Biocatalysts and methods for the synthesis of pregabalin intermediates

Abstract

The present disