571, 598, 600, and 626, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:2080. In some embodiments, the engineered galactose oxidase comprises at least one substitution or set of substitutions selected from the group consisting of 24R, 47D, 47L, 63T, 78L, 95R, 119Q, 121G, 197E, 197G, 197H, 197L, 197M, 197Q, 197R, 197S, 197W, 207Q, 214L, 219V, 220Q, 220R, 249N, 294K, 324G, 365H, 408A, 414L, 437N, 437R, 480L, 485L, 520L, 556S, 571S, 598T, 600D, and 626W, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 2080. In some embodiments, the engineered galactose oxidase comprises at least one substitution or set of substitutions selected from the group consisting of S24R, N47D, N47L, V63T, N78L, V95R, T119Q, P121G, P197E, P197G, P197H, P197L, P197M, P197Q, P197R, P197S, P197W, S207Q , I214L, T219V, S220Q, S220R, K249N, G294K, A324G, D365H, D408A, N414L, L437N, L437R, E480L, Y485L, E520L, V556S, A571S, N598T, G600D and S626W, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:2080.

[0027] In some embodiments, the engineered galactose oxidase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 2080, and wherein the engineered galactose oxidase comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 14 / 130 / 257 / 472, 14 / 257, 24, 29, 29 / 136, 29 / 136 / 197 / 436, 29 / 136 / 436, 29 / 197 / 436, 29 / 136 / 436 / 453, 29 / 197 / 257 / 436, 29 / 136 / 436 / 453. 36 / 453, 29 / 197 / 453, 29 / 436, 29 / 436 / 472, 29 / 453, 29 / 472, 43, 63, 95, 119, 130 / 421, 136, 136 / 197 / 436, 136 / 197 / 436 / 453, 136 / 436, 144, 197, 197 / 436, 197 / 436 / 453, 197 / 436 / 472, 197 / 453, 214, 219, 249, 257, 257 / 472, 297, 359, 436, 437, 460, 485, 495, 520, 556, 560, 567 and 592, wherein the amino acid positions of the polypeptide sequences refer to SEQ ID NO:2080.In some embodiments, the engineered galactose oxidase comprises at least one substitution or set of substitutions selected from the group consisting of: 14K / 257E, 14T / 130M / 257Q / 472L, 14T / 257Q, 24Q, 29I, 29I / 136G, 29I / 136G / 197D / 436M, 29I / 136G / 436M, 29I / 197D, 29I / 197D / 342R / 436M, 29I / 197D / 436M、29I / 197D / 436M / 453T、29I / 197D / 453T、29I / 436M、29S / 136G / 197D / 436M、29S / 136G / 436 M / 453T, 29S / 197D, 29S / 197D / 436M, 29S / 197D / 436M / 453T, 29S / 436M, 29S / 436M / 472L, 29S / 453T , 29S / 472L, 43G, 63E, 63T, 95R, 119M, 130V / 421N, 136G, 136G / 197D / 436M, 136G / 197D / 436M / 453 T, 136G / 436M, 144V, 197D, 197D / 436M, 197D / 436M / 453T, 197D / 436M / 472L, 197D / 453T, 197E, 197 : 197A, 197A, 197B, 197C, 197D, 197E, 197F, 197F, 197G, 197G, 197G, 197G, 197S, 197S, 197G ...In some embodiments, the engineered galactose oxidase comprises at least one substitution or set of substitutions selected from the group consisting of: N14K / S257E, N14T / I130M / S257Q / F472L, N14T / S257Q, S24Q, N29I, N29I / Q136G, N29I / Q136G / P197D / L436M, N29I / Q136G / L436M, N29I / P197D, N29I / P197D / K342R / L436M, N29I / P197D / L436M, 36M, N29I / P197D / L436M / V453T, N29I / P197D / V453T, N29I / L436M, N29S / Q136G / P197D / L436M, N29S / Q136G / L436M / V453T, N29S / P197D, N29S / P197D / L436M, N29S / P197D / L436M / V453T, N29S / L436M, N29S / L436M / F472L, N29S / V453T , N29S / F472L, Q43G, V63E, V63T, V95R, T119M, I130V / G421N, Q136G, Q136G / P197D / L436M, Q136G / P197D / L436M / V4 53T, Q136G / L436M, I144V, P197D, P197D / L436M, P197D / L436M / V453T, P197D / L436M / F472L, P197D / V453T, P197E, P 197G, P197H, P197L, P197M, P197Q, P197R, P197S, P197W, I214A, T219V, K249N, S257A, S257A / F472L, E297T, Y359L, L436M, L437G, L437R, L437Y, Q460G, Y485R, A495T, E520Y, V556S, W560G, W560I, M567S and G592H, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:2080.

[0028] In some embodiments, the engineered galactose oxidase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 2300, and wherein the engineered galactose oxidase comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 24, 24 / 51 / 63 / 197 / 359, 24 / 119 / 197, 24 / 197 / 249. / 437, 43 / 197 / 359, 43 / 249, 63, 63 / 67 / 197 / 571, 63 / 67 / 214 / 556, 63 / 119 / 197, 63 / 119 / 197 / 207 / 214, 63 / 119 / 197 / 339 / 341, 63 / 119 / 197 / 556, 63 / 119 / 197 / 556 / 571, 63 / 119 / 556, 63 / 197, 63 / 197 / 207 / 556, 63 / 197 / 207 / 556 / 571、63 / 197 / 214 / 571、63 / 197 / 249 / 495、63 / 197 / 556 / 571、95 / 197、95 / 219 / 359、119、119 / 197、119 / 197 / 207 / 571、119 / 197 / 214、119 / 197 / 214 / 556、119 / 197 / 214 / 571、119 / 197 / 339、119 / 197 / 556, 119 / 197 / 556 / 571, 119 / 197 / 571, 119 / 207 / 556 / 571, 197, 197 / 207, 197 / 207 / 214 / 471, 197 / 214, 197 / 219, 197 / 339 / 556 / 571, 197 / 556, 197 / 556 / 571, 197 / 571, 214 / 249 / 359, 219 and 556, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:2300. In some embodiments, the engineered galactose oxidase comprises at least one substitution or set of substitutions selected from the group consisting of 24Q, 24Q / 51Q / 63T / 197R / 359L, 24Q / 119M / 197R, 24Q / 197Q / 249N / 437R, 43G / 197R / 359L, 43G / 249N, 63T, 63T / 67K / 1 97S / 571S, 63T / 67K / 214A / 556G, 63T / 119M / 197G / 556G, 63T / 119M / 197L / 207D / 2 14A, 63T / 119M / 197L / 339V / 341R, 63T / 119M / 197S, 63T / 119M / 197S / 556G / 571S,63T / 119M / 556G, 63T / 197G, 63T / 197M / 556G / 571S, 63T / 197R / 249N / 495T, 63T / 197S, 63T / 1 97S / 207D / 556G, 63T / 197S / 207D / 556G / 571S, 63T / 197S / 214A / 571S, 95R / 197R, 95R / 219V / 3 59L, 119M, 119M / 197G / 207D / 571S, 119M / 197G / 214A / 556G, 119M / 197L / 214A / 556G, 119M / 19 7L / 571S, 119M / 197M, 119M / 197M / 214A / 571S, 119M / 197M / 339V, 119M / 197M / 556G, 119M / 197 S, 119M / 197S / 214A, 119M / 197S / 556G, 119M / 197S / 556G / 571S, 119M / 197S / 571S, 119M / 207D / 556G / 571S、197G / 207D / 214A / 471I、197H / 214A、197L、197L / 207D、197L / 556G、197M、197M / 214A, 197M / 339V / 556G / 571S, 197M / 556G / 571S, 197R / 219V, 197S, 197S / 214A, 197S / 219I, 197S / 556G, 197S / 556G / 571S, 197S / 571S, 214A / 249N / 359L, 219V and 556G, wherein the amino acid positions of the polypeptide sequences are numbered with reference to SEQ ID NO:2300. In some embodiments, the engineered galactose oxidase comprises at least one substitution or set of substitutions selected from the group consisting of S24Q, S24Q / K51Q / V63T / D197R / Y359L, S24Q / T119M / D197R, S24Q / D197Q / K249N / L437R, Q43G / D197R / Y359L, Q43G / K249N, V63T, V63T / N67K / D197S / A571S, V63T / N67K / I214A / V571S 56G, V63T / T119M / D197G / V556G, V63T / T119M / D197L / S207D / I214A, V63T / T119M / D197L / F339V / W341R, V63T / T 119M / D197S, V63T / T119M / D197S / V556G / A571S, V63T / T119M / V556G, V63T / D197G, V63T / D197M / V556G / A571S,V63T / D197R / K249N / A495T, V63T / D197S, V63T / D197S / S207D / V556G, V63T / D197S / S207D / V556G / A571 S, V63T / D197S / I214A / A571S, V95R / D197R, V95R / T219V / Y359L, T119M, T119M / D197G / S207D / A571S, T1 19M / D197G / I214A / V556G, T119M / D197L / I214A / V556G, T119M / D197L / A571S, T119M / D197M, T119M / D1 97M / I214A / A571S, T119M / D197M / F339V, T119M / D197M / V556G, T119M / D197S, T119M / D197S / I214A, T11 9M / D197S / V556G, T119M / D197S / V556G / A571S, T119M / D197S / A571S, T119M / S207D / V556G / A571S, D19 7G / S207D / I214A / V471I, D197H / I214A, D197L, D197L / S207D, D197L / V556G, D197M, D197M / I214A, D197 M / F339V / V556G / A571S, D197M / V556G / A571S, D197R / T219V, D197S, D197S / I214A, D197S / T219I, D197S / V556G, D197S / V556G / A571S, D197S / A571S, I214A / K249N / Y359L, T219V and V556G, wherein the amino acid positions of the polypeptide sequences are numbered with reference to SEQ ID NO: 2300. 、

[0029] In some embodiments, the engineered galactose oxidase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:2300, and wherein the engineered galactose oxidase comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 194 / 330 / 495, 196, 246 / 408 / 442 / 462, 246 / 442, 292, 327, 327 / 329, 330, 407, 442, 442 / 462 / 515, 462 / 583, 465, 498, and 583, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:2300. In some embodiments, the engineered galactose oxidase comprises at least one substitution or set of substitutions selected from the group consisting of 194G / 330H / 495S, 196Q, 196R, 246Q / 408N / 442Y / 462A, 246Q / 442Y, 246S / 442Y, 292R, 327K, 327R, 327R / 329W, 330H, 407K, 407R, 442Y, 442Y / 462A / 515M, 462A / 583A, 465R, 498C, 583G, and 583S, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:2300. In some embodiments, the engineered galactose oxidase comprises at least one substitution or set of substitutions selected from the group consisting of A194G / Y330H / A495S, E196Q, E196R, N246Q / D408N / F442Y / G462A, N246Q / F442Y, N246S / F442Y, S292R, Q327K, Q327R, Q327R / L329W, Y330H, Q407K, Q407R, F442Y, F442Y / G462A / L515M, G462A / T583A, L465R, S498C, T583G, and T583S, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:2300.

[0030] In some embodiments, the engineered galactose oxidase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 2424, and wherein the engineered galactose oxidase comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 14, 14 / 24 / 36 / 96, 14 / 24 / 36 / 296 / 424 / 560, 14 / 24 / 78 / 120 / 258, 14 / 24 / 92 / 96 / 99 / 408, 14 / 24 / 96 / 258 / 626, 14 / 24 / 258 / 560, 14 / 24 / 78 / 120 / 258 8 / 120 / 258 / 488 / 560 / 626、14 / 92 / 96 / 99、14 / 92 / 96 / 99 / 120 / 537、14 / 92 / 96 / 120 / 376、14 / 92 / 99 / 120 / 537、14 / 95 / 120 / 296 / 480 / 560、14 / 120 / 480 / 626、14 / 258、14 / 258 / 296 / 560、14 / 376 / 560、14 / 408、23 / 36 / 92 / 95 / 96、23 / 36 / 92 / 95 / 99 / 408 / 596、23 / 36 / 408 / 428、23 / 36 / 537 / 596、23 / 218 / 537、23 / 408 / 596、24、24 / 36 / 46 / 99 / 426 / 532 / 549、24 / 36 / 95 / 96、24 / 36 / 95 / 99 / 404 / 426 / 485、24 / 36 / 96 / 99 / 532 / 549、24 / 36 / 99 / 404 / 426 / 532 / 549 / 600、24 / 36 / 120 / 296 / 480 / 560、24 / 36 / 404 / 426 / 532、24 / 36 / 404 / 480 / 485 / 532 / 560 / 600、24 / 46 / 92 / 404 / 426 / 532、24 / 46 / 92 / 426 / 532、24 / 46 / 92 / 426 / 549、24 / 46 / 95 / 99 / 426 / 532、24 / 46 / 99 / 426 / 549 / 600、24 / 46 / 404 / 426 / 485 / 532、24 / 96 / 404 / 426、24 / 99、24 / 99 / 404 / 485 / 532 / 600、24 / 296、24 / 296 / 324 / 480、24 / 404 / 426 / 532、24 / 404 / 480 / 485、24 / 404 / 480 / 532 / 549 / 560、36、36 / 92 / 95 / 99 / 404 / 426 / 560、36 / 92 / 95 / 428 / 596、36 / 92 / 96 / 408 / 428 / 540 / 596、36 / 92 / 485、36 / 258 / 296、36 / 258 / 296 / 324 / 433 / 626、36 / 404、36 / 404 / 426 / 549 / 600、36 / 408 / 537 / 596、36 / 408 / 596、36 / 426 / 485 / 600、46、46 / 92 / 560、92、92 / 95 / 485 / 532 / 549 / 560、92 / 99 / 218 / 560、95、95 / 120 / 296 / 626、95 / 40 4 / 426 / 532, 96, 96 / 99, 96 / 258 / 560 / 626, 99, 99 / 404 / 426 / 560, 99 / 426 / 480 / 485, 120, 120 / 324 / 480 / 560, 218, 218 / 408, 296 / 324, 296 / 324 / 560, 324 / 560, 404, 404 / 485 / 600, 408, 480, 485, 532, 537, 537 / 640, 549, 549 / 560, 560, 596 and 600, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:2424. In some embodiments, the engineered galactose oxidase comprises at least one substitution or set of substitutions selected from the group consisting of: 14A / 24V / 36P / 96G, 14A / 24V / 36P / 296R / 424W / 560M, 14A / 24V / 78I / 120L / 258V, 14A / 24V / 96G / 258V / 626G, 14A / 24V / 258V / 560M、14A / 78I / 120L / 258V / 488T / 560M / 626G、14A / 95R / 120L / 296R / 480R / 560M、 14A / 120L / 480R / 626G, 14A / 258V, 14A / 258V / 296R / 560M, 14L, 14L / 92V / 96S / 99L, 1 4L / 92V / 96S / 99L / 120S / 537C, 14L / 92V / 96S / 120S / 376M, 14R, 14R / 24P / 92C / 96S / 99L / 408R, 14R / 92V / 99L / 120S / 537C, 14R / 376M / 560I, 14R / 408Q, 23A / 36L / 92C / 95 F / 96G, 23A / 36L / 92C / 95F / 99F / 408L / 596V, 23A / 36L / 408L / 428H, 23A / 36L / 537W / 596Q, 23A / 218G / 537W, 23A / 408L / 596V, 24P, 24P / 36L / 46E / 99V / 426W / 532G / 549L,24P / 36L / 95S / 99V / 404A / 426W / 485C、24P / 36L / 96M / 99V / 532G / 549L、24P / 3 6L / 99V / 404A / 426W / 532G / 549L / 600N、24P / 36L / 404A / 426W / 532G、24P / 36L / 404A / 480L / 485C / 532G / 560E / 600N、24P / 46E / 92G / 404A / 426W / 532G、24P / 4 6E / 92G / 426W / 532G、24P / 46E / 92G / 426W / 549L、24P / 46E / 95S / 99V / 426W / 532 G、24P / 46E / 99V / 426W / 549L / 600N、24P / 46E / 404A / 426W / 485C / 532G、24P / 9 6M / 404A / 426W、24P / 99V、24P / 99V / 404A / 485C / 532G / 600N、24P / 404A / 426W / 532G、24P / 404A / 480L / 485C、24P / 404A / 480L / 532G / 549L / 560E、24V / 36P / 9 5R / 96G、24V / 36P / 120L / 296R / 480R / 560M、24V / 296R、24V / 296R / 324F / 480R、 36L、36L / 92C / 95F / 428H / 596Q、36L / 92C / 96G / 408L / 428H / 540R / 596V、36L / 92G / 95S / 99V / 404A / 426W / 560E、36L / 92G / 485C、36L / 404A、36L / 404A / 426W / 549L / 600N、36L / 408L / 537W / 596Q、36L / 408L / 596Q、36L / 426W / 485C / 600N、 36P / 258V / 296R、36P / 258V / 296R / 324F / 433G / 626G、46P、46P / 92V / 560I、92C ,92G,92G / 95S / 485C / 532G / 549L / 560E,92V,92V / 99L / 218M / 560I,95A,95F,95R / 120L / 296R / 626G,95S / 404A / 426W / 532G,96G,96L / 258V / 560M / 626G,9 6M、96S、96S / 99L、99L、99V、99V / 404A / 426W / 560E、99V / 426W / 480L / 485C、12 0L / 324F / 480R / 560M、120S、218G / 408L、218M、296R / 324F、296R / 324F / 560M、324F / 560M, 404A, 404A / 485C / 600N, 408L, 408Q, 480L, 480R, 485C, 532G, 537C, 537W / 640R, 549L, 549L / 560E, 560I, 596V and 600N, wherein the amino acid positions of the polypeptide sequences are numbered with reference to SEQ ID NO:2424. In some embodiments, the engineered galactose oxidase comprises at least one substitution or set of substitutions selected from the group consisting of: N14A / S24V / K36P / N96G, N14A / S24V / K36P / V296R / G424W / W560M, N14A / S24V / N78I / R120L / S258V, N14A / S24V / N96G / S258V / S626G, N14A / S24V / S258V / W560M, N14A / N78I / R120L / S258V / N488T / W560M / S626G, N14A / S24V / S258V / N488T / W560M / S626G, V95R / R120L / V296R / E480R / W560M, N14A / R120L / E480R / S626G, N14A / S258V, N14A / S258V / V296R / W560M, N14L, N14L / S92V / N96S / S99L、N14L / S92V / N96S / S99L / R120S / S537C、N14L / S92V / N96S / R120S / R376M、N14R、N14R / S24P / S92C / N96S / S99L / D408R、N14R / S92V / S99L / R120S / S537C, N14R / R376M / W560I, N14R / D408Q, Q23A / K36L / S92C / V95F / N96G, Q23A / K36L / S92C / V95F / S99F / D408L / T596V, Q23A / K36L / D408L / N428H, Q23A / K36L / S537W / T596Q, Q23A / R218G / S537W, Q23A / D408L / T596V, S24P, S24P / K36L / V46E / S 99V / S426W / N532G / R549L, S24P / K36L / V95S / S99V / P404A / S426W / Y485C, S24P / K36L / N96M / S99V / N532G / R549L, S24P / K36L / S99 V / P404A / S426W / N532G / R549L / G600N, S24P / K36L / P404A / S426W / N532G, S24P / K36L / P404A / E480L / Y485C / N532G / W560E / G600N,S24P / V46E / S92G / P404A / S426W / N532G、S24P / V46E / S92G / S426W / N532G、S24P / V46E / S92G / S426W / R549L、S24P / V46E / V95S / S99V / S426W / N532G、S24P / V46E / S99V / S426W / R549L / G600N、S24P / V46E / P404A / S426W / Y485C / N532G、S24P / N96M / P404A / S426W、S24P / S99V、S24P / S99V / P404A / Y485C / N532G / G600N、S24P / P404A / S426W / N532G、S24P / P404A / E480L / Y485C、S24P / P404A / E480L / N532G / R549L / W560E、S24V / K36P / V95R / N96G、S24V / K36P / R120L / V296R / E480R / W560M、S24V / V296R、S24V / V296R / A324F / E480R、K36L、K36L / S92C / V95F / N428H / T596Q、K36L / S92C / N96G / D408L / N428H / N540R / T596V、K36L / S92G / V95S / S99V / P404A / S426W / W560E、K36L / S92G / Y485C、K36L / P404A、K36L / P404A / S426W / R549L / G600N、K36L / D408L / S537W / T596Q、K36L / D408L / T596Q、K36L / S426W / Y485C / G600N、K36P / S258V / V296R、K36P / S258V / V296R / A324F / S433G / S626G、V46P、V46P / S92V / W560I、S92C、S92G、S92G / V95S / Y485C / N532G / R549L / W560E、S92V、S92V / S99L / R218M / W560I、V95A、V95F、V95R / R120L / V296R / S626G、V95S / P404A / S426W / N532G、N96G、N96L / S258V / W560M / S626G、N96M、N96S、N96S / S99L、S99L、S99V、S99V / P404A / S426W / W560E、S99V / S426W / E480L / Y485C、R120L / A324F / E480R / W560M、R120S、R218G / D408L、R218M、V296R / A324F, V296R / A324F / W560M, A324F / W560M, P404A, P404A / Y485C / G600N, D408L, D408Q, E480L, E480R, Y485C, N532G, S537C, S537W / Q640R, R549L, R549L / W560E, W560I, T596V and G600N, wherein the amino acid positions of the polypeptide sequences are numbered with reference to SEQ ID NO: 2424.

[0031] In some embodiments, the engineered galactose oxidase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 2424, and wherein the engineered galactose oxidase comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 14, 14 / 24 / 36 / 96, 14 / 24 / 36 / 296 / 424 / 560, 14 / 24 / 78 / 120 / 258, 14 / 24 / 99 / 218 / 408 / 537 / 560, 14 / 24 / 258 / 560, 14 / 46 / 47 / 376, 14 / 46 / 9 6 / 99 / 560、14 / 92 / 96 / 99 / 376 / 560、14 / 92 / 96 / 120 / 376、14 / 92 / 96 / 376、14 / 92 / 99 / 120 / 218 / 408、14 / 92 / 99 / 120 / 537、14 / 92 / 99 / 218 / 408、14 / 92 / 218 / 408、14 / 95 / 120 / 296 / 480 / 560、14 / 376、14 / 376 / 537、14 / 376 / 560、14 / 408、14 / 537、23 / 36、23 / 36 / 92 / 95 / 96、23 / 36 / 92 / 95 / 99 / 408 / 596、23 / 36 / 96 / 408 / 596 / 640、23 / 36 / 408 / 428、23 / 36 / 537 / 540 / 640、23 / 36 / 537 / 596、23 / 218 / 596 / 640、24、24 / 36 / 46 / 99 / 426 / 532 / 549、24 / 36 / 95 / 96、24 / 36 / 95 / 99 / 404 / 426 / 485、24 / 36 / 96 / 99 / 532 / 549、24 / 36 / 120 / 296 / 480 / 560、24 / 36 / 404 / 426 / 532、24 / 36 / 404 / 480 / 485 / 532 / 560 / 600、24 / 46 / 92 / 404 / 4 26 / 532、24 / 46 / 92 / 426 / 532、24 / 46 / 92 / 426 / 549、24 / 46 / 95 / 99 / 426 / 532、24 / 46 / 99 / 426 / 549 / 600、24 / 46 / 404 / 426 / 485 / 532、24 / 96 / 404 / 426、24 / 96 / 404 / 426 / 560、24 / 99、24 / 296、24 / 404 / 426 / 532、24 / 404 / 480 / 485、24 / 532、36、36 / 92 / 95 / 428 / 596、36 / 95 / 96、36 / 99 / 426 / 485 / 600、36 / 258 / 296、36 / 258 / 296 / 324 / 433 / 626、36 / 404、36 / 404 / 426 / 549 / 600、36 / 408、36 / 408 / 537 / 596、36 / 426 / 485 / 600、46 / 92 / 560、78、92、92 / 95 / 485 / 532 / 549 / 560、92 / 96、92 / 99 / 120、92 / 99 / 218 / 560、92 / 218、92 / 404、95、96、96 / 99、99、99 / 426 / 480 / 485、99 / 640、 120, 120 / 324 / 480 / 560, 120 / 376, 218 / 537 / 596, 218 / 596, 258, 296, 296 / 324, 296 / 324 / 560, 296 / 480 / 560, 324, 361, 404, 404 / 426 / 485, 408 / 596, 424, 426 / 485, 426 / 532 / 549, 480, 532, 537 / 640, 549, 549 / 560, 560, 600, 626 and 640, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:2424. In some embodiments, the engineered galactose oxidase comprises at least one substitution or set of substitutions selected from the group consisting of: 14A / 24V / 36P / 96G, 14A / 24V / 36P / 296R / 424W / 560M, 14A / 24V / 78I / 120L / 258V, 14A / 24V / 258V / 560M, 14A / 95R / 120L / 296R / 480R / 5 60M, 14L / 92C / 218M / 408Q, 14L / 92V / 96S / 120S / 376M, 14L / 92V / 99L / 120S / 218M / 408R, 1 4L / 376M, 14L / 376M / 537C, 14L / 376M / 560I, 14R, 14R / 24P / 99L / 218M / 408Q / 537C / 560I, 14R / 46P / 47P / 376M, 14R / 46P / 96S / 99L / 560I, 14R / 92C / 96S / 376M, 14R / 92V / 96S / 99L / 376M / 560I, 14R / 92V / 99L / 120S / 537C, 14R / 92V / 99L / 218M / 408Q, 14R / 408Q, 14R / 537C, 23A / 36L, 23A / 36L / 92C / 95F / 96G, 23A / 36L / 92C / 95F / 99F / 408L / 596V, 23A / 36L / 96G / 40 8L / 596V / 640R, 23A / 36L / 408L / 428H, 23A / 36L / 537W / 540R / 640R, 23A / 36L / 537W / 596Q,23A / 218G / 596Q / 640R、24P、24P / 36L / 46E / 99V / 426W / 532G / 549L、24P / 36L / 95S / 99V / 404A / 426W / 485C、24P / 36L / 96M / 99V / 532G / 549L、24P / 36L / 404A / 426W / 532G、24P / 36L / 404A / 480L / 485C / 532G / 560E / 600N、24P / 46E / 92G / 4 04A / 426W / 532G、24P / 46E / 92G / 426W / 532G、24P / 46E / 92G / 426W / 549L、24P / 46E / 95S / 99V / 426W / 532G、24P / 46E / 99V / 426W / 549L / 600N、24P / 46E / 404A / 426W / 485C / 532G、24P / 96M / 404A / 426W、24P / 96M / 404A / 426W / 560E、24P / 9 9V、24P / 404A / 426W / 532G、24P / 404A / 480L / 485C、24P / 532G、24V、24V / 36P / 95R / 96G、24V / 36P / 120L / 296R / 480R / 560M、24V / 296R、36L、36L / 92C / 95F / 4 28H / 596Q、36L / 95F / 96G、36L / 99V / 426W / 485C / 600N、36L / 404A、36L / 404A / 426W / 549L / 600N、36L / 408L、36L / 408L / 537W / 596Q、36L / 426W / 485C / 600N 、36P / 258V / 296R、36P / 258V / 296R / 324F / 433G / 626G、46P / 92V / 560I、78I、9 2G、92G / 95S / 485C / 532G / 549L / 560E、92G / 404A、92V / 96S、92V / 99L / 120S、9 2V / 99L / 218M / 560I、92V / 218M、95A、95F、96G、96M、96S / 99L、99L / 640R、99V、99V / 426W / 480L / 485C、120L、120L / 324F / 480R / 560M、120S / 376M、218G / 5 37W / 596V、218G / 596Q、258V、296R、296R / 324F、296R / 324F / 560M、296R / 480 R / 560M、324F、361P、404A、404A / 426W / 485C、408L / 596Q、424W、426W / 485C、426W / 532G / 549L, 480L, 480R, 532G, 537W / 640R, 549L, 549L / 560E, 560M, 600N, 626G and 640R, wherein the amino acid positions of the polypeptide sequences are numbered with reference to SEQ ID NO:2424. In some embodiments, the engineered galactose oxidase comprises at least one substitution or set of substitutions selected from the group consisting of: N14A / S24V / K36P / N96G, N14A / S24V / K36P / V296R / G424W / W560M, N14A / S24V / N78I / R120L / S258V, N14A / S24V / S258V / W560M, N14A / V95R / R120L / V296R / E480R / W560M, N14L / S92C / R218M / D408Q, N14L / S92V / N96S / R120S / R376M, N14L / S92V / S99L / R120S / R218M / D408R, N14L / R376M, N14L / R376M / S537C, N14L / R376M / W560I, N14R, N14R / S24P / S99L / R21 8M / D408Q / S537C / W560I, N14R / V46P / N47P / R376M, N14R / V46P / N96S / S99L / W560I, N14R / S92C / N96S / R376M, N14R / S92V / N96S / S99L / R376M / W560I, N14R / S92V / S99L / R120S / S537C, N14R / S92V / S99L / R218M / D408Q, N14R / D408Q, N14R / S537C, Q23A / K36L, Q23A / K36 L / S92C / V95F / N96G, Q23A / K36L / S92C / V95F / S99F / D408L / T596V, Q23A / K36L / N96G / D408L / T596V / Q640R, Q23A / K36L / D408L / N428H , Q23A / K36L / S537W / N540R / Q640R, Q23A / K36L / S537W / T596Q, Q23A / R218G / T596Q / Q640R, S24P, S24P / K36L / V46E / S99V / S426W / N5 32G / R549L, S24P / K36L / V95S / S99V / P404A / S426W / Y485C, S24P / K36L / N96M / S99V / N532G / R549L, S24P / K36L / P404A / S426W / N532G,S24P / K36L / P404A / E480L / Y485C / N532G / W560E / G600N、S24P / V46E / S92G / P404A / S426W / N532G、S24P / V46E / S92G / S426W / N532G、S24P / V46E / S92G / S426W / R549L、S24P / V46E / V95S / S99V / S426W / N532G、S24P / V46E / S99V / S426W / R549L / G600N、S24P / V46E / P404A / S426W / Y485C / N532G、S24P / N96M / P404A / S426W、S24P / N96M / P404A / S426W / W560E、S24P / S99V、S24P / P404A / S426W / N532G、S24P / P404A / E480L / Y485C、S24P / N532G、S24V、S24V / K36P / V95R / N96G、S24V / K36P / R120L / V296R / E480R / W560M、S24V / V296R、K36L、K36L / S92C / V95F / N428H / T596Q、K36L / V95F / N96G、K36L / S99V / S426W / Y485C / G600N、K36L / P404A、K36L / P404A / S426W / R549L / G600N、K36L / D408L、K36L / D408L / S537W / T596Q、K36L / S426W / Y485C / G600N、K36P / S258V / V296R、K36P / S258V / V296R / A324F / S433G / S626G、V46P / S92V / W560I、N78I、S92G、S92G / V95S / Y485C / N532G / R549L / W560E、S92G / P404A、S92V / N96S、S92V / S99L / R120S、S92V / S99L / R218M / W560I、S92V / R218M、V95A、V95F、N96G、N96M、N96S / S99L、S99L / Q640R、S99V、S99V / S426W / E480L / Y485C、R120L、R120L / A324F / E480R / W560M、R120S / R376M、R218G / S537W / T596V、R218G / T596Q、S258V、V296R、V296R / A324F、V296R / A324F / W560M、V296R / E480R / W560M、A324F、S361P、P404A、P404A / S426W / Y485C, D408L / T596Q, G424W, S426W / Y485C, S426W / N532G / R549L, E480L, E480R, N532G, S537W / Q640R, R549L, R549L / W560E, W560M, G600N, S626G and Q640R, wherein the amino acid positions of the polypeptide sequences are numbered with reference to SEQ ID NO: 2424.

[0032] In yet other embodiments, the engineered galactose oxidase comprises a polypeptide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence of at least one engineered galactose oxidase variant listed in Table 4.1, Table 5.1, Table 6.1, Table 7.1, Table 11.1, Table 12.1, Table 13.1, Table 14.1, Table 14.2, Table 15.1, Table 16.1, Table 17.1, Table 18.1, Table 19.1, Table 22.1, Table 23.1, Table 25.1, Table 26.1, Table 27.1, Table 28.1, Table 29.2, Table 30.1 and / or Table 31.1. In yet other embodiments, the engineered galactose oxidase comprises a polypeptide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence of at least one engineered galactose oxidase variant set forth in SEQ ID NO: 2, 4, 166, 272, 928, 932, 1264, 1416, 1598, 1866, 1912, 2080, 2300 and / or 2424. In some embodiments, the engineered galactose oxidase is a variant engineered polypeptide set forth in SEQ ID NO: 2, 4, 166, 272, 928, 932, 1264, 1416, 1598, 1866, 1912, 2080, 2300 and / or 2424. In some further embodiments, the engineered galactose oxidase comprises a polypeptide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence of at least one engineered galactose oxidase variant listed in the even-numbered sequences in SEQ ID NO: 2-2860. In yet some further embodiments, the engineered galactose oxidase comprises a polypeptide sequence listed in the even-numbered sequences in SEQ ID NO: 2-2860. In some further embodiments, the engineered galactose oxidase comprises at least one improved property compared to the wild-type F. graminearium galactose oxidase. In some embodiments, the improved properties include improved activity on substrates. In some further embodiments, the substrates include primary alcohols. In some further embodiments, the improved properties include improved stereoselectivity. In yet other embodiments, the engineered galactose oxidase is purified.

[0033] The present invention also provides compositions comprising at least one engineered galactose oxidase provided herein. In some embodiments, the composition comprises one engineered galactose oxidase provided herein.

[0034] The present invention also provides polynucleotide sequences encoding the engineered galactose oxidase provided herein. In some embodiments, the polynucleotide sequence encodes more than one engineered galactose oxidase provided herein. The present invention also provides polynucleotide sequences encoding at least one engineered galactose oxidase, wherein the polynucleotide sequence comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 1, 3, 165, 271, 927, 931, 1263, 1415, 1597, 1865, 1911, 2079, 2299 and / or 2423, wherein the polynucleotide sequence of the engineered galactose oxidase comprises at least one substitution at one or more positions. In some additional embodiments, the polynucleotide sequence encoding at least one engineered galactose oxidase or a functional fragment thereof comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1, 3, 165, 271, 927, 931, 1263, 1415, 1597, 1865, 1911, 2079, 2299 and / or 2423. In some additional embodiments, the polynucleotide sequence is operably linked to a control sequence. In some additional embodiments, the polynucleotide sequence is codon optimized. In yet other embodiments, the polynucleotide comprises an odd-numbered sequence in SEQ ID NO: 1-2859. The present invention also provides an expression vector comprising at least one polynucleotide sequence encoding at least one galactose oxidase provided herein. The present invention also provides a host cell comprising at least one expression vector provided herein. The present invention also provides a host cell comprising at least one polynucleotide sequence encoding at least one galactose oxidase provided herein.

[0035] The present invention also provides a method for producing an engineered galactose oxidase in a host cell, the method comprising culturing the host cell under suitable conditions, thereby producing at least one engineered galactose oxidase provided herein. In some embodiments, the method further comprises recovering at least one engineered galactose oxidase from the culture and / or host cell. In some other embodiments, the method further comprises the step of purifying at least one engineered galactose oxidase provided herein.

[0036] Description of the invention

[0037] The present invention provides engineered galactose oxidase (GO enzyme), polypeptides having selective oxidation activity for primary alcohols (e.g., 2-ethynylglycerol), and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. These GO enzyme variants act in a selective manner, minimizing the need for functional group protection operations for non-target alcohols, and providing desired aldehyde stereoisomers (e.g., R-enantiomers). Methods for producing GO enzymes are also provided. The present invention also provides compositions comprising GO enzymes, and methods for using engineered GO enzymes. The present invention is particularly useful for the production of pharmaceutical compounds and other compounds.

[0038] 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 are 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.

[0039] 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.

[0040] 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.

[0041] abbreviation

[0042] Abbreviations for the genetically encoded amino acids are conventional and are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamic acid (Glu or E), glutamine (Gln or Q), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

[0043] When three-letter abbreviations are used, unless specifically preceded by "L" or "D", or it is clear from the context in which the abbreviation is used, an amino acid can be in the L-configuration or the D-configuration about the α-carbon (Cα). 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. 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 a mixture thereof), the sequence is presented as an amino (N) to carboxyl (C) direction according to conventional practice.

[0044] 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.

[0045] definition

[0046] 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 specifically defined otherwise. Accordingly, the following terms are intended to have the following meanings.

[0047] As used herein, 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.

[0048] Similarly, "comprise, comprises, comprising," "include, includes, and including" are interchangeable and are not intended to be limiting. Thus, as used herein, the term "comprising" and its cognates are used in their inclusive sense (i.e., equivalent to the term "including" and its corresponding cognates).

[0049] It should also be understood that where the term "comprising" is used in the description of various embodiments, those skilled in the art will understand that in some specific instances, the embodiments may alternatively be described using the language "consisting essentially of" or "consisting of."

[0050] As used herein, the term "about" means an acceptable error for a particular value. In some instances, "about" means within 0.05%, 0.5%, 1.0%, or 2.0% of a given value. In some instances, "about" means within 1, 2, 3, or 4 standard deviations of a given value.

[0051] As used herein, "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 they catalyze.

[0052] As used herein, "ATCC" refers to the American Type Culture Collection, whose biological deposit collections include genes and strains.

[0053] As used herein, "NCBI" refers to the National Center for Biological Information and the sequence databases provided therein.

[0054] As used herein, "galactose oxidase" ("GO enzymes"; EC 1.1.3.9) enzymes are copper-dependent enzymes that catalyze the oxidation of primary alcohols to the corresponding aldehydes in the presence of dimolecular oxygen. They act selectively in both a regio- and enantio-specific manner, resulting in synthetic methods that require little or no functional group protection and produce the desired stereoisomers. The oxidation is mild and controllable, so the activity does not lead to over-oxidation of the alcohol to its corresponding carboxylic acid.

[0055] As used herein, "horseradish peroxidase" (HRP, EC 1.11.1.7) enzymes are iron-dependent enzymes that activate and maintain GO enzyme catalytic activity by oxidizing the inactive redox state of the active site that occurs during the normal GO enzyme catalytic cycle. In the examples included herein, Type I HRP is specifically used in a catalytic manner, however this does not mean that it is unique in this role, as there are other isoforms of this enzyme class and chemical reagents that can perform this role.

[0056] As used herein, "catalase" refers to an iron-dependent enzyme (EC1.11.1.6) that acts on hydrogen peroxide, which is a byproduct of GO enzyme oxidation and can inactivate the GO enzyme when hydrogen peroxide exceeds a certain level. In the examples herein, catalase is specifically used as a catalytic maintenance enzyme, and in some embodiments, it can be replaced by other methods, such as by electrochemical decomposition of hydrogen peroxide.

[0057] "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.

[0058] As used herein, "hydrophilic amino acids or residues" refer to amino acids or residues having side chains that exhibit a hydrophobicity less than zero according to the normalized consensus hydrophobicity scale of Eisenberg et al. (Eisenberg et al., J. Mol. Biol., 179: 125-142

[1984] ). Genetically encoded hydrophilic amino acids include L-Thr (T), L-Ser (S), L-His (H), L-Glu (E), L-Asn (N), L-Gln (Q), L-Asp (D), L-Lys (K), and L-Arg (R).

[0059] As used herein, "acidic amino acids or residues" refer to hydrophilic amino acids or residues having side chains that exhibit a pKa value of less than about 6 when the amino acid is contained in a peptide or polypeptide. Acidic amino acids typically have negatively charged side chains at physiological pH due to the loss of hydrogen ions. Genetically encoded acidic amino acids include L-Glu (E) and L-Asp (D).

[0060] As used herein, "basic amino acid or residue" refers to a hydrophilic amino acid or residue having a side chain that exhibits a pKa value greater than about 6 when the amino acid is contained in a peptide or polypeptide. Basic amino acids generally have positively charged side chains at physiological pH due to association with hydronium ions. Genetically encoded basic amino acids include L-Arg (R) and L-Lys (K).

[0061] As used herein, "polar amino acid or residue" refers to a hydrophilic amino acid or residue having a side chain that is uncharged at physiological pH but has at least one bond in which a pair of electrons shared by two atoms is held more closely by one of the atoms. Genetically encoded polar amino acids include L-Asn (N), L-Gln (Q), L-Ser (S), and L-Thr (T).

[0062] As used herein, "hydrophobic amino acid or residue" refers to an amino acid or residue having a side chain that exhibits a hydrophobicity greater than zero according to the normalized consensus hydrophobicity scale of Eisenberg et al. (Eisenberg et al., J. Mol. Biol., 179: 125-142

[1984] ). Genetically encoded hydrophobic amino acids include L-Pro (P), L-Ile (I), L-Phe (F), L-Val (V), L-Leu (L), L-Trp (W), L-Met (M), L-Ala (A), and L-Tyr (Y).

[0063] As used herein, "aromatic amino acid or residue" refers to a hydrophilic or hydrophobic amino acid or residue having a side chain that includes at least one aromatic or heteroaromatic ring. Genetically encoded aromatic amino acids include L-Phe (F), L-Tyr (Y), and L-Trp (W). Although L-His (H) is sometimes classified as a basic residue due to the pKa of its heteroaromatic nitrogen atom, or as an aromatic residue because its side chain includes a heteroaromatic ring, histidine is classified as a hydrophilic residue or as a "constrained residue" (see below) herein.

[0064] As used herein, "constrained amino acid or residue" refers to an amino acid or residue with constrained geometry. Herein, constrained residues include L-Pro (P) and L-His (H). Histidine has a constrained geometry because it has a relatively small imidazole ring. Proline has a constrained geometry because it also has a five-membered ring.

[0065] As used herein, "non-polar amino acid or residue" refers to a hydrophobic amino acid or residue having a side chain that is uncharged at physiological pH and has a bond in which an electron pair shared by two atoms is usually equally held by each of the two atoms (i.e., the side chain is not polar). Genetically encoded non-polar amino acids include L-Gly (G), L-Leu (L), L-Val (V), L-Ile (I), L-Met (M), and L-Ala (A).

[0066] As used herein, "aliphatic amino acid or residue" refers to a hydrophobic amino acid or residue with an aliphatic hydrocarbon side chain. Genetically encoded aliphatic amino acids include L-Ala (A), L-Val (V), L-Leu (L) and L-Ile (I). It is worth noting that cysteine ​​(or "L-Cys" or "[C]") is unusual because it can form disulfide bridges with other L-Cys (C) amino acids or other sulfonyl or sulfhydryl-containing amino acids. "Cysteine-like residues" include cysteine ​​and other amino acids containing sulfhydryl moieties that can be used to form disulfide bridges. The ability of L-Cys (C) (and other amino acids with -SH side chains) to be present in a peptide in a reduced free-SH or oxidized disulfide-bridged form affects whether L-Cys (C) contributes a net hydrophobic or hydrophilic character to the peptide. Although L-Cys(C) exhibits a hydrophobicity of 0.29 according to Eisenberg's normalized consensus scale (Eisenberg et al., 1984, supra), it is understood that for the purposes of the present disclosure, L-Cys(C) is classified into its own unique group.

[0067] As used herein, "small amino acid or residue" refers to an amino acid or residue having a side chain comprising a total of three or fewer carbons and / or heteroatoms (excluding α-carbon and hydrogen). According to the above definition, small amino acids or residues can be further classified as aliphatic, non-polar, polar or acidic small amino acids or residues. Genetically encoded small amino acids include L-Ala (A), L-Val (V), L-Cys (C), L-Asn (N), L-Ser (S), L-Thr (T) and L-Asp (D).

[0068] As used herein, "hydroxyl-containing amino acid or residue" refers to an amino acid containing a hydroxyl (-OH) moiety. Genetically encoded hydroxyl-containing amino acids include L-Ser (S), L-Thr (T), and L-Tyr (Y).

[0069] As used herein, "polynucleotide" and "nucleic acid" refer to two or more nucleotides covalently linked together. A polynucleotide may be completely comprised of ribonucleotides (i.e., RNA), completely comprised of 2'deoxyribonucleotides (i.e., DNA), or a mixture of ribonucleotides and 2'deoxyribonucleotides. Although nucleosides are typically linked together via standard phosphodiester linkages, a polynucleotide may include one or more non-standard linkages. A polynucleotide may be single-stranded or double-stranded, or may include both single-stranded and double-stranded regions. In addition, although a polynucleotide typically comprises naturally occurring coding nucleobases (i.e., adenine, guanine, uracil, thymine, and cytosine), it may comprise one or more modified and / or synthesized nucleobases, such as, for example, inosine, xanthine, hypoxanthine, etc. In some embodiments, such modified or synthesized nucleobases are nucleobases encoding amino acid sequences.

[0070] As used herein, "coding sequence" refers to the portion of a nucleic acid (eg, a gene) that encodes the amino acid sequence of a protein.

[0071] As used herein, the terms "biocatalysis," "biocatalytic," "bioconversion," and "biosynthesis" refer to the use of enzymes to perform chemical reactions on organic compounds.

[0072] As used herein, "wild-type" and "naturally occurring" refer to the form found in nature. For example, a wild-type polypeptide or polynucleotide sequence is a sequence present in an organism that can be isolated from a natural source and has not been intentionally modified by human manipulation.

[0073] As used herein, "recombinant," "engineered," "variant," 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 exist in nature or to materials that correspond to the native or native form of the 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 through manipulation using 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.

[0074] The term "percentage (%) of sequence identity" is used herein to refer to the comparison between polynucleotides or polypeptides, and is determined by comparing two optimally aligned sequences in 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 optimal alignment of the two sequences. The percentage can be calculated as follows: determine 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, divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the percentage of sequence identity. Alternatively, the percentage can be calculated as follows: determine 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, divide the number of matching positions by the total number of positions in the comparison window, and multiply 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 align two sequences. Optimal alignment of sequences for comparison can be performed by any suitable method, including, but not limited to, 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 similarity search 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, 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 positive threshold score T when aligned with a word of the same length in the database sequence. T is referred to as the neighborhood word score threshold (see, 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 mismatched 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 drops by an amount X from its maximum achieved value; the cumulative score reaches 0 or less than 0 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.

[0075] As used herein, "reference sequence" refers to a determined sequence used as a basis for sequence and / or activity 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 a length of at least 20 nucleotides or amino acid residues, a length of at least 25 residues, a length of at least 50 residues, a length of at least 100 residues, or the total length of a nucleic acid or polypeptide. Because two polynucleotides or polypeptides can each (1) include a similar sequence (i.e., a part of a complete sequence) between the two sequences, and (2) can also include a different (divergent) sequence between 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 in a "comparison window" to identify and compare the sequence similarity of a local region. In some embodiments, a "reference sequence" can be based on a primary amino acid sequence, wherein a reference sequence is a sequence that can have one or more variations in a primary sequence.

[0076] As used herein, "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 with a reference sequence of at least 20 consecutive nucleotides or amino acids, and wherein the portion of the sequence in the comparison window can include 20% or less additions or deletions (i.e., gaps) compared to the reference sequence (which does not include additions or deletions) for optimal alignment of the two sequences. The comparison window can be longer than 20 consecutive residues, and optionally includes a window of 30, 40, 50, 100 or more.

[0077] As used herein, "corresponding to," "referenced to," or "relative to," when used in the context of numbering a given amino acid or polynucleotide sequence, means that the residues of a given reference sequence are numbered when the given amino acid or polynucleotide sequence is compared to the 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 position of the residues within the given amino acid or polynucleotide sequence. For example, given an amino acid sequence, such as an engineered galactose oxidase, the residue matching between the two sequences can be optimized by introducing gaps to align with the reference sequence. In these cases, the residues in the given amino acid or polynucleotide sequence are numbered with respect to the reference sequence to which it is aligned, despite the presence of the gaps.

[0078] 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.

[0079] As used herein, "amino acid difference" and "residue difference" refer to the difference of the amino acid residue at one position of the polypeptide sequence relative to the amino acid residue at the corresponding position in the reference sequence. In some cases, the reference sequence has a histidine tag, but the numbering remains unchanged relative to the equivalent reference sequence without a histidine tag. 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 X93 compared to SEQ ID NO:4" refers to the difference of the amino acid residue at the polypeptide position corresponding to position 93 of SEQ ID NO:4. Therefore, if the reference polypeptide of SEQ ID NO:4 has serine at position 93, "the residue difference at position X93 compared to SEQ ID NO:4" refers to the amino acid substitution of any residue except serine at the polypeptide position corresponding to position 93 of SEQ ID NO:4. 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 examples (e.g., in the tables presented in the Examples), 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 a residue position in a reference sequence, and B is a single-letter identifier for a residue substitution in the sequence of an engineered polypeptide. In some examples, a polypeptide of the present invention may comprise one or more amino acid residue differences relative to a reference sequence, indicated by a column of designated positions where residue differences exist relative to a reference sequence. In some embodiments, when more than one amino acid can be used in a specific residue position of a polypeptide, the various amino acid residues that can be used are separated by " / " (e.g., X307H / X307P or X307H / P). Slashes can also be used to indicate more than one substitution within a given variant (i.e., there is more than one substitution in a given sequence such as in a combinatorial variant). In some embodiments, the present invention includes engineered polypeptide sequences containing one or more amino acid differences, the amino acid differences comprising conservative amino acid substitutions or non-conservative amino acid substitutions. In some additional embodiments, the present invention provides engineered polypeptide sequences comprising both conservative amino acid substitutions and non-conservative amino acid substitutions.

[0080] As used herein, "conservative amino acid substitutions" refer to the replacement of a residue with a different residue having a similar side chain, and thus generally include the replacement of an amino acid in a polypeptide with an amino acid in the same or similar amino acid defined class. For example, but not limited to, in some embodiments, 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.

[0081] As used herein, "non-conservative substitution" refers to the substitution of an amino acid in a polypeptide with an amino acid having significantly different side chain properties. Non-conservative substitutions may use amino acids between, rather than within, the defined groups and affect (a) the structure of the peptide backbone in the area of ​​the substitution (e.g., proline for glycine), (b) charge or hydrophobicity, or (c) side chain bulk. 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.

[0082] As used herein, "deletion" refers to the modification of a polypeptide by removing one or more amino acids from a reference polypeptide. Deletion can include the 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 number of amino acids that make up the reference enzyme, or up to 20% of the total number of amino acids, while retaining enzymatic activity and / or retaining the improved properties of the engineered galactose oxidase. Deletion can involve internal portions and / or terminal portions of a polypeptide. In various embodiments, deletion can include continuous segments or can be discontinuous. Deletions in amino acid sequences are generally represented by "-".

[0083] As used herein, "insertion" refers to the modification of a polypeptide by adding one or more amino acids to a reference polypeptide. The insertion may be in an internal portion of the polypeptide or to the carboxyl or amino terminus. Insertions as used herein include fusion proteins as known in the art. Insertions may be continuous stretches of amino acids or separated by one or more amino acids in a naturally occurring polypeptide.

[0084] The term "amino acid substitution set" or "substitution set" refers to a set of amino acid substitutions in a polypeptide sequence compared to a reference sequence. A substitution set can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions. In some embodiments, a substitution set refers to a collection of amino acid substitutions present in any of the variant galactose oxidases listed in the tables provided in the Examples.

[0085] "Functional fragment" and "biologically active fragment" are used interchangeably herein and refer to polypeptides having an amino-terminal deletion and / or a carboxyl-terminal deletion and / or an internal deletion, but wherein the remaining amino acid sequence is identical to the corresponding positions in the sequence to which it is compared (e.g., the full-length engineered galactose oxidase of the present invention) and retains substantially all of the activity of the full-length polypeptide.

[0086] As used herein, "isolated polypeptide" refers to a polypeptide that is substantially separated from other contaminants (e.g., 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., within a host cell or via in vitro synthesis). The recombinant galactose oxidase polypeptide can be present in a cell, in a cell culture medium, or prepared in various forms (such as a lysate or an isolated preparation). Therefore, in some embodiments, the recombinant galactose oxidase polypeptide can be an isolated polypeptide.

[0087] As used herein, "substantially pure polypeptide" or "purified protein" refers to a composition in which the polypeptide species is the predominant species present (i.e., it is more abundant than any other individual macromolecular species in the composition on a molar or weight basis), and when the target species constitutes at least about 50% of the macromolecular species present by mole or % weight, it is generally a substantially purified composition. However, in some embodiments, the composition comprising galactose oxidase comprises less than 50% pure (e.g., about 10%, about 20%, about 30%, about 40%, or about 50%) galactose oxidase. Typically, a substantially pure galactose oxidase composition constitutes 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 species is purified to substantial homogeneity (i.e., contaminant species cannot be detected in the composition by conventional detection methods), wherein the composition consists essentially of a single macromolecular species. Solvent species, small molecules (<500 Daltons), and elemental ionic species are not considered macromolecular species.In some embodiments, the isolated recombinant galactose oxidase polypeptide is a substantially pure polypeptide composition.

[0088] As used herein, "improved enzyme properties" refers to at least one improved property of an enzyme. In some embodiments, the invention provides engineered galactose oxidase polypeptides that exhibit improvements in any enzyme property compared to a reference galactose oxidase polypeptide and / or a wild-type galactose oxidase polypeptide and / or another engineered galactose oxidase polypeptide. Thus, the level of "improvement" between various galactose oxidase polypeptides, including wild-type and engineered galactose oxidases, can be determined and compared. Improved properties include, but are not limited to, properties such as increased protein expression, increased thermoactivity, increased thermostability, increased pH activity, increased stability, increased enzyme activity, increased substrate specificity or affinity, increased specific activity, increased resistance to substrate or end product inhibition, increased chemical stability, improved chemical selectivity, improved solvent stability, increased tolerance to acidic pH, increased tolerance to proteolytic activity (i.e., reduced sensitivity to proteolysis), reduced aggregation, increased solubility, and altered temperature profiles. In other embodiments, the term is used to refer to at least one improved property of a galactose oxidase. In some embodiments, the invention provides engineered galactose oxidase polypeptides that exhibit improvements in any enzyme property compared to a reference galactose oxidase polypeptide and / or a wild-type galactose oxidase polypeptide and / or another engineered galactose oxidase polypeptide. Thus, the level of "improvement" between various galactose oxidase polypeptides, including wild-type and engineered galactose oxidases, can be determined and compared.

[0089] As used herein, "increased enzyme activity" and "enhanced catalytic activity" refer to improved properties of engineered polypeptides, which can be expressed as an increase in specific activity (e.g., product produced / time / weight protein) or an increase in the percentage of conversion of substrate to product (e.g., using a specified amount of enzyme in a specified time period, the percentage of conversion of a starting amount of substrate to product) compared to a reference enzyme. In some embodiments, the term refers to improved properties of engineered galactose oxidase polypeptides provided herein, which can be expressed as an increase in specific activity (e.g., product produced / time / weight protein) or an increase in the percentage of conversion of substrate to product (e.g., using a specified amount of galactose oxidase, the percentage of conversion of a starting amount of substrate to product in a specified time period) compared to a reference galactose oxidase. In some embodiments, these terms are used to refer to improved galactose oxidases provided herein. Exemplary methods for determining the enzymatic activity of engineered galactose oxidases of the present invention are provided in the Examples. Any property associated with enzyme activity can be affected, including typical enzyme properties. m 、V max or k cat, their alterations can result in increased enzymatic activity. For example, the improvement in enzymatic activity can be from about 1.1 times the enzymatic activity of the corresponding wild-type enzyme to up to 2 times, 5 times, 10 times, 20 times, 25 times, 50 times, 75 times, 100 times, 150 times, 200 times or more enzymatic activity compared to the naturally occurring galactose oxidase or another engineered galactose oxidase from which the galactose oxidase polypeptide is derived.

[0090] As used herein, "conversion" refers to the enzymatic conversion (or bioconversion) of one or more substrates into one or more corresponding products. "Percent conversion" refers to the percentage of substrates that are converted into products under specified conditions within a certain period of time. Therefore, the "enzyme activity" or "activity" of a galactose oxidase polypeptide can be expressed as the "percent conversion" of substrates into products within a specific period of time.

[0091] An enzyme with "generalist properties" (or "generalist enzymes") refers to an enzyme that exhibits improved activity on a wide range of substrates compared to the parent sequence. A generalist enzyme need not exhibit improved activity for every possible substrate. In some embodiments, the invention provides galactose oxidase variants with generalist properties in that they exhibit similar or improved activity on a wide range of sterically and electronically different substrates relative to the parent gene. In addition, the generalist enzymes provided herein are engineered to be improved across a wide range of differentiated molecules to increase the production of metabolites / products.

[0092] 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 a chaotropic agent. mValues ​​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., Nucl. Acids Res., 18:6409-6412

[1990] (erratum, Nucl. Acids Res., 26:6413 ... Res., 19:698

[1991] ); Sambrook et al., supra); Suggs et al., 1981, supra Developmental Biology Using Purified Genes In some embodiments, the polynucleotide encodes a polypeptide disclosed herein and hybridizes to the complement of a sequence encoding an engineered galactose oxidase of the invention under defined conditions, such as moderately stringent or highly stringent conditions.

[0093] As used herein, "hybridization stringency" refers to hybridization conditions, such as washing conditions, in nucleic acid hybridization. Typically, hybridization reactions are performed under conditions of lower stringency, followed by washings 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. mConditions 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 hybridization under conditions equivalent to 50% formamide, 5×Denhart solution, 5×SSPE, 0.2%SDS at 42°C, followed by washing in 0.1×SSPE and 0.1%SDS at 65°C. Another high stringency condition is hybridization under conditions equivalent to hybridization at 65°C in 5X SSC containing 0.1% (w / v) SDS and washing at 65°C in 0.1×SSC containing 0.1% SDS. Other high stringency hybridization conditions as well as medium stringency conditions are described in the references cited above.

[0094] As used herein, "codon optimized" refers to that the codons of the polynucleotide encoding the protein are changed to those codons that are 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. This codon usage bias may be higher for a given gene, a gene with a common function or ancestral origin, a high-expression protein versus a low-copy number protein, and an aggregated protein coding region of the genome of an organism. In some embodiments, codon optimization can be performed on the polynucleotide encoding galactose oxidase for optimized production in a host organism selected for expression.

[0095] As used herein, "preferred," "optimal," and "high codon usage bias" codons, when used alone or in combination, refer interchangeably to codons in a protein coding region that are used at a higher frequency 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 frequency in aggregating protein coding regions of an entire organism, codon frequency in aggregating protein coding regions of related organisms, or a combination thereof. Codons whose frequency increases with the level of gene expression are generally the optimal codons for expression. Various methods are known for determining codon frequency (e.g., codon usage, relative synonymous codon usage) and codon preference and the effective number of codons used in a gene in a particular organism, including multivariate analysis, such as using cluster analysis or correlation analysis (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] ; and Wright, Gene 87:23-29

[1990] ). Codon usage tables for many different organisms are available (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, supra). Escherichia coli and salmonella In, Neidhardt et al. (eds.), ASM Press, Washington DC, p. 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] ).

[0096] As used herein, "control sequences" 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, promoter sequences, signal peptide sequences, start sequences, and transcription terminators. At a minimum, control sequences include promoters and transcription and translation termination signals. Control sequences may be provided with joints 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.

[0097] "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.

[0098] "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.

[0099] The phrase "suitable reaction conditions" refers to those conditions in the enzymatic conversion reaction solution (e.g., ranges of enzyme loading, substrate loading, temperature, pH, buffer, cosolvents, etc.) under which the galactose oxidase polypeptide of the invention is able to convert the substrate into a desired product compound. Some exemplary "suitable reaction conditions" are provided herein.

[0100] As used herein, "loading," such as in "compound loading" or "enzyme loading," refers to the concentration or amount of a component in the reaction mixture at the start of the reaction.

[0101] As used herein, in the context of an enzymatic conversion reaction process, "substrate" refers to a compound or molecule that is acted upon by an engineered enzyme (eg, an engineered galactose oxidase polypeptide) provided herein.

[0102] As used herein, an "increased" yield of a product produced by a reaction (e.g., the R-enantiomer of 3-ethynylglyceraldehyde [EGA]) occurs when the presence of a particular component (e.g., galactose oxidase) during the reaction results in the production of more product compared to a reaction conducted under the same conditions with the same substrate and other substituents, but in the absence of the component of interest.

[0103] A reaction is said to be "substantially free" of a particular enzyme if the amount of that enzyme is less than about 2%, about 1%, or about 0.1% (wt / wt) compared to other enzymes involved in catalyzing the reaction.

[0104] As used herein, "fractionating" a liquid (e.g., a culture broth) means applying a separation process (e.g., salt precipitation, column chromatography, size exclusion, and filtration), or a combination of such processes, to provide a solution in which the desired protein is present as a percentage of the total protein in the solution that is greater than the percentage in the initial liquid product.

[0105] As used herein, "starting composition" refers to any composition comprising at least one substrate. In some embodiments, the starting composition comprises any suitable substrate.

[0106] As used herein, in the context of an enzymatic conversion process, "product" refers to the compound or molecule resulting from the action of the enzyme polypeptide on the substrate.

[0107] As used herein, "equilibrium" as used herein refers to the process of producing a steady-state concentration of a chemical species in a chemical or enzymatic reaction (e.g., the interconversion of two species A and B), including the interconversion of stereoisomers, as determined by the forward rate constant and reverse rate constant of the chemical or enzymatic reaction.

[0108] As used herein, "cofactor" refers to a non-protein compound that acts in conjunction with an enzyme in catalyzing a reaction.

[0109] As used herein, "alkyl" refers to a saturated hydrocarbon group having 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-C4) alkyl refers to an alkyl group of 1 to 4 carbon atoms).

[0110] As used herein, "alkenyl" refers to a group having 2 to 12 carbon atoms (inclusive), linear or branched, containing at least one double bond but optionally containing more than one double bond.

[0111] As used herein, "alkynyl" refers to a group having 2 to 12 carbon atoms (inclusive), straight or branched, containing at least one triple bond but optionally containing more than one triple bond, and further optionally containing one or more double bonded moieties.

[0112] As used herein, "heteroalkyl", "heteroalkenyl" and "heteroalkynyl" refer to alkyl, alkenyl and alkynyl groups 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 α -, -PH-, -S(O)-, -S(O)2-, -S(O)NR α -、-S(O)2NR α - etc., including combinations thereof, where each R α are independently selected from hydrogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl.

[0113] As used herein, "alkoxy" refers to the group -OR β , where R β is an alkyl group as defined above, including optionally substituted alkyl groups also as defined herein.

[0114] As used herein, "aryl" refers to an unsaturated aromatic carbocyclic group having 6 to 12 carbon atoms (including endpoints) with a single ring (e.g., phenyl) or more than one condensed ring (e.g., naphthyl or anthracenyl). Exemplary aryl groups include phenyl, pyridyl, naphthyl, etc.

[0115] As used herein, "amino" refers to the group -NH2. Substituted amino refers to the group -NHR δ NR δ R δ and NR δ R δ R δ , where each R δ Independently selected from substituted or unsubstituted alkyl, cycloalkyl, cycloheteroalkyl, alkoxy, aryl, heteroaryl, heteroarylalkyl, acyl, alkoxycarbonyl, sulfanyl, sulfinyl, sulfonyl, etc. Typical amino groups include, but are not limited to, dimethylamino, diethylamino, trimethylammonium, triethylammonium, methylsulfonylamino, furanyl-oxy-sulfonylamino, etc.

[0116] As used herein, "oxo" refers to =0.

[0117] As used herein, "oxy" refers to a divalent group -O-, which may have various substituents to form different oxy groups, including ethers and esters.

[0118] As used herein, "carboxyl" refers to -COOH.

[0119] As used herein, "carbonyl" refers to -C(O)-, which may have various substituents to form different carbonyl groups including acids, acyl halides, aldehydes, amides, esters, and ketones.

[0120] As used herein, "alkoxycarbonyl" refers to -C(O)OR ε , where R ε is alkyl as defined herein, which may be optionally substituted.

[0121] As used herein, "aminocarbonyl" refers to -C(O)NH2. Substituted aminocarbonyl refers to -C(O)NR δ R δ , where the amino group NR δ R δ As defined herein.

[0122] As used herein, "halogen" and "halo" refer to fluorine, chlorine, bromine and iodine.

[0123] As used herein, "hydroxy" refers to -OH.

[0124] As used herein, "cyano" refers to -CN.

[0125] As used herein, "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 may have a single ring (e.g., pyridyl or furyl) or more than one condensed ring (e.g., indolizinyl or benzothienyl).

[0126] As used herein, "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.

[0127] As used herein, "heteroarylalkenyl" refers to an alkenyl group substituted with a heteroaryl group (i.e., a heteroaryl-alkenyl- group), preferably having from 2 to 6 carbon atoms (inclusive) in the alkenyl portion and from 5 to 12 ring atoms (inclusive) in the heteroaryl portion.

[0128] As used herein, "heteroarylalkynyl" refers to an alkynyl group substituted with a heteroaryl group (i.e., a heteroaryl-alkynyl- group), preferably having 2 to 6 carbon atoms (inclusive) in the alkynyl portion and 5 to 12 ring atoms (inclusive) in the heteroaryl portion.

[0129] As used herein, "heterocycle", "heterocyclic" and interchangeably "heterocycloalkyl" refer to saturated or unsaturated groups having a single ring or more than one condensed ring, 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 condensed 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.

[0130] As used herein, "membered ring" is intended to include any cyclic structure. The number before the term "membered" represents 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.

[0131] Unless otherwise indicated, the positions occupied by hydrogen in the foregoing radicals may be further substituted by substituents 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, formamido, substituted formamido, alkylsulfonyl, alkylsulfinyl, alkylsulfonylamino, sulfonamido, substituted sulfonamido, cyano, amino , substituted amino, alkylamino, dialkylamino, aminoalkyl, acylamino, amidino, amidoximo, hydroxamoyl, phenyl, aryl, substituted aryl, aryloxy, arylalkyl, arylalkenyl, arylalkynyl, pyridyl, imidazolyl, heteroaryl, substituted heteroaryl, heteroaryloxy, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, substituted cycloalkyl, cycloalkyloxy, pyrrolidinyl, piperidinyl, morpholino, heterocycle, (heterocycle)oxy and (heterocycle)alkyl; and preferred heteroatoms are oxygen, nitrogen and sulfur. It is understood that where open valences exist on these substituents, they may be further substituted with alkyl, cycloalkyl, aryl, heteroaryl and / or heterocyclic groups, where such open valences exist on carbon, they may be further substituted with halogen and oxygen-, nitrogen- or sulfur-bonded substituents, and where more than one such open valence exists, these groups may be joined to form a ring by direct bond formation or by bond formation with new heteroatoms (preferably oxygen, nitrogen or sulfur). It is also understood that the above substitutions may be made provided that replacement of hydrogen with a substituent does not introduce unacceptable instability into the molecules of the invention and is otherwise chemically reasonable.

[0132] As used herein, the term "culturing" refers to the growth of a population of microbial cells under any suitable conditions (eg, using liquid, gel, or solid culture media).

[0133] Recombinant polypeptides can be produced using any suitable method known in the art. The gene encoding the wild-type polypeptide of interest can be cloned into a vector such as a plasmid and expressed in a desired host such as Escherichia coli. Variants of recombinant polypeptides can be produced by various methods known in the art. In fact, there are various different mutagenesis techniques well known to those skilled in the art. In addition, mutagenesis kits can also be obtained from many commercial molecular biology suppliers. The method can be used to make specific substitutions at a determined amino acid (site-directed), specificity (region-specificity) or random mutations in a local region of a gene, or random mutagenesis (e.g., saturation mutagenesis) in the entire gene. Many suitable methods for producing enzyme variants are known to those skilled in the art, including but not limited to, using PCR to single-stranded DNA or double-stranded DNA site-directed mutagenesis, cassette mutagenesis, gene synthesis, error-prone PCR, reorganization, and chemical saturation mutagenesis, or any other suitable method known in the art. Mutagenesis and directed evolution methods can be easily applied to polynucleotides encoding enzymes to produce variant libraries that can be expressed, screened and measured. Any suitable mutagenesis and directed evolution methods can be used in the present invention and 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、6,303,344、6,309,883、6,319,713、6,319,714、6,323,030、6,326,204、6,335,160、6,335,198、6,344,356、6,352,85 9, 6,355,484, 6,358,740, 6,358,742, 6,365,377, 6,365,408, 6,368,861, 6,372,497, 6,337,186, 6,376,246, 6,379,964, 6,387,702, 6,391,552, 6,391,640, 6,395,547, 6,406,855, 6,406,9 10. 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、6,902,922、6,917,882、6,946,296、6,961,664、6,995,017、7,024,312、7,0 58,515、7,105,297、7,148,054、7,220,566、7,288,375、7,384,387、7,421,347、7,430,477、7,462,469、7,534,564、7,620,500、7,620,502、7,629, 170、7,702,464、7,747,391、7,747,393、7,751,986、7,776,598、7,783,428、7,795,030、7,853,410、7,868,138、7,783,428、7,873,477、7,873,499、 Nos. 7,904,249, 7,957,912, 7,981,614, 8,014,961, 8,029,988, 8,048,674, 8,058,001, 8,076,138, 8,108,150, 8,170,806, 8,224,580, 8,377,681, 8,383,346, 8,457,903, 8,504,498, 8,589,085, 8,762,066, 8,768,871, 9,593,326, and all related U.S. and PCT and non-U.S. counterpart applications; Ling et al., Anal. Biochem., 254(2):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. Biotechnol., 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

[1996] ; Stemmer, Nature, 370:389-391

[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).

[0134] In some embodiments, the enzyme clones obtained after mutagenesis are screened by subjecting the enzyme preparation to a determined temperature (or other assay conditions) and measuring the amount of enzyme activity remaining after heat treatment or other suitable assay conditions. The clones containing the polynucleotide encoding the polypeptide are then isolated from the gene, sequenced to identify changes in the nucleotide sequence (if any), and used to express the enzyme in a host cell. Measuring the enzyme activity from the expression library can be performed using any suitable method known in the art (e.g., standard biochemical techniques, such as HPLC analysis).

[0135] After the variants are generated, they can be screened for any desired property (e.g., high or increased activity, or low or decreased activity, increased thermal activity, increased thermal stability, and / or acidic pH stability, etc.). In some embodiments, "recombinant galactose oxidase polypeptides" (also referred to herein as "engineered galactose oxidase polypeptides," "variant galactose oxidases," "galactose oxidase variants," and "galactose oxidase combinatorial variants") can be used. In some embodiments, "recombinant galactose oxidase polypeptides" (also referred to herein as "engineered galactose oxidase polypeptides," "variant galactose oxidases," "galactose oxidase variants," and "galactose oxidase combinatorial variants") can be used.

[0136] As used herein, "vector" is a DNA construct for introducing a DNA sequence into a cell. In some embodiments, a vector is an expression vector operably linked to a suitable control sequence capable of achieving expression of a polypeptide encoded in a DNA sequence in a suitable host. In some embodiments, an "expression vector" has a promoter sequence operably linked to a DNA sequence (e.g., a transgenic) to drive expression in a host cell, and in some embodiments, also comprises a transcription terminator sequence.

[0137] As used herein, the term "expression" includes any step involved in the production of the polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses secretion of the polypeptide from the cell.

[0138] As used herein, the term "production" refers to the production of proteins and / or other compounds from a cell. It is intended that the term encompass any step involved in the production of a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses secretion of a polypeptide from a cell.

[0139] As used herein, an amino acid or nucleotide sequence (e.g., a promoter sequence, a signal peptide, a terminator sequence, etc.) is "heterologous" if the two sequences are not associated in nature with another sequence to which it is operably linked. For example, a "heterologous polynucleotide" is any polynucleotide that is introduced into a host cell by laboratory techniques, and includes polynucleotides that are removed from a host cell, subjected to laboratory manipulation, and then reintroduced into a host cell.

[0140] As used herein, the terms "host cell" and "host strain" refer to suitable hosts for expression vectors containing DNA provided herein (e.g., polynucleotides encoding galactose oxidase variants). In some embodiments, the host cell is a prokaryotic or eukaryotic cell that has been transformed or transfected with a vector constructed using recombinant DNA techniques as known in the art.

[0141] The term "analog" means a polypeptide having more than 70% sequence identity, but less than 100% sequence identity (e.g., more than 75%, 78%, 80%, 83%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity) with a reference polypeptide. In some embodiments, an analog means a polypeptide comprising one or more non-naturally occurring amino acid residues (including but not limited to homoarginine, ornithine and norvaline) and naturally occurring amino acids. In some embodiments, an analog also includes one or more D-amino acid residues and non-peptide connections between two or more amino acid residues.

[0142] The term "effective amount" means an amount sufficient to produce the desired result. One of ordinary skill in the art can determine what the effective amount is by using routine experimentation.

[0143] The terms "isolated" and "purified" are used to refer to a molecule (e.g., an isolated nucleic acid, polypeptide, etc.) or other component that is removed from at least one other component with which it is naturally associated. The term "purified" does not require absolute purity, but is intended as a relative definition.

[0144] As used herein, "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 superior to that of another 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, the art generally reports it as enantiomeric excess ("ee") (usually a percentage) calculated therefrom according to the following formula: [major enantiomer-minor enantiomer] / [major enantiomer+minor enantiomer]. In the case where 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), which is generally optionally reported as diastereomeric excess ("de"). Enantiomeric excess and diastereomeric excess are types of stereoisomer excess.

[0145] As used herein, the terms "regioselectivity" and "regioselective reaction" refer to reactions in which one direction of bond formation or breaking occurs in preference to all other possible directions. A reaction can be completely (100%) regioselective if the discrimination is complete, substantially regioselective (at least 75%) if the reaction product at one site is preferred over the reaction products at other sites, or partially regioselective (x%, where the percentage is set depending on the reaction of interest).

[0146] As used herein, "chemoselectivity" refers to the preferential formation of one product over another in a chemical or enzymatic reaction.

[0147] As used herein, "pH stable" refers to a galactose oxidase polypeptide that maintains similar activity (e.g., more than 60% to 80%) after exposure to high or low pH (e.g., 4.5-6 or 8 to 12) for a period of time (e.g., 0.5-24 hours) compared to the untreated enzyme.

[0148] As used herein, "thermostable" refers to a galactose oxidase polypeptide that maintains similar activity (e.g., more than 60% to 80%) after exposure to the same elevated temperature for a period of time (e.g., 0.5h-24h) compared to a wild-type enzyme exposed to the same elevated temperature (e.g., 40°C to 80°C).

[0149] As used herein, "solvent stable" refers to a galactose oxidase polypeptide that maintains similar activity (e.g., more than 60% to 80%) after exposure to the same solvent at the same concentration for a period of time (e.g., 0.5h-24h) compared to the wild-type enzyme exposed to different concentrations (e.g., 5%-99%) of the solvent (ethanol, isopropanol, dimethyl sulfoxide [DMSO], tetrahydrofuran, 2-methyltetrahydrofuran, acetone, toluene, butyl acetate, methyl tert-butyl ether, etc.).

[0150] As used herein, "thermostable and solvent stable" refers to a galactose oxidase polypeptide that is both thermostable and solvent stable.

[0151] As used herein, "optional" and "optionally" mean that the subsequently described event or circumstance may or may not occur, and that the description includes instances when the event or circumstance occurs and instances in which the event or circumstance does not occur. One of ordinary skill in the art will understand that for any molecule described as containing one or more optional substituents, only sterically feasible and / or synthetically feasible compounds are intended to be included.

[0152] As used herein, "optionally substituted" refers to all subsequent modifiers in one or a series of chemical groups. For example, in the term "optionally substituted arylalkyl", the "alkyl" part and the "aryl" part of the molecule may be substituted or may not be substituted, and for a series of "optionally substituted alkyl, cycloalkyl, aryl and heteroaryl", the alkyl group, cycloalkyl group, aryl group and heteroaryl group may be substituted or may not be substituted independently of each other.

[0153] As used herein, "protecting group" refers to a group of atoms that masks, reduces or prevents the reactivity of a functional group when connected to a reactive functional group in a molecule. Typically, the protecting group can be selectively removed as desired during the synthesis process. Examples of protecting groups are well known in the art. Functional groups that may have protecting groups include, but are not limited to, hydroxyl, amino and carboxyl groups. Representative amino protecting groups include, but are not limited to, formyl, acetyl, trifluoroacetyl, benzyl, benzyloxycarbonyl ("CBZ"), tert-butoxycarbonyl ("Boc"), trimethylsilyl ("TMS"), 2-trimethylsilyl-ethanesulfonyl ("SES"), trityl and substituted trityl groups, allyloxycarbonyl, 9-fluorenylmethoxycarbonyl ("FMOC"), nitro-veratryloxycarbonyl ("NVOC"), etc. Representative hydroxy protecting groups include, but are not limited to, those in which the hydroxy group is acylated (e.g., methyl and ethyl esters, acetate or propionate groups or glycol esters) or those in which the hydroxy group is alkylated, such as benzyl ether and trityl ether, as well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers (e.g., TMS or TIPPS groups) and allyl ethers. Other protecting groups can be found in the references described herein. DETAILED DESCRIPTION OF THE INVENTION

[0155] The present invention provides engineered galactose oxidase (GO enzyme), polypeptides having GO enzyme activity and polynucleotides encoding these enzymes, and vectors and host cells comprising these polynucleotides and polypeptides. Also provided are methods for producing GO enzymes. The present invention also provides compositions comprising GO enzymes, and methods for using engineered GO enzymes. The present invention is particularly useful for the production of pharmaceutical compounds and other compounds.

[0156] Galactose oxidase (GO enzyme) from Fusarium graminearum is a naturally occurring copper-dependent enzyme that is able to oxidize substrates containing primary alcohols under mild reaction conditions. In addition to copper, the enzyme is dependent on a cofactor formed post-translationally as a result of bound copper and molecular oxygen-mediated cross-linking of active site residues tyrosine and cysteine. The enzyme is then active and able to catalyze the oxidation of primary alcohols by reducing oxygen and generating aldehydes and hydrogen peroxide via a free radical mechanism.

[0157] Scenario 1: Oxidation of primary alcohols by galactose oxidase

[0158]

[0159] Early directed evolution work was performed that focused on evolving GO enzyme variants with improved selectivity and activity for 3-ethynylglycerol (EGO) to produce the corresponding aldehyde. The initially evolved variants showed only a slight enrichment for the S-enantiomer of 3-ethynylglyceraldehyde (EGA). This enzyme was further evolved into variants with enantioselectivity favoring the formation of the R-enantiomer (see Scheme 2). Additional directed evolution is required to further enhance the R-enantiomer selectivity and improve the oxidation activity under process conditions.

[0160] Scenario 2: Oxidation of 3-ethynylglycerol by galactose oxidase.

[0161]

[0162] Further directed evolution work was performed, which focused on evolving GO enzyme variants with improved activity on ethynylglycerolphosphate (EGP) to produce the corresponding phosphorylated aldehyde (Compound P) (see Scheme 3).

[0163] Solution 3: Oxidation of ethynylglycerolphosphate by galactose oxidase

[0164]

[0165] Engineered GO enzyme peptides

[0166] The present invention provides engineered GO enzyme polypeptides, polynucleotides encoding the polypeptides, methods for preparing the polypeptides, and methods for using the polypeptides. When describing a polypeptide, it should be understood that it also describes a polynucleotide encoding the polypeptide. In some embodiments, the present invention provides an engineered, non-naturally occurring GO enzyme with improved properties compared to a wild-type GO enzyme. Any suitable reaction conditions can be used in the present invention. In some embodiments, methods are used to analyze the improved properties of an oxidation reaction of an engineered polypeptide. In some embodiments, as further described below and in the examples, reaction conditions are changed according to the concentration or amount of an engineered GO enzyme, one or more substrates, one or more buffers, one or more solvents, cofactors, pH, conditions including temperature and reaction time, and / or conditions for fixing an engineered GO enzyme polypeptide on a solid support.

[0167] In some embodiments, additional reaction components or additional techniques are utilized to supplement reaction conditions. In some embodiments, these include taking steps to stabilize the enzyme or prevent enzyme inactivation, reduce product inhibition, shift the reaction equilibrium to desired product formation.

[0168] In some other embodiments, any of the above-described methods for converting a substrate compound into a product compound may also include one or more steps selected from the following: extraction, separation, purification, crystallization, filtration and / or lyophilization of one or more product compounds. Methods, techniques and protocols for extracting, separating, purifying and / or crystallizing one or more products from a biocatalytic reaction mixture produced by the methods provided herein are known to those of ordinary skill and / or obtainable by routine experimentation. In addition, illustrative methods are provided in the examples hereinafter.

[0169] Engineered GO enzyme polynucleotides encoding engineered polypeptides, expression vectors and host cells

[0170] The present invention provides polynucleotides encoding engineered enzyme polypeptides described herein. In some embodiments, the polynucleotides are operably linked to one or more heterologous regulatory sequences controlling gene expression to produce a recombinant polynucleotide capable of expressing a polypeptide. In some embodiments, an expression construct comprising at least one heterologous polynucleotide encoding one or more engineered enzyme polypeptides is introduced into an appropriate host cell to express one or more corresponding enzyme polypeptides.

[0171] As will be apparent to the skilled person, the availability of protein sequences and the knowledge of the codons corresponding to various amino acids provide a description of all polynucleotides capable of encoding the subject polypeptide. The degeneracy of the genetic code, in which the same amino acid is encoded by optional or synonymous codons, allows the preparation of a very large number of nucleic acids, all of which encode engineered enzymes (e.g., GO enzymes) polypeptides. Therefore, the present invention provides methods and compositions for selecting and combining each and every possible variation of the preparable enzyme polynucleotides for producing the enzyme polypeptides described herein based on possible codon options, and all such variations are considered to be specifically disclosed for any polypeptide described herein, including the amino acid sequences presented in the embodiments (e.g., in each table).

[0172] In some embodiments, codons are preferably optimized to be utilized by the selected host cell for protein production. For example, preferred codons used in bacteria are typically used for expression in bacteria. Therefore, the codon-optimized polynucleotides encoding engineered enzyme polypeptides comprise preferred codons at about 40%, 50%, 60%, 70%, 80% or greater than 90% of the codon positions in the full-length coding region.

[0173] In some embodiments, the enzyme polynucleotide encodes an engineered polypeptide having enzyme activity and properties disclosed herein, wherein the polypeptide comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to an amino acid sequence selected from a reference sequence of a SEQ ID NO provided herein, or any variant (e.g., those provided in the Examples), and one or more residue differences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residue positions) compared to the amino acid sequence of one or more reference polynucleotides or any variant as disclosed in the Examples. In some embodiments, the reference polypeptide sequence is selected from SEQ ID NO: 2, 4, 166, 272, 928, 932, 1264, 1416, 1598, 1866, 1912, 2080, 2300 and / or 2424.

[0174] In some embodiments, polynucleotide can be selected from any polynucleotide sequence provided herein or its complementary sequence or the polynucleotide sequence of any variant enzyme polypeptide provided herein and hybridize under high stringency conditions.In some embodiments, the polynucleotide encoding that can hybridize under high stringency conditions comprises the enzyme polypeptide that has the amino acid sequence of one or more residue differences compared with the reference sequence.

[0175] In some embodiments, the polynucleotide of any separation in the engineered enzyme polypeptide of encoding this paper is manipulated in various ways to promote the expression of enzyme polypeptide.In some embodiments, the polynucleotide of encoding enzyme polypeptide constitutes expression vector, wherein there is one or more control sequence to regulate the expression of enzyme polynucleotide and / or polypeptide.According to the expression vector used, the manipulation of the polynucleotide of separation before the polynucleotide of separation is inserted into the vector can be desired or necessary.The technology of utilizing recombinant DNA method to modify polynucleotide and nucleotide sequence is well known in the art.In some embodiments, control sequence comprises, among others, promoter, leader sequence, polyadenylation sequence, propeptide sequence, signal peptide sequence and transcription terminator.In some embodiments, suitable promoter is selected based on the selection of host cell. For bacterial host cells, suitable promoters for directing transcription of the nucleic acid constructs of the present disclosure include, but are not limited to, promoters obtained from the Escherichia coli lac operon, the Streptomyces coelicolor agarase gene (dagA), the Bacillus subtilis levansucrase gene (sacB), the Bacillus licheniformis alpha-amylase gene (amyL), the Bacillus stearothermophilus maltogenic amylase gene (amyM), the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), the Bacillus licheniformis penicillinase gene (penP), the 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, but are not limited to, promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid-stable alpha-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 / 054585). 96 / 00787), and the NA2-tpi promoter (a hybrid of the promoters from the Aspergillus niger neutral α-amylase gene and the Aspergillus oryzae triosephosphate isomerase gene), and mutants, truncated, 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] ).

[0176] In some embodiments, the control sequence is also a suitable transcription terminator sequence (i.e., a sequence recognized by the host cell to terminate transcription). In some embodiments, the terminator sequence is operably connected to the 3' end of the nucleic acid sequence encoding the enzyme polypeptide. Any suitable terminator that is functional in the selected host cell can be used in the present invention. Exemplary transcription terminators 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. Exemplary terminators 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 et al., above).

[0177] In some embodiments, the control sequence is also a suitable leader sequence (i.e., the non-translated region of the mRNA important for translation by the host cell). In some embodiments, the leader sequence is operably linked to the 5' end of the nucleic acid sequence encoding the enzyme polypeptide. Any suitable leader sequence that is functional in the selected host cell can be used in the present invention. Exemplary leaders for filamentous fungal host cells are obtained from the following genes: Aspergillus oryzae TAKA amylase and Aspergillus nidulans triosephosphate isomerase. Suitable leaders for yeast host cells are 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).

[0178] In some embodiments, the control sequence is also a polyadenylation sequence (i.e., a sequence operably linked to the 3' end of the nucleic acid sequence and which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to the transcribed mRNA). Any suitable polyadenylation sequence that is functional in the host cell of choice may be used in the present invention. Exemplary polyadenylation sequences for filamentous fungal host cells include, but are not limited to, the following genes: 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 known (see, e.g., Guo and Sherman, Mol. Cell. Biol., 15:5983-5990

[1995] ).

[0179] In some embodiments, the control sequence is also a signal peptide (i.e., a coding region encoding an amino acid sequence that is linked to the amino terminus of a polypeptide and directs the encoded polypeptide to the secretory pathway of the cell). In some embodiments, the 5' end of the coding sequence of the nucleic acid sequence inherently contains a signal peptide coding region that is naturally linked in translation reading frame to a segment of the coding region encoding the secreted polypeptide. Alternatively, in some embodiments, the 5' end of the coding sequence contains a signal peptide coding region that is foreign to the coding sequence. Any suitable signal peptide coding region that directs the expressed polypeptide to the secretory pathway of the selected host cell can be used for expression of one or more engineered polypeptides. Effective signal peptide coding regions for bacterial host cells include, but are not limited to, those obtained from the following genes: Bacillus NClB 11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-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] ). In some embodiments, 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 α-factor and Saccharomyces cerevisiae invertase.

[0180] In some embodiments, the control sequence is also 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 "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 can be obtained from any suitable source including but 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., WO 95 / 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.

[0181] In some embodiments, regulatory sequences are also utilized. These sequences promote the regulation of polypeptide expression relative to host cell growth. Examples of regulatory systems are those that cause the expression of genes 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.

[0182] On the other hand, the present invention relates to a polynucleotide comprising an engineered enzyme polypeptide, and one or more expression regulatory regions such as promoters and terminators, replication origins, etc., according to the type of host to be introduced. In some embodiments, various nucleic acids and control sequences described herein are linked together to produce a recombinant expression vector, which includes one or more convenient restriction sites to allow insertion or replacement of the nucleic acid sequence encoding the enzyme polypeptide at such a site. Alternatively, in some embodiments, the nucleic acid sequence of the present invention is expressed by inserting the nucleic acid sequence or a nucleic acid construct comprising the sequence into a suitable vector for expression. In some embodiments involving the generation of an expression vector, the coding sequence is located in a vector so that the coding sequence is operably connected to a suitable control sequence for expression.

[0183] The recombinant expression vector can be any suitable vector (e.g., plasmid or virus) that can be easily subjected to recombinant DNA procedures and cause expression of the enzyme polynucleotide sequence. The choice of vector generally depends 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.

[0184] 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 selectable embodiments, the vector is a vector that, when introduced into a host cell, is integrated into the genome and replicated with one or more chromosomes into which it is integrated. In addition, in some embodiments, a single vector or plasmid is utilized, or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, and / or a transposon.

[0185] In some embodiments, the expression vector comprises one or more selectable markers that allow easy selection of transformed cells. A "selectable marker" is a gene whose product provides antimicrobial or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, etc. Examples of bacterial selectable 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; e.g., from A. nidulans or A. orzyae), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase; e.g., from S. Hygroscopicus), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase; e.g., from A. nidulans or A. oryzae), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), and equivalents thereof.

[0186] On the other hand, the present invention provides a host cell, the host cell comprising at least one polynucleotide encoding at least one engineered enzyme polypeptide of the present invention, the polynucleotide being operably connected to one or more control sequences for expressing one or more engineered enzymes in the host cell. The host cell suitable for expressing the polypeptide encoded by the expression vector of the present invention is well known in the art, and includes but is 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 or Pichia pastoris (Pichia pastoris) (ATCC accession number 201178)); insect cells, such as Drosophila (Drosophila) S2 and Spodoptera (Spodoptera) Sf9 cells; animal cells, such as CHO, COS, BHK, 293 and Bowes melanoma cells; and plant cells. Exemplary host cells also include various Escherichia coli strains (e.g., W3110 (ΔfhuA) and BL21). Examples of bacterial selection markers include, but are not limited to, the dal gene from Bacillus subtilis or Bacillus licheniformis, or markers that confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol and / or tetracycline resistance.

[0187] In some embodiments, the expression vectors of the invention contain elements that allow the vector to be integrated into the genome of the host cell or that allow the vector to replicate autonomously in the cell independent of the genome. In some embodiments involving integration into the host cell genome, the vector relies on the nucleic acid sequence encoding the polypeptide or any other element of the vector for integration of the vector into the genome by homologous or nonhomologous recombination.

[0188] In some alternative embodiments, the expression vector comprises other nucleotide sequences for guiding integration into the genome of the host cell by homologous recombination. Other nucleotide sequences enable the carrier to be integrated into the host cell genome at one or more precise positions in one or more chromosomes. In order to increase the possibility of integration at the precise position, the integration element preferably comprises a sufficient number of nucleotides, such as 100 to 10,000 base pairs, preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000 base pairs, which are highly homologous to the corresponding target sequence, to improve the possibility of homologous recombination. The integration element can be any sequence homologous to the target sequence in the genome of the host cell. In addition, the integration element can be a non-coding or encoding nucleic acid sequence. On the other hand, the carrier can be integrated into the genome of the host cell by non-homologous recombination.

[0189] For autonomous replication, the vector may also include an origin of replication so that the vector can replicate autonomously in the host cell in question. Examples of bacterial origins of replication are the P15A ori that allows replication in Escherichia coli, or the origin of replication of plasmids pBR322, pUC19, pACYCl77 (the plasmid has a P15A ori) or pACYC184 and the origin of replication of pUB110, pE194 or pTA1060 that allows replication in Bacillus (Bacillus). Examples of origins of replication for use in yeast host cells are the 2 μm origin of replication, ARS1, ARS4, a combination of ARS1 and CEN3, and a combination of ARS4 and CEN6. The origin of replication may be an origin of replication with a mutation that allows it to function temperature-sensitively in a host cell (see, e.g., Ehrlich, Proc. Natl. Acad. Sci. USA 75: 1433

[1978] ).

[0190] In some embodiments, more than one copy of the nucleic acid sequence of the present invention is inserted into the host cell to increase the production of the gene product. The increase in the number of nucleic acid sequence copies can be obtained by integrating at least one additional copy of the sequence into the host cell genome, or by including an amplifiable selection marker gene in the nucleic acid sequence, wherein cells containing an amplified copy of the selection marker gene and thereby containing an additional copy of the nucleic acid sequence can be selected by culturing cells in the presence of a suitable selection agent.

[0191] Many expression vectors useful in the present invention are commercially available. Suitable commercial expression vectors include, but are not limited to, p3xFLAG™ TM Expression vector (Sigma-Aldrich Chemicals), which includes a CMV promoter and hGH polyadenylation site for expression in mammalian host cells and a pBR322 origin of replication and an ampicillin resistance marker for amplification in E. coli. Other suitable expression vectors include, but are not limited to, pBluescriptII SK(-) and pBK-CMV (Stratagene), and plasmids derived from pBR322 (Gibco BRL), pUC (Gibco BRL), pREP4, pCEP4 (Invitrogen), or pPoly (see, e.g., Lathe et al., Gene 57:193-201

[1987] ).

[0192] Therefore, in some embodiments, a vector comprising a sequence encoding at least one variant galactose oxidase is transformed into a host cell to allow the propagation of the vector and the expression of one or more variant galactose oxidases. In some embodiments, the variant galactose oxidase is post-translationally modified to remove the signal peptide, and can be cleaved after secretion in some cases. In some embodiments, the host cell of the transformation described above is cultured in a suitable nutrient medium under conditions that allow expression of one or more variant galactose oxidases. Any suitable culture medium that can be used to culture host cells can be used for the present invention, including but not limited to a minimal culture medium or a complex culture medium containing a suitable supplement. In some embodiments, host cells are grown in HTP culture medium. Suitable culture medium can be obtained from various commercial suppliers, or can be prepared according to a disclosed formula (e.g., in the catalog of the American Type Culture Collection).

[0193] On the other hand, the present invention provides a host cell comprising a polynucleotide encoding an improved galactose oxidase polypeptide provided herein, the polynucleotide being operably connected to one or more control sequences for expressing galactose oxidase in a host cell. Host cells for expressing galactose oxidase polypeptides encoded by the expression vector of the present invention are well known in the art, and include but are not limited to bacterial cells such as Escherichia coli, Bacillus megaterium, Lactobacillus kefir, Streptomyces and Salmonella typhimurium cells; Fungal cells such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC accession number 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. Suitable culture media and growth conditions for the host cells described above are well known in the art.

[0194] Polynucleotides for expressing galactose oxidase can be introduced into cells by various methods known in the art. Techniques include, among others, electroporation, biolistic particle bombardment, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion. Various methods for introducing polynucleotides into cells are known to those skilled in the art.

[0195] In some embodiments, the host cell is a eukaryotic cell. Suitable eukaryotic host cells include, but are not limited to, fungal cells, algae cells, insect cells, and plant cells. Suitable fungal host cells include, but are not limited to, Ascomycota, Basidiomycota, Deuteromycota, Zygomycota, and Fungi imperfecti. In some embodiments, the fungal host cell is a yeast cell and a filamentous fungal cell. The filamentous fungal host cell of the present invention includes all filamentous forms of Eumycotina and Oomycota. The feature of filamentous fungi is a vegetative mycelium, in which the cell wall is composed of chitin, cellulose, and other complex polysaccharides. The filamentous fungal host cell of the present invention is morphologically different from yeast.

[0196] In some embodiments of the invention, the filamentous fungal host cell is of any suitable genus and species, including, but not limited to, Achlya, Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Cephalosporium, Chrysosporium, Cochliobolus, Corynascus, Cryphonectria, Cryptococcus, Coprinus, Coriolus, Diplodia, Endothia, Fusarium, Gibberella, Gliocladium, Humicola, Hypocrea. , Myceliophthora, Mucor, Neurospora, Penicillium, Podospora, Phlebia, Piromyces, Pyricularia, Rhizomucor, Rhizopus, Schizophyllum, Scytalid ium, Sporotrichum, Talaromyces, Thermoascus, Thielavia, Trametes, Tolypocladium, Trichoderma, Verticillium, and / or Volvariella, and / or sexual or asexual forms, and synonyms, basal synonyms, or taxonomic equivalents thereof.

[0197] In some embodiments of the invention, the host cell is a yeast cell, including but not limited to a cell of a Candida, Hansenula, Saccharomyces, Schizosaccharomyces, Pichia, Kluyveromyces, or Yarrowia species. In some embodiments of the invention, the yeast cell is Hansenula polymorpha, Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Saccharomyces diastaticus, Saccharomyces norbensis, Saccharomyces kluyveri, Schizosaccharomyces pombe, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia kodamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia quercuum, Pichia pijperi, Pichia stipitis, Pichia methanolica, Pichia angusta, Kluyveromyces lactis, Pichia spp. lactis, Candida albicans or Yarrowia lipolytica.

[0198] In some embodiments of the invention, the host cell is an algal cell, such as Chlamydomonas (eg, C. reinhardtii) and Phormidium (Phormidium sp. ATCC 29409).

[0199] In some other embodiments, the host cell is a prokaryotic cell. Suitable prokaryotic cells include, but are not limited to, Gram-positive, Gram-negative, and Gram-variable bacterial cells. Any suitable bacterial organism can be used in the present invention, including but not limited to Agrobacterium, Alicyclobacillus, Anabaena, Anacystis, Acinetobacter, Acidothermus, Arthrobacter, Azobacter, Bacillus, Bifidobacterium, Brevibacterium ), Butyrivibrio, Buchnera, Campestris, Campylobacter, Clostridium, Corynebacterium, Chromatium, Coprococcus, Escherichia, Enterococcus, Enterobacter, Erwinia, Fuso bacterium, Faecalibacterium, Francisella, Flavobacterium, Geobacillus, Haemophilus, Helicobacter, Klebsiella, Lactobacillus, Lactococcus, Ilyobacter, Micrococcus, Microbar Microbacterium, Mesorhizobium, Methylobacterium, Methylobacterium, Mycobacterium, Neisseria, Pantoea, Pseudomonas, Prochlorococcus, Rhodobacter, Rhodopseudomonas, Rhodopseudomonas, Roseburia,Rhodospirillum, Rhodococcus, Scenedesmus, Streptomyces, Streptococcus, Synecoccus, Saccharomonospora, Staphylococcus, Serratia, Salmonella, Shigella, Thermoanaerobacterium, Tropheryma, Tularensis, Temecula, Thermosynechococcus, Thermococcus, Ureaplasma, Xanthomonas, Xylella, Yersinia, and Zymomonas. In some embodiments, the host cell is a species of: Agrobacterium, Acinetobacter, Azotobacter, Bacillus, Bifidobacterium, Buchnera, Geobacillus, Campylobacter, Clostridium, Corynebacterium, Escherichia, Enterococcus, Erwinia, Flavobacterium, Lactobacillus, Lactococcus, Pantoea, Pseudomonas, Staphylococcus, Salmonella, Streptococcus, Streptomyces or Zymomonas. In some embodiments, the bacterial host strain is non-pathogenic to humans. In some embodiments, the bacterial host strain is an industrial strain. Many industrial strains of bacteria are known and suitable for the present invention. In some embodiments of the present invention, the bacterial host cell is an Agrobacterium species (e.g., A. radiobacter, A. rhizogenes and A. rubi). In some embodiments of the invention, the bacterial host cell is an Arthrobacter species (e.g., A. aurescens, A. citreus, A. globiformis, A. hydrocarboglutamicus, A. mysorens, A. nicotianae, A. paraffineus, A. protophonniae, A. roseoparqffinus, A. sulfurus, and A. ureafaciens). In some embodiments of the invention, the bacterial host cell is a Bacillus species (e.g., B. thuringensis, B. anthracis,In some embodiments, the host cell is an industrial Bacillus strain, including but not limited to Bacillus subtilis, Bacillus pumilus, Bacillus licheniformis, Bacillus megaterium, Bacillus brevis, Bacillus firmus, B. alkaophius, B. licheniformis, B. clausii, B. stearothermophilus, B. halodurans, and B. amyloliquefaciens. In some embodiments, the host cell is an industrial Bacillus strain, including but not limited to Bacillus subtilis, Bacillus pumilus, B. licheniformis, B. megaterium, B. pumilus, B. lautus, B. coagulans, B. brevis, B. firmus, B. alkaophius, B. licheniformis, B. clausii, B. stearothermophilus, B. halodurans, and B. amyloliquefaciens. In some embodiments, the bacillus host cell is bacillus subtilis, bacillus licheniformis, bacillus megaterium, bacillus stearothermophilus and / or bacillus amyloliquefaciens. In some embodiments, the bacterial host cell is a clostridium species (for example, acetobutylicum clostridium (C.acetobutylicum), tetanus clostridium E88 (C.tetani E88), ivory coast clostridium (C.lituseburense), C.saccharobutylicum, perfringens clostridium (C.perfringens) and Bai Shi clostridium (C.beijerinckii)). In some embodiments, the bacterial host cell is a coryneform bacteria species (for example, glutamicum coryneform bacteria (C.glutamicum) and acetoacetic acid coryneform bacteria (C.acetoacidophilum)). In some embodiments, the bacterial host cell is an Escherichia species (for example, Escherichia coli). In some embodiments, the host cell is Escherichia coli W3110. In some embodiments, the bacterial host cell is an Erwinia species (e.g., Erwinia uredovora, Erwinia carotovora, Erwinia ananas, Erwinia herbicola, E. punctata, and E. terreus). In some embodiments, the bacterial host cell is a Pantoea species (e.g., Pantoea citrea and Pantoea agglomerans). In some embodiments, the bacterial host cell is a Pseudomonas species (e.g., Pseudomonas putida, Pseudomonas aeruginosa,In some embodiments, the bacterial host cell is a Streptococcus species (e.g., S. equisimiles, S. pyogenes, and S. uberis). In some embodiments, the bacterial host cell is a Streptomyces species (e.g., S. ambofaciens, S. achromogenes, S. avermitilis, S. coelicolor, S. aureofaciens, S. aureus, S. fungicidicus, S. griseus, and S. lividans). In some embodiments, the bacterial host cell is a Zymomonas species (eg, Z. mobilis and Z. lipolytica).

[0200] Many prokaryotic and eukaryotic strains that can be used in the present invention are readily available to the public from a number of culture collections, such as the American Type Culture Collection (ATCC), the German Collection of Microorganisms and Fungi (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, DSM), the Netherlands Central Agricultural Research Center (Centraalbureau Voor Schimmelcultures, CBS), and the U.S. Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

[0201] In some embodiments, host cells are genetically modified to have improved protein secretion, protein stability features and / or protein expression and / or other properties desired for secretion.Genetic modification can be achieved by genetic engineering techniques and / or typical microbiological techniques (e.g., chemical or UV mutagenesis and subsequent selection).In fact, in some embodiments, the combination of recombinant modification and typical selection techniques is used to produce host cells.Using recombinant techniques, nucleic acid molecules can be introduced, lacked, suppressed or modified in a manner that causes an increase in the output of one or more galactose oxidase variants in the host cell and / or in the culture medium.For example, knocking out Alp1 function produces cells with protease defects, and knocking out pyr5 function produces cells with pyrimidine defect phenotypes.In a genetic engineering method, homologous recombination is used to induce targeted genetic modification by specific targeting genes in vivo to suppress the expression of coded proteins.In alternative methods, siRNA, antisense and / or ribozyme technology can be used to suppress gene expression.Various methods of reducing protein expression in cells known in the art include but are not limited to the disappearance of all or part of the gene encoding the protein, and site-specific mutagenesis to destroy the expression or activity of the gene product. (See, e.g., Chaveroche et al., Nucl. Acids Res., 28:22e97

[2000] ; Cho et al., Molec. Plant Microbe Interact., 19:7-15

[2006] ; Maruyama and Kitamoto, Biotechnol Lett., 30:1811-1817

[2008] ; Takahashi et al., Mol. Gen. Genom., 272:344-352

[2004] ; and You et al., Arch. Microbiol., 191:615-622

[2009] , each of which is incorporated herein by reference). Random mutagenesis followed by screening for desired mutations may also be used (See, e.g., Combier et al., FEMS Microbiol. Lett., 220:141-8

[2003] ; and Firon et al., Eukary. Cell. 2:247-55

[2003] , both incorporated by reference).

[0202] The introduction of a vector or DNA construct into a host cell can be accomplished using any suitable method known in the art, including but not limited to calcium phosphate transfection, DEAE-dextran mediated transfection, PEG-mediated transformation, electroporation, or other commonly used techniques known in the art. In some embodiments, the E. coli expression vector pCK100900i (see, U.S. Pat. No. 9,714,437, incorporated herein by reference) can be used.

[0203] In some embodiments, the engineered host cells of the invention (i.e., "recombinant host cells") are cultured in conventional nutrient media modified appropriately to activate promoters, select transformants, or amplify galactose oxidase polynucleotides. Culture conditions, such as temperature, pH, etc., are those previously used with the host cells selected for expression and are well known to those skilled in the art. As described, many standard references and textbooks are available for the culture and production of many cells, including those of bacterial, plant, animal (especially mammalian) and archaeal origin.

[0204] In some embodiments, cells expressing variant galactose oxidase polypeptides of the present invention are grown under batch or continuous fermentation conditions. Typical "batch fermentation" is a closed system in which the composition of the culture medium is set at the beginning of the fermentation and is not affected by artificial changes during the fermentation. A variation of the batch system is "fed-batch fermentation", which can also be used in the present invention. In this variation, substrates are added in increments as the fermentation proceeds. Fed-batch systems are useful when catabolite repression may inhibit the metabolism of the cell and when a limited amount of substrate is desired in the culture medium. Batch fermentation and fed-batch fermentation are common and well known in the art. "Continuous fermentation" is an open system in which a determined fermentation medium is continuously added to a bioreactor and an equal amount of conditioned medium is taken out simultaneously for processing. Continuous fermentation generally maintains the culture at a constant high density, in which the cells are primarily in logarithmic phase growth. Continuous fermentation systems attempt to maintain steady-state growth conditions. Methods for regulating nutrients and growth factors for continuous fermentation processes and techniques for maximizing product formation rates are well known in the field of industrial microbiology.

[0205] In some embodiments of the invention, a cell-free transcription / translation system can be used to produce one or more variant galactose oxidases. Several systems are commercially available and methods are well known to those skilled in the art.

[0206] The present invention provides methods for preparing variant galactose oxidase polypeptides or biologically active fragments thereof. In some embodiments, the method comprises: providing a host cell transformed with a polynucleotide encoding an amino acid sequence having at least about 70% (or at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%) sequence identity to SEQ ID NO: 2, 70, 122, 514, 426 and comprising at least one mutation provided herein; culturing the transformed host cell in a culture medium under conditions in which the host cell expresses the encoded variant galactose oxidase polypeptide; and optionally recovering or isolating the expressed variant galactose oxidase polypeptide, and / or recovering or isolating the culture medium containing the expressed variant galactose oxidase polypeptide. In some embodiments, the method further provides optionally lysing the transformed host cell after expressing the encoded galactose oxidase polypeptide, and optionally recovering and / or isolating the expressed variant galactose oxidase polypeptide from the cell lysate. The present invention also provides a method for preparing a variant galactose oxidase polypeptide, the method comprising culturing a host cell transformed with a variant galactose oxidase polynucleotide under conditions suitable for producing a variant galactose oxidase polypeptide, and recovering the variant galactose oxidase polypeptide. Typically, the galactose oxidase polypeptide is recovered or separated from the host cell culture medium, the host cell, or both using protein recovery techniques well known in the art, including those described herein. In some embodiments, the host cells are collected by centrifugation, destroyed by physical or chemical means, and the resulting crude extract is retained for further purification. Microbial cells for protein expression can be destroyed by any convenient method, including but not limited to freeze-thaw cycles, sonication, mechanical destruction, and / or the use of cell lysing agents, as well as many other suitable methods well known to those skilled in the art.

[0207] The engineered galactose oxidase expressed in the host cells can be recovered from the cells and / or culture medium using any one or more of the techniques known in the art for protein purification, including, among others, lysozyme treatment, sonication, filtration, salting out, ultracentrifugation, and chromatography. A suitable solution for lysis and efficient protein extraction from bacteria such as E. coli is available under the trade name CelLytic B TM(Sigma-Aldrich) is commercially available. Therefore, in some embodiments, the obtained polypeptide is recovered / separated and optionally purified by any of various methods known in the art. For example, in some embodiments, the polypeptide is separated from the nutrient medium by conventional procedures, including but not limited to centrifugation, filtration, extraction, spray drying, evaporation, chromatography (e.g., ion exchange, affinity, hydrophobic interaction, chromatofocusing and size exclusion) or precipitation. In some embodiments, as required, a protein refolding step is used to complete the construction of the mature protein. In addition, in some embodiments, high performance liquid chromatography (HPLC) is used in the final purification step. For example, in some embodiments, methods known in the art can be used in the present invention (see, for example, Parry et al., Biochem. J., 353: 117

[2001] ; and Hong et al., Appl. Microbiol. Biotechnol., 73: 1331

[2007] , both of which are incorporated herein by reference). In fact, any suitable purification method known in the art can be used in the present invention.

[0208] Chromatographic techniques used to separate galactose oxidase polypeptides include, but are not limited to, reverse phase chromatography, high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, and affinity chromatography. The conditions used to purify a particular enzyme depend in part on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc., and are known to those skilled in the art.

[0209] In some embodiments, affinity techniques can be used to isolate improved galactose oxidase. For affinity chromatography purification, any antibody that specifically binds to a galactose oxidase polypeptide can be used. In order to produce antibodies, various host animals can be immunized by injection of galactose oxidase, including but not limited to rabbits, mice, rats, etc. The galactose oxidase polypeptide can be attached to a suitable carrier such as BSA by means of a side chain functional group or a linker attached to a side chain functional group. Depending on the host species, various adjuvants can be used to enhance the immune response, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpets. keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacillus Calmette-Guérin) and Corynebacterium parvum.

[0210] In some embodiments, the galactose oxidase variant is prepared and used in the form of cells expressing the enzyme, as a crude extract, or as an isolated or purified preparation. In some embodiments, the galactose oxidase variant is prepared as a lyophilized agent, a powder form (e.g., acetone powder), or as an enzyme solution. In some embodiments, the galactose oxidase variant is in the form of a substantially pure preparation.

[0211] In some embodiments, the galactose oxidase polypeptide is attached to any suitable solid substrate. Solid substrates include, but are not limited to, solid phases, surfaces, and / or membranes. Solid supports include, but are not limited to, organic polymers such as polystyrene, polyethylene, polypropylene, polyfluoroethylene, polyethyleneoxy, and polyacrylamide, as well as copolymers and grafts thereof. Solid supports 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 substrate can be in the form of beads, balls, particles, granules, gels, membranes, 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 characteristics. The solid support can be configured in the form of a hole, depression, or other container, vessel, feature, or position. More than one support can be configured on the array at various positions, and the position can be addressed by automatic delivery of reagents or by detection methods and / or instruments.

[0212] In some embodiments, immunological methods are used to purify galactose oxidase variants. In one method, antibodies against variant galactose oxidase polypeptides (e.g., polypeptides comprising any of SEQ ID NOs: 2, 70, 122, 514, and / or 426, and / or immunogenic fragments thereof) produced using conventional methods are immobilized on beads, mixed with cell culture medium under conditions where variant galactose oxidase is bound, and precipitated. In a related method, immunochromatography can be used.

[0213] In some embodiments, the variant galactose oxidase is expressed as a fusion protein comprising a non-enzyme portion. In some embodiments, the variant galactose oxidase sequence is fused to a purification facilitating domain. As used herein, the term "purification facilitating domain" refers to a domain that mediates the purification of a polypeptide fused thereto. Suitable purification domains include, but are not limited to, metal chelating peptides, histidine-tryptophan modules that allow purification on immobilized metals, sequences that bind glutathione (e.g., GST), hemagglutinin (HA) tags (corresponding to epitopes derived from influenza hemagglutinin proteins; see, e.g., Wilson et al., Cell 37: 767

[1984] ), maltose binding protein sequences, FLAG epitopes used in FLAGS extension / affinity purification systems (e.g., systems available from ImmunexCorp), etc. An expression vector contemplated for use in the compositions and methods described herein provides expression of a fusion protein comprising a polypeptide of the present invention fused to a polyhistidine region separated by an enterokinase cleavage site. The histidine residues facilitate purification on IMIAC (immobilized metal ion affinity chromatography; see, e.g., Porath et al., Prot. Exp. Purif., 3:263-281

[1992] ), while the enterokinase cleavage site provides a means for separating the variant galactose oxidase polypeptide from the fusion protein. The pGEX vector (Promega) can also be used to express exogenous polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption to ligand-agarose beads (e.g., glutathione-agarose in the case of GST-fusion proteins) followed by elution in the presence of free ligand.

[0214] Therefore, in another aspect, the invention provides a method for producing an engineered enzyme polypeptide, wherein the method comprises culturing a host cell capable of expressing a polynucleotide encoding an engineered enzyme polypeptide under conditions suitable for expression of the polypeptide. In some embodiments, the method further comprises the step of separating and / or purifying the enzyme polypeptide as described herein.

[0215] Suitable culture media and growth conditions for host cells are well known in the art. It is contemplated that any suitable method for introducing a polynucleotide for expressing an enzyme polypeptide into a cell can be used in the present invention. Suitable techniques include, but are not limited to, electroporation, biolistic particle bombardment, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion.

[0216] Various features and embodiments of the invention are illustrated in the following representative examples, which are intended to be illustrative rather than limiting.

[0217] experiment

[0218] The following examples are provided, including the results of experiments and acquisitions, for illustrative purposes only and should not be construed as limiting the present invention. In fact, many reagents and equipment described below have various suitable sources. It is not intended that the present invention be limited to any particular source of any reagent and equipment item.

[0219] In the experimental disclosure hereinafter, the following abbreviations are used: 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); psi and PSI (pounds per square inch); °C (degrees Celsius); RT and rt (room temperature); RH (relative humidity); CV (coefficient of variation); CAM and cam (chloramphenicol); PMBS (polymyxin B sulfate); IPTG (isopropyl β-Dl-thiogalactopyranoside); LB (Luria broth); TB (terrific broth, broth); SFP (shake flask powder); CDS (coding sequence); DNA (deoxyribonucleic acid); RNA (ribonucleic acid); nt (nucleotide; polynucleotide); aa (amino acid; polypeptide); Escherichia coli W3110 (a commonly used laboratory strain of E. coli, available from Coli Genetic Stock Center [CGSC], New Haven, CT); HTP (high throughput); HPLC (high pressure liquid chromatography); HPLC-UV (HPLC-ultraviolet visible detector); 1H NMR (proton nuclear magnetic resonance spectroscopy); FIOPC (fold improvement over positive control); Sigma and Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO); Difco (Difco Laboratories, BDDiagnostic Systems, Detroit, MI); Microfluidics (Microfluidics, Westwood, MA); Life Technologies (Life Technologies, Fisher Scientific, part of Waltham, MA); Amresco (Amresco, LLC, Solon, OH); Carbosynth (Carbosynth, Ltd., Berkshire, UK); Varian (Varian Medical Systems, Palo Alto, CA); Agilent (Agilent Technologies, Inc., Santa Clara, CA); Infors (Infors USA Inc., Annapolis Junction, MD); and Thermotron (Thermotron, Inc., Holland, MI). .

[0220] Example 1

[0221] Improvements of GOA compared to SEQ ID NO: 2 for enantioselective production of EGA

[0222] The parent gene codons of the GOA (SEQ ID NO:2) enzyme for producing variants of the present invention are optimized to be expressed in Escherichia coli, and synthesized and cloned into pET-30a vectors. BL21 (DE3) Escherichia coli cells were transformed with the corresponding plasmid containing the GOA coding gene, and plated on Luria broth (LB) agar plates containing 1% glucose and 50 μg / mL kanamycin (KAN), and grown overnight at 37°C. Monoclonal colonies were selected and inoculated into 96-well shallow-well microtiter plates of 180 μL LB containing 1% glucose and 50 μg / mL KAN. The plate was sealed with an O2 permeable seal, and the culture was grown overnight at 30°C, 200rpm and 85% relative humidity (RH). Then, 10 μL of each cell culture was transferred to a well of a 96-well deep-well plate containing 390 μL TB, 50 μg / mL KAN and 0.5 mM CuSO4. The deep-well plate was sealed with an O2-permeable seal and incubated at 30 °C, 250 rpm, and 85% RH until an OD of 600 0.6-0.8. The cell culture was then induced by isopropylthioglycoside (IPTG) to a final concentration of 1 mM and incubated overnight at 25°C, 270 rpm. The cells were then pelleted using centrifugation at 4000 rpm for 10 min. The supernatant was discarded and the pellet was frozen at -80°C before lysis.

[0223] The frozen precipitate was lysed with 200 μL of lysis buffer containing 50 mM sodium phosphate (NaPi) buffer, pH 7.4, 1 mg / mL lysozyme, 0.5 mg / mL PMBS, 17 mg / mL horseradish peroxidase (HRP) and 17 mg / mL catalase. The lysis mixture was shaken at room temperature (RT) for 2.5 hours. The plate was then centrifuged at 4000 rpm and 4 ° C for 10 min. The supernatant was then used as a clarified lysate for biocatalytic reactions to determine the activity level.

[0224] The library of SEQ ID NO: 2 was generated using well established techniques (eg recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced with HTP and clarified lysates were generated as described above.

[0225] Each 100 μL reaction was carried out in a 96-well deep well (2 mL volume) plate with 50 μL of clarified lysate solution, 50 g / L Compound X (2-ethynylglycerol, EGO), 50 mM NaPi buffer, 50 μM CuSO4 at pH 7.4. The plate was sealed with an O2 permeable seal and incubated at 30°C and shaken at 300 rpm in a 50 mm throw Infors shaker maintained at 85% RH overnight for 20 hours.

[0226] The wells were derivatized by taking a 50 μL aliquot and adding 10 μL (R)-(+)-1-amino-2-(methoxymethyl)pyrrolidine (R-AMP) and incubating with shaking in a 96-well round bottom (0.3 mL volume) plate for ~30 minutes at RT. The samples were quenched by adding 200 μL acetonitrile (MeCN), shaking briefly to mix, and centrifuging at 4000 rpm for 5 minutes at 4°C. The supernatant was transferred to a 96-well shallow well plate and heat sealed for analysis by Analytical Method 8.1.

[0227] Variant activity relative to the activity of SEQ ID NO: 2 was determined by the abundance of the 2-ethynylglyceraldehyde (EGA) peak in the SIM MS (227.3 m / z) of the data sample relative to the abundance of the corresponding SIM MS product peak of SEQ ID NO: 2. The amount of EGA was quantified by multiplying the MS product peak area by a factor calculated using a standard dose-response curve for EGA on MS using Analytical Method 8.1. The 10 samples with the most improved product production were selected for analysis using Analytical Method 9.1 to identify improved enantioselective variants.

[0228] The enantioselectivity relative to SEQ ID NO: 2 was calculated as the enantiomeric excess of the R-enantiomer of EGA formed (%ee R-compound Y) relative to the %ee R of the corresponding SEQ ID NO: 2. The enantiomeric selectivity was quantified by subtracting compound Y from the S-enantiomer of EGA (compound Z) and dividing the difference by the sum of the R-enantiomer and S-enantiomer product peaks determined by HPLC analysis.

[0229]

[0230] Example 2

[0231] Preparation of Galactose Oxidase (GOA) Wet Cell Pellet

[0232] The parent gene codons of the GOA (SEQ ID NO: 2) enzyme used to produce the variant of the present invention were optimized for expression in E. coli, and synthesized and cloned into the pCK900 vector (see, e.g., U.S. Patent No. 9,714,437, incorporated herein by reference). W3110 E. coli cells were transformed with the corresponding plasmid containing the GOA encoding gene and plated on LB agar plates containing 1% glucose and 30 μg / mL CAM and grown overnight at 37°C. Monoclonal colonies were selected and inoculated into 96-well shallow microtiter plates in 180 μL LB containing 1% glucose and 30 μg / mL CAM. The plate was sealed with an O2 permeable seal and the culture was grown overnight at 30°C, 200 rpm and 85% RH. Then, 10 μL of each cell culture was transferred to a well of a 96-well deep well plate containing 390 μL TB and 30 μg / mL CAM. The deep-well plate was sealed with an O2-permeable seal and incubated at 30 °C, 250 rpm, and 85% RH until an OD of 600 0.6-0.8. The cell culture was then induced by IPTG to a final concentration of 1 mM and incubated overnight at 30°C, 250 rpm. The cells were then pelleted using centrifugation at 4000 rpm for 10 min. The supernatant was discarded and the pellet was frozen at -80°C before lysis.

[0233] Example 3

[0234] Preparation of cell lysate containing HTP-GOA

[0235] The cryopreservation prepared as specified in Example 2 was lysed with 400 μL of lysis buffer containing 50 mM NaPi buffer, pH 7.4, 1 mg / mL lysozyme, 0.5 mg / mL PMBS. The lysis mixture was shaken at RT for 2 hours. The plate was then centrifuged at 4000 rpm and 4°C for 15 min. The supernatant was then used as a clarified lysate in the experiment described below for biocatalytic reactions to determine the activity level.

[0236] Example 4

[0237] Preparation of cell lysate containing Retest GOA

[0238] The cryopreservation prepared as specified in Example 2 was lysed with 150 μL of lysis buffer containing 50 mM NaPi buffer, pH 7.4, 1 mg / mL lysozyme, 0.5 mg / mL PMBS. The lysis mixture was shaken at RT for 2 hours. The plate was then centrifuged at 4000 rpm and 4°C for 15 min. The supernatant was then used as a clarified lysate in the experiment described below for biocatalytic reactions to determine the activity level.

[0239] Example 5

[0240] Improvements of GOA compared to SEQ ID NO: 4 for enantioselective production of compound Y

[0241] Select SEQ ID NO:4 as the parent enzyme for the next round of directed evolution. Use well-established technology (e.g., saturation mutagenesis, and the recombination of previously identified beneficial mutations) to produce a library of engineered genes. Produce polypeptides of every kind of gene encoding with HTP as described in Example 2, and produce clarified lysates as described in Example 3.

[0242] Each 100 μL reaction was performed in a 96-well deep well (2 mL volume) plate with 50 μL of clarified lysate, 20 g / L compound X, 50 mM NaPi buffer, 25 μM CuSO4, 0.25 g / L horseradish peroxidase (HRP), 0.25 g / L catalase at pH 7.4. The plate was sealed with an O2 permeable seal and incubated at 30°C and shaken at 300 rpm in a 50 mm throw Kuhner shaker maintained at 85% RH overnight.

[0243] The variants in the wells were derivatized by taking a 50 μL aliquot and adding 10 μL (R)-(+)-1-amino-2-(methoxymethyl)pyrrolidine (R-AMP) and incubating in a 96-well half-deep well (1 mL volume) plate with shaking for ~30 minutes at RT. The samples were quenched by adding 200 μL acetonitrile (MeCN), shaking briefly to mix, and centrifuging at 4000 rpm for 5 minutes at 4°C. The supernatant was transferred to a 96-well shallow well plate and heat sealed for analysis by analytical method 8.1 as described in Example 8.

[0244] The activity of each variant relative to the activity of SEQ ID NO: 4 was calculated as the % conversion of the product (compound Y+Z) formed / the corresponding conversion percentage of SEQ ID NO: 4. The conversion percentage was quantified by multiplying the HPLC product peak area by a factor calculated using a standard dose-response curve of compound Y on HPLC using analytical method 8.1. Variants with an activity of a fold improvement (FIOP) greater than 0.7 relative to the positive control were selected to identify improved enantioselective variants using analytical method 9.1 as described in Example 9.

[0245] The enantioselectivity of each variant relative to SEQ ID NO: 4 was calculated as the %ee R with respect to the enantiomeric excess of the compound Y product formed relative to the corresponding SEQ ID NO: 4. The enantioselectivity was quantified by subtracting compound Y from compound Z and dividing the difference by the sum of the Y and Z product peaks determined by HPLC analysis. The results are provided below.

[0246]

[0247]

[0248]

[0249] Example 6

[0250] Improvements of GOA compared to SEQ ID NO: 4 for enantioselective production of compound Y

[0251] Select SEQ ID NO:4 as the parent enzyme for this round of directed evolution. Use well-established techniques (e.g., saturation mutagenesis, and the recombination of previously identified beneficial mutations) to generate a library of engineered genes. Generate polypeptides encoded by each gene as described in Example 2 with HTP, and generate clarified lysates as described in Example 4.

[0252] Each 100 μL reaction was performed in a 96-well deep well plate with 50 μL of clarified lysate, 20 g / L compound X, 50 mM NaPi buffer, 25 μM CuSO4, 0.25 g / L HRP, 0.25 g / L catalase at pH 7.4. The plate was sealed with an O2 permeable seal and incubated at 30°C and shaken at 300 rpm in a 50 mm throw Kuhner shaker maintained at 85% RH overnight.

[0253] The enzyme variants in the wells were derivatized by taking a 50 μL aliquot and adding 10 μL R-AMP and incubating at RT in a 96-well half-deep well plate with shaking for ˜30 minutes. The samples were quenched by adding 200 μL MeCN, shaking briefly to mix, and centrifuging at 4000 rpm for 5 minutes at 4° C. The supernatant was transferred to a 96-well shallow well plate and heat sealed for analysis by analytical method 8.1.

[0254] The activity of each enzyme variant relative to the activity of SEQ ID NO: 4 was calculated as the % conversion of the product formed (compound Y+Z) / the corresponding conversion percentage of SEQ ID NO: 4. The conversion percentage was quantified by multiplying the HPLC product peak area by a factor calculated using a standard dose-response curve of compound Y on HPLC using analytical method 8.1. Variants with an activity of greater than 0.7 were selected for analysis using analytical method 9.1 to identify improved enantioselective variants.

[0255] The enantioselectivity of each variant relative to SEQ ID NO: 4 was calculated as the %ee ER EGA formed relative to the corresponding %ee R, EGA formed of SEQ ID NO: 4, as in Example 5. The results are provided below.

[0256]

[0257]

[0258] Example 7

[0259] Improvement of GOA compared to SEQ ID NO: 166 for enantioselective production of compound Y

[0260] Select SEQ ID NO:166 as the parent enzyme for this round of directed evolution. Use well-established technology (such as saturation mutagenesis, and the restructuring of previously identified beneficial mutations) to produce a library of engineered genes. Produce polypeptides of every kind of gene encoding with HTP as described in Example 2, and produce clarified lysates as described in Example 4.

[0261] Each 100 μL reaction was performed in a 96-well deep well plate with 45 μL of clarified lysate, 20 g / L Compound X, 50 mM NaPi buffer, 50 μM CuSO4, 0.25 g / L HRP, 0.25 g / L catalase at pH 7.4. The plate was sealed with an O2 permeable seal and incubated at 30°C and shaken at 300 rpm in a 50 mm throw Kuhner shaker maintained at 85% RH overnight.

[0262] The enzyme variants in the wells were derivatized by taking a 50 μL aliquot and adding 10 μL R-AMP and incubating in a 96-well half-deep well plate with shaking for ~30 minutes at RT. In the case of samples analyzed by analytical method 10.1 (as described in Example 10), the samples were quenched by adding 60 μL ethanol and then mixing, and further subsequently 20 μL of the diluted sample was transferred to a 96-well shallow well plate containing 120 μL water. The plate was shaken briefly and then analyzed.

[0263] In the case of samples analyzed by analytical method 8.1, samples were quenched by adding 200 μL MeCN, shaking briefly to mix, and centrifuging at 4000 rpm for 5 minutes at 4° C. The supernatant was transferred to a 96-well shallow plate and heat sealed for analysis.

[0264] The activity of each variant relative to the activity of SEQ ID NO: 166 was calculated as the ultraviolet (UV) absorbance of the R-AMP derivatized product formed at 247 nm / the corresponding UV absorbance of SEQ ID NO: 166 using assay method 10.1. 247 Absorbance. Variants with an activity of FIOP greater than 0.75 were selected for analysis using Analytical Method 9.1 (Example 9) to identify improved enantioselective variants.

[0265] The enantioselectivity of each variant relative to SEQ ID NO: 166 was calculated as the % ee R EGA formed relative to the corresponding % ee R EGA of SEQ ID NO: 166, as in Example 5.

[0266]

[0267]

[0268]

[0269]

[0270] Example 8

[0271] Analysis and Detection of 2-Ethynylglyceraldehyde Derivatized with R-AMP

[0272] The data described in Examples 1, 5, 6, 7, 11, 14, and 16 were collected using the analytical methods in Table 8.1. The methods provided herein can be used to analyze variants generated using the present invention. However, it is not intended that the present invention be limited to the methods described herein, as other suitable methods are known in the art that can be applied to analyze variants provided herein and / or generated using the methods provided herein.

[0273]

[0274]

[0275] Example 9

[0276] Analysis and Detection of Enantiomers of 2-Ethynylglyceraldehyde Derivatized with R-AMP

[0277] The data described in Examples 1, 5, 6, 7, 11, 14, 15, 17, and 18 were collected using the analytical methods provided in Table 9.1. The methods provided herein can be used to analyze variants generated using the present invention. However, it is not intended that the present invention be limited to the methods described herein, as other suitable methods are known in the art that can be applied to analyze variants provided herein and / or generated using the methods provided herein.

[0278]

[0279] Example 10

[0280] Spectrophotometric Detection of 2-Ethynylglyceraldehyde Derivatized with R-AMP

[0281] The data described in Example 7 were collected using the analysis methods provided in Table 10.1. The methods provided herein can be used to analyze variants generated using the present invention. However, it is not intended that the present invention be limited to the methods described herein, as other suitable methods are known in the art that can be applied to analyze variants provided herein and / or generated using the methods provided herein.

[0282]

[0283] Embodiment 11

[0284] Improvement of GOA over SEQ ID NO: 272 for enantioselective production of compound Y

[0285] Select SEQ ID NO:272 as the parent enzyme for this round of directed evolution. Use well-established technology (such as saturation mutagenesis, and the restructuring of previously identified beneficial mutations) to produce a library of engineered genes. As described in Example 2, produce every kind of gene-encoded polypeptide with HTP, and as described in Example 4, produce a clarified lysate.

[0286] Each 100 μL reaction was performed in a 96-well deep well plate with 50 μL of clarified lysate, 20 g / L compound X, 50 mM NaPi buffer, 25 μM CuSO4, 0.25 g / L HRP, 0.25 g / L catalase at pH 7.4. The plate was sealed with an O2 permeable seal and incubated at 30°C and shaken at 300 rpm in a 50 mm throw Kuhner shaker maintained at 85% RH overnight.

[0287] The enzyme variants in the wells were derivatized by taking a 50 μL aliquot and adding 10 μL of a 100 g / L solution of R-AMP in water and incubating at RT in a 96-well half-deep well plate with shaking for ~30 minutes. The samples were quenched by adding 200 μL MeCN, shaking briefly to mix, and centrifuging at 4000 rpm for 5 minutes at 4°C. The supernatant was transferred to a 96-well shallow well plate and heat sealed for analysis by analytical method 8.1.

[0288] The activity of each enzyme variant relative to the activity of SEQ ID NO: 272 was calculated as the % conversion of the product (compound Y+Z) formed / the corresponding conversion percentage of SEQ ID NO: 272. The conversion percentage was quantified by multiplying the HPLC product peak area by a factor calculated using a standard dose-response curve of compound Y on HPLC using analytical method 8.1. Variants with an activity of greater than 0.7 were selected for analysis using analytical method 9.1 to identify improved enantioselective variants.

[0289] The enantioselectivity of each variant relative to SEQ ID NO: 272 was calculated as the %ee R EGA formed relative to the corresponding %ee R EGA formed of SEQ ID NO: 272, as in Example 5. The results are provided below.

[0290]

[0291]

[0292]

[0293]

[0294]

[0295] Example 12

[0296] Improvement of GOA over SEQ ID NO: 272 for enantioselective production of compound Y

[0297] Select SEQ ID NO:272 as the parent enzyme for this round of directed evolution. Use well-established technology (such as saturation mutagenesis, and the restructuring of previously identified beneficial mutations) to produce a library of engineered genes. As described in Example 2, produce every kind of gene-encoded polypeptide with HTP, and as described in Example 4, produce a clarified lysate.

[0298] Each 100 μL reaction was performed in a 96-well deep well plate with 50 μL of clarified lysate, 20 g / L compound X, 50 mM NaPi buffer, 25 μM CuSO4, 0.25 g / L HRP, 0.25 g / L catalase at pH 7.4. The plate was sealed with an O2 permeable seal and incubated at 30°C and shaken at 300 rpm in a 50 mm throw Kuhner shaker maintained at 85% RH overnight.

[0299] The enzyme variants in the wells were derivatized by taking a 50 μL aliquot and adding 10 μL of a 100 mg / mL solution of R-AMP in water and incubating in a 96-well half-deep well plate with shaking for 30 minutes at RT. In the case of samples analyzed by analytical method 20.1 (as described in Example 20), the samples were quenched by adding 60 μL of ethanol and then mixing, and further subsequently 20 μL of the diluted samples were transferred to a 96-well shallow well plate containing 120 μL of water. The plate was shaken briefly and then analyzed.

[0300] The activity of each variant relative to the activity of SEQ ID NO: 272 was calculated as the ultraviolet (UV) absorbance of the R-AMP derivatized product formed at 340 nm / the corresponding UV absorbance of SEQ ID NO: 272 using Assay 20.1. 340 Absorbance.

[0301]

[0302]

[0303]

[0304]

[0305] Embodiment 13

[0306] Improvement of GOA over SEQ ID NO: 928 for enantioselective production of compound Y

[0307] In this round of directed evolution, the strep tag was removed from the C-terminus of SEQ ID NO: 908 and a His tag was added. The resulting sequence SEQ ID NO: 928 was selected as the parent enzyme for this round of directed evolution. Libraries of engineered genes were generated using well-established techniques (e.g., saturation mutagenesis, and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were generated with HTP as described in Example 2, and clarified lysates were generated as described in Example 4.

[0308] Each 100 μL reaction was performed in a 96-well deep well plate with 10 μL of clarified lysate, 30 g / L compound X, 50 mM MES buffer, 200 μM CuSO4, 0.20 g / L HRP, 0.20 g / L catalase at pH 7.4. The plate was sealed with an O2 permeable seal and incubated at 30°C and shaken at 300 rpm in a 50 mm throw Kuhner shaker maintained at 85% RH overnight.

[0309] The enzyme variants in the plate wells are derivatized by taking 50 μL aliquots and adding 10 μL of a 100 mg / mL solution of R-AMP in water and incubating in a 96-well semi-deep well plate with shaking for 30 minutes at RT. In the case of analyzing samples by analytical method 20.1 (as described in Example 20), the samples are quenched by adding 60 μL of ethanol and then mixing, and further subsequently 20 μL of the diluted samples are transferred to a 96-well shallow well plate containing 120 μL of water. The samples are further diluted in the assay plate to prepare a 100-fold final dilution. The plate is briefly shaken and then analyzed.

[0310] The activity of each variant relative to the activity of SEQ ID NO: 928 was calculated as the ultraviolet (UV) absorbance of the R-AMP derivatized product formed at 340 nm / the corresponding UV absorbance of SEQ ID NO: 928 using Assay 20.1. 340 Absorbance.

[0311]

[0312]

[0313]

[0314]

[0315] Embodiment 14

[0316] Improvement of GOA over SEQ ID NO: 928 for enantioselective production of compound Y

[0317] Select SEQ ID NO:928 as the parent enzyme for this round of directed evolution. Use well-established technology (such as saturation mutagenesis, and the restructuring of previously identified beneficial mutations) to produce a library of engineered genes. As described in Example 2, produce the polypeptide of every kind of gene encoding with HTP, and as described in Example 4, produce the lysate of clarification.

[0318] Each 100 μL reaction was performed in a 96-well deep well plate with 15 μL of clarified lysate, 21 g / L compound X, 9 g / L compound Y, 50 mM MES buffer, 200 μM CuSO4, 0.20 g / L HRP, 0.20 g / L catalase at pH 7.4. The plate was sealed with an O2 permeable seal and incubated at 30°C and shaken at 300 rpm in a 50 mm throw Kuhner shaker maintained at 85% RH overnight.

[0319] The enzyme variants in the plate wells were diluted 3 times with acetonitrile by adding 20uL of the reaction to a 96-well shallow well plate containing 40uL of acetonitrile. 20μL of this 3-fold diluted reaction was derivatized by adding 10μL of a 100g / L solution of R-AMP in acetonitrile and incubating in a 96-well half-deep well plate at 30°C with shaking for ~45 minutes. The sample was quenched by adding 170μL of MeCN, briefly shaken to mix, and centrifuged at 4000rpm for 5 minutes at 4°C. The supernatant was transferred to a 96-well shallow well plate and heat sealed for analysis by analytical method 8.1 (Table 14.1) or 9.1 (Table 14.2).

[0320]

[0321]

[0322]

[0323] Embodiment 15

[0324] Improvement of GOA over SEQ ID NO: 932 for enantioselective production of compound Y

[0325] Select SEQ ID NO:932 as the parent enzyme for this round of directed evolution. Use well-established technology (e.g., saturation mutagenesis, and the reorganization of previously identified beneficial mutations) to produce a library of engineered genes. As described in Example 2, produce every kind of gene-encoded polypeptide with HTP, and produce a clarified lysate as described in Example 4.

[0326] Each 100 μL reaction was performed in a 96-well deep well plate with 7.5 μL of clarified lysate from a total lysate volume of 200 uL, 30 g / L Compound X, 50 mM MES buffer, 200 μM CuSO4, 5 g / L HRP, 0.2 g / L catalase at pH 7.4. The plate was sealed with an O2 permeable seal and incubated at 30°C and shaken at 300 rpm in a 50 mm throw Kuhner shaker maintained at 85% RH overnight.

[0327] The enzyme variants in the wells were diluted 3-fold with acetonitrile by adding 20uL of the reaction to a 96-well shallow well plate containing 40uL of acetonitrile. 20μL of this 3-fold diluted reaction was derivatized by adding 10μL of a 100g / L solution of R-AMP in acetonitrile and incubating in a 96-well half-deep well plate at 30°C with shaking for ~45 minutes. The sample was quenched by adding 170μL of MeCN, briefly shaking to mix, and centrifuged at 4000rpm for 5 minutes at 4°C. The supernatant was transferred to a 96-well shallow well plate and heat sealed for analysis by analytical method 9.1.

[0328] The enantioselectivity of each variant relative to SEQ ID NO: 932 was calculated as the %ee R EGA formed relative to the corresponding %ee R EGA of SEQ ID NO: 932, as in Example 5. The results are provided below.

[0329]

[0330] Example 16

[0331] Improvement of GOA over SEQ ID NO: 1264 for enantioselective production of compound Y

[0332] Select SEQ ID NO:1264 as the parent enzyme for this round of directed evolution. Use the technology of good establishment (for example saturation mutagenesis, and the restructuring of the previously identified beneficial mutation) to produce the library of engineered genes. As described in Example 2, produce the polypeptide of every kind of gene encoding with HTP, and as described in Example 4, produce the lysate of clarification, but use 300ul every hole lysis volume.

[0333] Each 100 μL reaction was performed in a 96-well deep well plate with 50 μL of clarified lysate from a total lysis volume of 300 uL, 18 g / L Compound X, 12 g / L Compound Y, ˜80 mM NaPi buffer, 200 μM CuSO 4 , 0.20 g / L HRP, 0.20 g / L Catalase at pH 7.4. The plate was sealed with an O 2 permeable seal and incubated at 30° C. and shaken at 300 rpm in a 50 mm throw Kuhner shaker maintained at 85% RH overnight.

[0334] The enzyme variants in the plate wells were diluted 2-fold with acetonitrile by adding 35uL of the reaction to a 96-well shallow well plate containing 35uL of acetonitrile. 20μL of this 2-fold diluted reaction was derivatized by adding 10μL of a 100g / L solution of R-AMP in acetonitrile and incubating in a 96-well half-deep well plate at 30°C with shaking for ~45 minutes. The sample was quenched by adding 200μL of MeCN, briefly shaking to mix, and centrifuged at 4000rpm for 5 minutes at 4°C. The supernatant was transferred to a 96-well shallow well plate and heat sealed for analysis by analytical method 8.1.

[0335]

[0336]

[0337] Embodiment 17

[0338] Improvement of GOA over SEQ ID NO: 1264 for enantioselective production of compound Y

[0339] Select SEQ ID NO:1264 as the parent enzyme for this round of directed evolution. Use the technology of good establishment (for example saturation mutagenesis, and the restructuring of the previously identified beneficial mutation) to produce the library of engineered genes. As described in Example 2, produce the polypeptide by every kind of gene encoding with HTP, and as described in Example 4, produce the lysate of clarification, but use 200uL every hole lysis volume.

[0340] Each 100 μL reaction was performed in a 96-well deep well plate with 10 μL of clarified lysate from a total lysis volume of 200 uL, 30 g / L Compound X, ~100 mM NaPi buffer, 200 μM CuSO4, 5 g / L HRP, 0.20 g / L Catalase at pH 7.4. The plate was sealed with an O2 permeable seal and incubated at 30°C and shaken at 300 rpm in a 50 mm throw Kuhner shaker maintained at 85% RH overnight.

[0341] The enzyme variants in the plate wells were diluted 2-fold with acetonitrile by adding 35uL of the reaction to a 96-well shallow well plate containing 35uL of acetonitrile. 20μL of this 2-fold diluted reaction was derivatized by adding 10μL of a 100g / L solution of R-AMP in acetonitrile and incubating in a 96-well half-deep well plate at 30°C with shaking for ~45 minutes. The sample was quenched by adding 200μL of MeCN, briefly shaking to mix, and centrifuged at 4000rpm for 5 minutes at 4°C. The supernatant was transferred to a 96-well shallow well plate and heat sealed for analysis by analytical method 9.1.

[0342]

[0343]

[0344] Embodiment 18

[0345] Improvement of GOA over SEQ ID NO: 1416 for enantioselective production of compound Y

[0346] Select SEQ ID NO:1416 as the parent enzyme for this round of directed evolution. Use the technology of good establishment (for example saturation mutagenesis, and the restructuring of the previously identified beneficial mutation) to produce the library of engineered genes. As described in Example 2, produce the polypeptide of every kind of gene encoding with HTP, and as described in Example 4, produce the lysate of clarification, but use 200uL every hole lysis volume.

[0347] Each 100 μL reaction was performed in a 96-well deep well plate with 40 μL of clarified lysate from a total lysis volume of 200 uL, 30 g / L Compound X, ~200 mM NaPi buffer, 200 μM CuSO4, 0.2 g / L HRP, 0.20 g / L Catalase at pH 6.5. The plate was heat sealed and incubated at 30°C and shaken at 300 rpm for 4 hours in a 50 mm throw Kuhner shaker maintained at 85% RH.

[0348] The enzyme variants in the plate wells were diluted 2-fold with acetonitrile by adding 35uL of the reaction to a 96-well shallow well plate containing 35uL of acetonitrile. 20μL of this 2-fold diluted reaction was derivatized by adding 10μL of a 100g / L solution of R-AMP in acetonitrile and incubating in a 96-well half-deep well plate at 30°C with shaking for ~45 minutes. The sample was quenched by adding 200μL of MeCN, briefly shaking to mix, and centrifuged at 4000rpm for 5 minutes at 4°C. The supernatant was transferred to a 96-well shallow well plate and heat sealed for analysis by analytical method 9.1.

[0349]

[0350]

[0351]

[0352]

[0353] Embodiment 19

[0354] Improvement of GOA over SEQ ID NO: 1598 for enantioselective production of compound Y

[0355] Select SEQ ID NO:1598 as the parent enzyme for this round of directed evolution. Use well-established technology (such as saturation mutagenesis, and the restructuring of previously identified beneficial mutations) to produce a library of engineered genes. Produce polypeptides of every kind of gene encoding with HTP as described in Example 2, and produce clarified lysates as described in Example 4.

[0356] Each 100 μL reaction was performed in a 96-well deep well plate with 40 μL of clarified lysate from a total lysis volume of 200 uL, 30 g / L Compound X, ˜200 mM NaPi buffer, 200 μM CuSO 4 , 0.2 g / L HRP, 0.20 g / L Catalase at pH 6.5. The plate was sealed with an O 2 permeable seal and incubated at 30° C. and shaken at 300 rpm for 4 hours in a 50 mm throw Kuhner shaker maintained at 85%.

[0357] The enzyme variants in the plate wells were diluted 2-fold with acetonitrile by adding 35uL of the reaction to a 96-well shallow well plate containing 35uL of acetonitrile. 20μL of this 2-fold diluted reaction was derivatized by adding 10μL of a 100g / L solution of S-AMP in acetonitrile and incubating in a 96-well half-deep well plate at 30°C with shaking for ~45 minutes. The sample was quenched by adding 200μL of MeCN, briefly shaken to mix, and centrifuged at 4000rpm for 5 minutes at 4°C. The supernatant was transferred to a 96-well shallow well plate and heat sealed for analysis by analytical method 21.1.

[0358]

[0359]

[0360]

[0361] Embodiment 20

[0362] Spectrophotometric Detection of 2-Ethynylglyceraldehyde Derivatized with R-AMP

[0363] The data described in Examples 12 and 13 were collected using the analytical methods provided in Table 20.1. The methods provided herein can be used to analyze variants generated using the present invention. However, it is not intended that the present invention be limited to the methods described herein, as other suitable methods are known in the art that may be applicable to analyzing variants provided herein and / or generated using the methods provided herein.

[0364]

[0365] Embodiment 21

[0366] Analysis and Detection of Enantiomers of 2-Ethynylglyceraldehyde Derivatized with R-AMP

[0367] The data described in Examples 19, 22, 28, and 30 were collected using the analytical methods provided in Table 21.1. The methods provided herein can be used to analyze variants generated using the present invention. However, it is not intended that the present invention be limited to the methods described herein, as other suitable methods are known in the art that may be applicable to analyzing variants provided herein and / or generated using the methods provided herein.

[0368]

[0369] Embodiment 22

[0370] Improvement of GOA over SEQ ID NO: 1866 for enantioselective production of compound Y

[0371] Select SEQ ID NO:1866 as the parent enzyme for this round of directed evolution. Use well-established technology (such as saturation mutagenesis, and the restructuring of previously identified beneficial mutations) to produce a library of engineered genes. As described in Example 2, produce every kind of gene-encoded polypeptide with HTP, and as described in Example 4, produce a clarified lysate.

[0372] Each 100 μL reaction was performed in a 96-well deep well plate with 40 μL of clarified lysate from a total lysis volume of 200 uL, 30 g / L Compound X, ˜200 mM NaPi buffer, 200 μM CuSO 4 , 0.2 g / L HRP, 0.20 g / L Catalase at pH 6.5. The plate was sealed with an O 2 permeable seal and incubated at 30° C. and shaken at 300 rpm for 4 hours in a 50 mm throw Kuhner shaker maintained at 85% RH.

[0373] The enzyme variants in the plate wells were diluted 2-fold with acetonitrile by adding 35uL of the reaction to a 96-well shallow well plate containing 35uL of acetonitrile. 20μL of this 2-fold diluted reaction was derivatized by adding 10μL of a 100g / L solution of S-AMP in acetonitrile and incubating in a 96-well half-deep well plate at 30°C with shaking for ~45 minutes. The sample was quenched by adding 200μL of MeCN, briefly shaken to mix, and centrifuged at 4000rpm for 5 minutes at 4°C. The supernatant was transferred to a 96-well shallow well plate and heat sealed for analysis by analytical method 21.1.

[0374]

[0375]

[0376] Embodiment 23

[0377] Improvement of GOA over SEQ ID NO: 1912 for enantioselective production of compound Y

[0378] Select SEQ ID NO:1912 as the parent enzyme for this round of directed evolution. Use well-established technology (such as saturation mutagenesis, and the restructuring of previously identified beneficial mutations) to produce a library of engineered genes. As described in Example 2, produce the polypeptide of every kind of gene encoding with HTP, and as described in Example 4, produce the lysate of clarification.

[0379] Each 100 μL reaction was performed in a 96-well deep well plate with 20 μL of clarified lysate from a total lysis volume of 200 uL, 30 g / L Compound X, ˜200 mM NaPi buffer, 200 μM CuSO 4 , 0.2 g / L HRP, 0.20 g / L Catalase at pH 6.5. The plate was sealed with an O 2 permeable seal and incubated at 30° C. and shaken at 300 rpm for 4 hours in a 50 mm throw Kuhner shaker maintained at 85% RH.

[0380] The enzyme variants in the plate wells were diluted 10-fold with 0.04% TFA in acetonitrile by adding 20uL of the reaction to a 96-deep well plate containing 180uL of 0.04% TFA in acetonitrile. The samples were centrifuged at 4000rpm for 5 minutes at 4°C. The supernatant was diluted 4-fold with DI water by adding 50uL of the supernatant to a 96-shallow well plate containing 150uL of deionized (DI) water. The plate was heat sealed for analysis by analytical method 24.1.

[0381]

[0382]

[0383] Embodiment 24

[0384] Analysis and detection of the conversion of compound X to compound Y

[0385] The data described in Examples 23, 25, 26, 27, and 31 were collected using the analytical methods provided in Table 24.1. The methods provided herein can be used to analyze variants generated using the present invention. However, it is not intended that the present invention be limited to the methods described herein, as other suitable methods are known in the art that may be applicable to analyzing variants provided herein and / or generated using the methods provided herein.

[0386]

[0387]

[0388] Embodiment 25

[0389] Improvement of GOA over SEQ ID NO: 1912 for enantioselective production of compound Y

[0390] Select SEQ ID NO:1912 as the parent enzyme for this round of directed evolution. Use well-established technology (such as saturation mutagenesis, and the restructuring of previously identified beneficial mutations) to produce a library of engineered genes. As described in Example 2, produce the polypeptide of every kind of gene encoding with HTP, and as described in Example 4, produce the lysate of clarification.

[0391] Each 100 μL reaction was performed in a 96-well deep well plate with 60 μL of clarified lysate from a total lysis volume of 200 uL, 30 g / L Compound X, ˜200 mM NaPi buffer, 200 μM CuSO 4 , 0.2 g / L HRP, 0.20 g / L Catalase at pH 6.5. The plate was sealed with an O 2 permeable seal and incubated at 30° C. and shaken at 300 rpm for 22 hours in a 50 mm throw Kuhner shaker maintained at 85% RH.

[0392] The enzyme variants in the plate wells were diluted 10-fold with 0.04% TFA in acetonitrile by adding 20uL of reaction to a 96-deep well plate containing 180uL of 0.04% TFA in acetonitrile. The samples were centrifuged at 4000rpm for 5 minutes at 4°C. The supernatant was diluted 4-fold with DI water by adding 50uL of supernatant to a 96-shallow well plate containing 150uL of DI water. The plates were heat sealed for analysis by analytical method 24.1.

[0393]

[0394]

[0395]

[0396] Embodiment 26

[0397] Improvements of GOA compared to SEQ ID NO: 2080 for enantioselective production of compound Y

[0398] Select SEQ ID NO:2080 as the parent enzyme for this round of directed evolution. Use well-established technology (such as saturation mutagenesis, and the restructuring of previously identified beneficial mutations) to produce a library of engineered genes. As described in Example 2, produce the polypeptide of every kind of gene encoding with HTP, and as described in Example 4, produce the lysate of clarification.

[0399] Each 100 μL reaction was performed in a 96-well deep well plate with 30 μL of clarified lysate from a total lysis volume of 200 uL, 30 g / L Compound X, ˜200 mM NaPi buffer, 200 μM CuSO 4 , 0.2 g / L HRP, 0.20 g / L Catalase at pH 6.5. The plate was sealed with an O 2 permeable seal and incubated at 30° C. and shaken at 300 rpm for 2 hours in a 50 mm throw Kuhner shaker maintained at 85% RH.

[0400] The enzyme variants in the plate wells were diluted 10-fold with 0.04% TFA in acetonitrile by adding 20uL of reaction to a 96-deep well plate containing 180uL of 0.04% TFA in acetonitrile. The samples were centrifuged at 4000rpm for 5 minutes at 4°C. The supernatant was diluted 4-fold with DI water by adding 50uL of supernatant to a 96-shallow well plate containing 150uL of DI water. The plates were heat sealed for analysis by analytical method 24.1.

[0401]

[0402] Embodiment 27

[0403] Improvements of GOA compared to SEQ ID NO: 2080 for enantioselective production of compound Y

[0404] Select SEQ ID NO:2080 as the parent enzyme for this round of directed evolution. Use well-established technology (such as saturation mutagenesis, and the restructuring of previously identified beneficial mutations) to produce a library of engineered genes. As described in Example 2, produce the polypeptide of every kind of gene encoding with HTP, and as described in Example 4, produce the lysate of clarification.

[0405] Each 100 μL reaction was performed in a 96-well deep well plate with 60 μL of clarified lysate from a total lysis volume of 200 uL, 30 g / L Compound X, ˜200 mM NaPi buffer, 200 μM CuSO 4 , 0.2 g / L HRP, 0.20 g / L Catalase at pH 6.5. The plate was sealed with an O 2 permeable seal and incubated at 30° C. and shaken at 300 rpm for 22 hours in a 50 mm throw Kuhner shaker maintained at 85% RH.

[0406] The enzyme variants in the plate wells were diluted 10-fold with 0.04% TFA in acetonitrile by adding 20uL of reaction to a 96-deep well plate containing 180uL of 0.04% TFA in acetonitrile. The samples were centrifuged at 4000rpm for 5 minutes at 4°C. The supernatant was diluted 4-fold with DI water by adding 50uL of supernatant to a 96-shallow well plate containing 150uL of DI water. The plates were heat sealed for analysis by analytical method 24.1.

[0407]

[0408]

[0409]

[0410] Embodiment 28

[0411] Improvements of GOA compared to SEQ ID NO: 2300 for enantioselective production of compound Y

[0412] Select SEQ ID NO:2300 as the parent enzyme for this round of directed evolution. Use well-established technology (such as saturation mutagenesis, and the restructuring of previously identified beneficial mutations) to produce a library of engineered genes. As described in Example 2, produce the polypeptide of every kind of gene encoding with HTP, and as described in Example 4, produce the lysate of clarification.

[0413] Each 100 μL reaction was performed in a 96-well deep well plate with 60 μL of clarified lysate from a total lysis volume of 200 uL, 30 g / L Compound X, ˜200 mM NaPi buffer, 200 μM CuSO 4 , 0.2 g / L HRP, 0.20 g / L Catalase at pH 6.5. The plate was sealed with an O 2 permeable seal and incubated at 30° C. and shaken at 300 rpm for 22 hours in a 50 mm throw Kuhner shaker maintained at 85% RH.

[0414] The enzyme variants in the plate wells were diluted 2-fold with acetonitrile by adding 35uL of the reaction to a 96-well shallow well plate containing 35uL of acetonitrile. 20μL of this 2-fold diluted reaction was derivatized by adding 10μL of a 100g / L solution of S-AMP in acetonitrile and incubating in a 96-well half-deep well plate at 30°C with shaking for ~45 minutes. The sample was quenched by adding 200μL of MeCN, briefly shaken to mix, and centrifuged at 4000rpm for 5 minutes at 4°C. The supernatant was transferred to a 96-well shallow well plate and heat sealed for analysis by analytical method 21.1.

[0415]

[0416]

[0417] Embodiment 29

[0418] Improved Compound P production, GOA compared to SEQ ID NO: 2300

[0419] Directed evolution efforts were performed focusing on evolving GO enzyme variants with improved activity on ethynylglycerolphosphate (EGP) to produce the corresponding phosphorylated aldehyde (Compound P) (see Scheme 3 above).

[0420] Select SEQ ID NO:2300 as the parent enzyme for this round of directed evolution. Use well-established technology (such as saturation mutagenesis, and the restructuring of previously identified beneficial mutations) to produce a library of engineered genes. As described in Example 2, produce the polypeptide of every kind of gene encoding with HTP, and as described in Example 4, produce the lysate of clarification.

[0421] Each 100 μL reaction was performed in a 96-well deep well plate with 20 μL of 2-fold diluted clarified lysate from a total lysis volume of 200 uL, 10 g / L ethynyl glycerol phosphate, 200 μM CuSO4, 1 g / L HRP, 0.20 g / L catalase, 50 mM PIPES buffer at pH 7.0. The plate was heat sealed and shaken at 400 rpm at 30°C for 3 hours.

[0422] After 3 hours, the sample was diluted with 200 μL 50 mM potassium phosphate, pH 7.5. In a separate plate, 50 μL of the diluted sample was transferred and mixed with 150 μL of a 10 g / L solution of o-benzylhydroxylamine in methanol. The plate was sealed and shaken at 400 rpm at 25° C. for 20-30 minutes. The derivatized samples were diluted 2x in methanol and then subjected to UPLC analysis as described in Table 29.1.

[0423]

[0424]

[0425]

[0426] Embodiment 30

[0427] Improvement of GOA over SEQ ID NO: 2424 for enantioselective production of compound Y

[0428] Select SEQ ID NO:2424 as the parent enzyme for this round of directed evolution. Use well-established technology (such as saturation mutagenesis, and the restructuring of previously identified beneficial mutations) to produce a library of engineered genes. As described in Example 2, produce the polypeptide of every kind of gene encoding with HTP, and as described in Example 4, produce the lysate of clarification.

[0429] Each 100 μL reaction was performed in a 96-well deep well plate with 60 μL of clarified lysate from a total lysis volume of 200 uL, 30 g / L Compound X, 50 mM PIPES buffer, 200 μM CuSO4, 0.2 g / L HRP, 0.20 g / L Catalase at pH 6.5. The plate was sealed with an O2 permeable seal and incubated at 30°C and shaken at 300 rpm for 22 hours in a 50 mm throw Kuhner shaker maintained at 85% RH.

[0430] The enzyme variants in the wells were diluted 2-fold with acetonitrile by adding 35uL of the reaction to a 96-well shallow well plate containing 35uL of acetonitrile. 20μL of this 2-fold diluted reaction was derivatized by adding 10μL of a 100g / L solution of S-AMP in acetonitrile and incubating with shaking at 30°C in a 96-well half-deep well plate for ~45 minutes. The sample was quenched by adding 200μL of a MeCN:heptane (1:1) mixture, shaking briefly to mix, and centrifuging at 4000rpm for 5 minutes at 4°C. The supernatant was transferred to a 96-well shallow well plate and heat sealed for analysis by analytical method 21.1.

[0431]

[0432]

[0433]

[0434]

[0435] Embodiment 31

[0436] Improvement of GOA over SEQ ID NO: 2424 for enantioselective production of compound Y

[0437] Select SEQ ID NO:2424 as the parent enzyme for this round of directed evolution. Use well-established technology (such as saturation mutagenesis, and the restructuring of previously identified beneficial mutations) to produce a library of engineered genes. As described in Example 2, produce the polypeptide of every kind of gene encoding with HTP, and as described in Example 4, produce the lysate of clarification.

[0438] Each 100 μL reaction was performed in a 96-well deep well plate with 60 μL of clarified lysate from a total lysis volume of 200 uL, 30 g / L Compound X, 50 mM PIPES buffer, 200 μM CuSO4, 0.2 g / L HRP, 0.20 g / L Catalase at pH 6.5. The plate was sealed with an O2 permeable seal and incubated at 30°C and shaken at 300 rpm for 22 hours in a 50 mm throw Kuhner shaker maintained at 85% RH.

[0439] The enzyme variants in the plate wells were diluted 10-fold with 0.04% TFA in acetonitrile by adding 20uL of reaction to a 96-deep well plate containing 180uL of 0.04% TFA in acetonitrile. The samples were centrifuged at 4000rpm for 5 minutes at 4°C. The supernatant was diluted 4-fold with DI water by adding 50uL of supernatant to a 96-shallow well plate containing 150uL of DI water. The plates were heat sealed for analysis by analytical method 24.1.

[0440]

[0441]

[0442]

[0443] 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.

[0444] 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 galactose oxidase, wherein the amino acid residues of the engineered galactose oxidase differ from those of SEQ ID NO: 2 by K331R, F406Y and F465A / Q, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:

2.

2. An engineered galactose oxidase, wherein the amino acid residues of the engineered galactose oxidase differ from those of SEQ ID NO: 2 by K331R, F406Y, F465A and E407Q, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 2, and wherein the amino acid residues of the engineered galactose oxidase differ from those of the wild-type Fusarium graminearum ( F. graminearium ) galactose oxidase comprises improved stereoselectivity compared to galactose oxidase.

3. An engineered galactose oxidase, wherein the amino acid residue difference between the polypeptide sequence of the engineered galactose oxidase and SEQ ID NO: 4 is 1 selected from I463K / V / R, E407V, L204S / Q / V, G197K, T520V / L / P / G / S, I202T / C, R191A, G517S / L / D / E / M, P199T / G / R, F296A / W / S / L, E466R, V220P / E / R, A324G / S, T243V / C, N192I / Q / M, A248T / E , T205A, G294N / K / S, L515T, D247G, N522S, A173S / C, D193T, T521S / V / G / P, V269Q / Y, A465G, I170L, S198A / T / G, V171A / L / C, S252T, V493G, M227L, A194V, S332R and G197S, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 4, and wherein the engineered galactose oxidase comprises improved stereoselectivity compared to wild-type Fusarium graminearum galactose oxidase.

4. An engineered galactose oxidase, wherein the polypeptide sequence of the engineered galactose oxidase is the same as SEQ ID NO:4 has an amino acid residue difference of one selected from V295N / G / R / E / S, A465T / M / G, E407M / I / F, S252R / V / M / T, T223N / L / H / M, V220P / C / S / E / M, G517K / S / D, S332Q, A173S, T521Y / A / V / Q / G, T203V, P199S / N / A, T243A / S, G197S / T / K, G294E / Q / S, F296S, V171A, L515V, E466R / G, T520A / S, S188T, N192Q and V493T, wherein the amino acid position of the polypeptide sequence refers to SEQ ID NO: 4, and wherein the engineered galactose oxidase comprises improved stereoselectivity compared to wild-type Fusarium graminearum galactose oxidase.

5. An engineered galactose oxidase, wherein the amino acid residue difference between the polypeptide sequence of the engineered galactose oxidase and SEQ ID NO: 166 is 1-7 amino acid residue differences selected from V220E / S / C / M, S252V, S332Q, E407I / V / Q / M, F296V / S, A465T / G / M, V171C / L, T520A, T521G / Q, R295E / G / S / N, T243V / A, G517R / H / N, L204A / S, A277T, A173S, G294E / S, N192Q / T, T221I, M227N, L515V, V284I and S198R / A, wherein the amino acid positions of the polypeptide sequence refer to SEQ ID NO: 166, and wherein the engineered galactose oxidase comprises improved stereoselectivity compared to wild-type Fusarium graminearum galactose oxidase.

6. An engineered galactose oxidase, wherein the amino acid residue difference between the polypeptide sequence of the engineered galactose oxidase and SEQ ID NO: 272 is 1-10 selected from S220E, R295E / T / S / Q / D, T520A, T521G, N192Q, T243S, T465G / F, A16S, Q553S, V222D / T / Y, N29Y / T, M279T / L, S426L / A / P, A16E, S24E / A / P / Q, Y56I / F, Q148A / R, S92V / D, E196D, S304C, V296T / E / L, T63V , S257D, R560W / T, R637L, I499F / V, A46V, K36P, R549G / Q / W, S8V / I, S258L, S363E / L, N134H, F43Q / A / E, N597D, A4Q, S567M, S103I, A194E / G, N319S / R, Q481D, F228W, R460A / Q, N598E, A194G and K571A, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 272, and wherein the engineered galactose oxidase comprises improved stereoselectivity compared to wild-type Fusarium graminearum galactose oxidase.

7. An engineered galactose oxidase, wherein the amino acid residue difference between the polypeptide sequence of the engineered galactose oxidase and SEQ ID NO: 272 is 1-10 selected from S24P, S92D / V, V222D / Y, M279L / T, N319S / R, R637L, F43Q / A / E, Q148A / R, R560W / T, I499F / V, K36P, R295E / D / Q / S / T, S363L / E, S220E, S304C, A16E / S, T63V, V296E / T, S426L / The engineered galactose oxidase comprises an amino acid residue difference of: A194E / G, A243S, B194E / G, B243S, C329A, C551A, C571A, N598E / L, N192Q, R460A / Q, S257D / N, and A4Q, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 272, and wherein the engineered galactose oxidase comprises improved stereoselectivity compared to wild-type Fusarium graminearum galactose oxidase.

8. An engineered galactose oxidase, wherein the amino acid residue difference between the polypeptide sequence of the engineered galactose oxidase and SEQ ID NO: 928 is 1-10 selected from A4Q, F43Q / E / A, A46V, S426L, R549G / W, R560W, T63V, S258L, R295Q / T, S567M, K571A, E196D, N319S / R, I56F / V, Q148A, M279L / T, S363L, N598E / L, A16S / E, S92D, Q192N, R637L, K36P / V, N597D , N29T / Y / H, S220E, V222D, S24P, V296S, E407I, T465G, G294E, T520A, N237D, N538D, S537G, N134A, T483R, S568E / P, K486P, K556A / V / S, S564W / D / E / T, T95E, S609D, S433G, K224D and K343G, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 928, and wherein the engineered galactose oxidase comprises improved stereoselectivity compared to wild-type Fusarium graminearum galactose oxidase.

9. An engineered galactose oxidase, wherein the amino acid residue difference between the polypeptide sequence of the engineered galactose oxidase and SEQ ID NO:928 is 1-6 amino acid residue differences selected from the group consisting of A4Q, F43Q, A46V, S426L, R549G, R560W, T520A, N597D, K36V, T63V, N598E, T562D, T483R, S568D / P, T95V and K394A, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:928, and wherein the engineered galactose oxidase comprises improved stereoselectivity compared to wild-type Fusarium graminearum galactose oxidase.

10. An engineered galactose oxidase, wherein the amino acid residue difference between the polypeptide sequence of the engineered galactose oxidase and SEQ ID NO: 928 is 1-4 amino acid residue differences selected from the group consisting of G294E, E407M / V, T465G, S220M / E, V296S, S332Q, V638A, T63A, E196L / A / R / G / Q / V / I, G197R / Q / P / A / E, A194R / W / V, S198T / G, V447I, S290G / A, Q327R, S198G, A173V / S, S189A and S292G, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 928, and wherein the engineered galactose oxidase comprises improved stereoselectivity compared to wild-type Fusarium graminearum galactose oxidase.

11. An engineered galactose oxidase, wherein the amino acid residue difference between the polypeptide sequence of the engineered galactose oxidase and SEQ ID NO: 932 is 1-10 selected from the group consisting of A16E, Q43A / E, S220M, L258S, N538D, R637L, L426S, T465G, R549W / G, E407I, G294E, Q295S, N319S, M267T, V63T, T95E / V, A173S, K343G, S564D / W, S568P, S609D, K556V, T18K, S24P, V222D, T520A, N597D, N237D, P265S, M279L and A194R, wherein the amino acid positions of the polypeptide sequence refer to SEQ ID NO: NO: 932, and wherein the engineered galactose oxidase comprises improved stereoselectivity compared to wild-type Fusarium graminearum galactose oxidase.

12. An engineered galactose oxidase, wherein the amino acid residue difference between the polypeptide sequence of the engineered galactose oxidase and SEQ ID NO: 1264 is 1-6 selected from Q43E / F, N237D, T520A / N / E, N597D, M279L, G294E, R549G / E, N538D, N598E, K51P, T55W / M / R, T111Q, S150P, K367I, W564D / K, K371D / A / P, T594Q / C, T18K, V95E, Q327R, T548M, S258H, C229S, S243K / L, K342R / S, T6 The engineered galactose oxidase comprises an amino acid residue difference of: 35K, S468N, S604M / G, S568A, F291V, K36N, S312T, R183D, K224G, C384G, K61E, S570K, R637N / W, Q295T, S99H, M567G, C28S / P, V46A, I56Y, V63T, R191V, R544P, K343S, Y485L, H335R, S252G, S198R and T596G, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 1264, and wherein the engineered galactose oxidase comprises improved stereoselectivity compared to wild-type Fusarium graminearum galactose oxidase.

13. An engineered galactose oxidase, wherein the amino acid residue difference between the polypeptide sequence of the engineered galactose oxidase and SEQ ID NO: 1264 is 1-5 amino acid residue differences selected from the group consisting of C28P, S99H, T520E, R637N, S403P, K224G, K61E, K343S, R637W, Q295T, K342S, S568A, T594C and T55R, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 1264, and wherein the engineered galactose oxidase comprises improved stereoselectivity compared to wild-type Fusarium graminearum galactose oxidase.

14. An engineered galactose oxidase, wherein the amino acid residue difference between the polypeptide sequence of the engineered galactose oxidase and SEQ ID NO: 1416 is 1-6 selected from Q274G / N, A439G, Y437C / V / R / G / K / L, Q156V / L, T429V, I262V, A336P, N375L, F605L, N35D, L253V, V393T / G / D / P, I561S / T, N488L / T, A354D / T, G200A, S105R, V478L / M, A627R, G45V, S568K, P380H / R / L / K, A154H, N13H / K / A, Q239M, L595W, N315G, Y359F, G328K / R / L, V366T, Q373T, V241I, D37M / Y / I / V, P263S, G559S, Y89R, F438S, L541R, T550S, D217P, T441I, N26M, Q606S, G641D and G224W, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 1416, and wherein the engineered galactose oxidase comprises improved stereoselectivity compared to wild-type Fusarium graminearum galactose oxidase.

15. An engineered galactose oxidase, wherein the amino acid residue difference between the polypeptide sequence of the engineered galactose oxidase and SEQ ID NO: 1598 is 1-11 selected from N13K, P470L, G559S, I563A, I262V, P263S, Q43F, V46A, I56Y, V63T, S99H, Q156L, S403P, G641D, L437V / C / R, P380K, S257R, K30E, T565S, L615I, Y254L, A175G, I287L, I177L, S409R / H, P592K / G, W 1598, wherein the engineered galactose oxidase comprises an improved stereoselectivity compared to the wild-type Fusarium graminearum galactose oxidase.

16. An engineered galactose oxidase, wherein the polypeptide sequence of the engineered galactose oxidase is the same as SEQ ID NO: NO: 1866, wherein the amino acid residue differences between the polypeptide sequence and 1866 are 1-5 selected from the group consisting of M234L, Q373D, E466V, T62Q / D / G, I569L, G197A, T596S, N597A, C384N, V184L, I463V, P592G, T565S, N29V, I177L, S280N, T594M, N601L, A194Q, A149N / R, K546E, T251V, T399V, L615I, T286C, W208F, I417L, D259N and T278L, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 1866, and the engineered galactose oxidase comprises improved stereoselectivity compared to wild-type Fusarium graminearum galactose oxidase.

17. An engineered galactose oxidase, wherein the polypeptide sequence of the engineered galactose oxidase is the same as SEQ ID NO: The amino acid residue difference between 1912 is 1 amino acid residue difference selected from P50T / V / I / H / D, Q43G / T / D / P, P197L / D, K486I / P / L / V / R / A, S3K, T42F, W40P, K30N / L / R, A142H / C / V / S / G, T38M, Q156T / L, Y44H, R161V / Q, A9L, N26T / H / C, Q79P / S / A, T18S, N601L, N29T / M / V / A / Y, L159G / S / K, Q75N, G135D, Q136A / G, G48P / C and A4K, wherein the amino acid position of the polypeptide sequence refers to SEQ ID NO: 1912, and wherein the engineered galactose oxidase comprises improved stereoselectivity compared to wild-type Fusarium graminearum galactose oxidase.

18. An engineered galactose oxidase, wherein the amino acid residue difference between the polypeptide sequence of the engineered galactose oxidase and SEQ ID NO: 1912 is 1-8 amino acid residue differences selected from the group consisting of F472L, L436M, T465G, N29S / I / A / T, Q407D, K486S / I / A, K30N / R, Q136G, P50V / T, N65A, Q156C / M, V478F, Q79S / E, V430I, Q43D, L615I and R161A, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 1912, and wherein the engineered galactose oxidase comprises improved stereoselectivity compared to wild-type Fusarium graminearum galactose oxidase.

19. An engineered galactose oxidase, wherein the amino acid residue difference between the polypeptide sequence of the engineered galactose oxidase and SEQ ID NO: 2080 is 1 amino acid residue difference selected from the group consisting of P197M / L / R / E / S / H / Q / W, G600D, N78L, V556S, D365H, S220Q / R, Y485L, S24R, V95R, S207Q, E520L, N47D, A571S, T219V, K249N, V63T, G294K and L437N, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 2080, and wherein the engineered galactose oxidase comprises improved stereoselectivity compared to wild-type Fusarium graminearum galactose oxidase.

20. An engineered galactose oxidase, wherein the polypeptide sequence of the engineered galactose oxidase is the same as SEQ ID NO: The amino acid residue differences between the polypeptides 2080 are 1-4 selected from the group consisting of P197G / E / S / Q / R / H / D / L / M / W, V556S, V95R, T219V, V63T / E, N29I / S, K342R, L436M, Q136G, V453T, F472L, Y359L, I144V, Q43G, E520Y, N14T / K, I130M / V, S257Q / A / E, Y485R, K249N, A495T, G592H, L437R / G / Y, T119M, S24Q, I214A, M567S, E297T, W560I / G, Q460G and G421N. The amino acid positions of the polypeptide sequences are referenced to SEQ ID NO: 2080, and wherein the engineered galactose oxidase comprises improved stereoselectivity compared to wild-type Fusarium graminearum galactose oxidase.

21. An engineered galactose oxidase, wherein the amino acid residue difference between the polypeptide sequence of the engineered galactose oxidase and SEQ ID NO: 2300 is 1-4 amino acid residue differences selected from the group consisting of D197L / M / S / R / H / G / Q, S207D, T119M, F339V, T219V / I, V63T, V556G, A571S, I214A, K249N, Y359L, N67K, A495T, S24Q, Q43G, W341R, V95R, L437R, V471I and K51Q, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 2300, and wherein the engineered galactose oxidase comprises improved stereoselectivity compared to wild-type Fusarium graminearum galactose oxidase.

22. An engineered galactose oxidase, wherein the amino acid residue difference between the polypeptide sequence of the engineered galactose oxidase and SEQ ID NO: 2300 is 1-4 amino acid residue differences selected from E196R / Q, Q327R, Q407R / K, L465R, Y330H, F442Y, N246Q / S, T583S / G / A, Q327K, D408N, G462A, S292R, S498C, L329W, L515M, A194G and A495S, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 2300.

23. An engineered galactose oxidase, wherein the amino acid residue difference between the polypeptide sequence of the engineered galactose oxidase and SEQ ID NO: 2424 is 1-8 selected from D408Q / R / L, E480L / R, G600N, K36L / P, N14L / R / A, N532G, N96M / S / G / L, P404A, R120S / L, R218M / G, R549L, S24P / V, S537C / W, S92C / G / V, S99L / V / F, V46P / E , V95A / S / R / F, W560I / E / M, Y485C, S426W, R376M, V296R, S626G, N78I, S258V, A324F, N488T, G424W, S433G, T596Q / V, Q23A, N428H, Q640R and N540R, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 2424, and wherein the engineered galactose oxidase comprises improved stereoselectivity compared to wild-type Fusarium graminearum galactose oxidase.

24. An engineered galactose oxidase, wherein the amino acid residue difference between the polypeptide sequence of the engineered galactose oxidase and SEQ ID NO: 2424 is 1-8 selected from S24P / V, V46E / P, S92G / V / C, S426W, R549L, K36L / P, S99V / L / F, N532G, V95S / A / R / F, Y485C, W560E / M / I, P404A, E480L / R, G600N, N96M / G / S, V296R, R120L / S, N78I, S258V, S361P, S626G, A324F, N14R / A / L, R218M / G, D408Q / R / L, S537C / W, R376M, Q23A, T596V / Q, Q640R, N47P, G424W, N540R, S433G and N428H, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 2424, and wherein the engineered galactose oxidase comprises improved stereoselectivity compared to wild-type Fusarium graminearum galactose oxidase.

25. An engineered galactose oxidase, wherein the engineered galactose oxidase is an engineered galactose oxidase variant set forth in the even-numbered sequences of SEQ ID NO: 4-8 or 14-2860.

26. The engineered galactose oxidase of any one of claims 1-25, wherein the engineered galactose oxidase comprises improved activity towards a substrate compared to a wild-type Fusarium graminearum galactose oxidase.

27. The engineered galactose oxidase of claim 26, wherein the substrate comprises a primary alcohol.

28. The engineered galactose oxidase of any one of claims 1-25, wherein the engineered galactose oxidase is purified.

29. A composition comprising at least one engineered galactose oxidase according to any one of claims 1-25.

30. A polynucleotide encoding at least one engineered galactose oxidase according to any one of claims 1-27.

31. The polynucleotide of claim 30, wherein the polynucleotide is operably linked to a control sequence.

32. The polynucleotide of claim 30, wherein the polynucleotide is codon optimized.

33. The polynucleotide of claim 30, wherein the polynucleotide is an odd-numbered sequence of SEQ ID NO: 3-7 or 13-2859.

34. An expression vector comprising at least one polynucleotide according to any one of claims 30-33.

35. A host cell comprising at least one expression vector according to claim 34.

36. A host cell comprising at least one polynucleotide according to any one of claims 30-33.

37. A method for producing an engineered galactose oxidase in a host cell, the method comprising culturing the host cell according to claim 35 or 36 under suitable conditions, thereby producing at least one engineered galactose oxidase.

38. The method of claim 37, further comprising recovering at least one engineered galactose oxidase from the culture and / or the host cell.

39. The method of claim 37 or 38, further comprising the step of purifying the at least one engineered galactose oxidase.

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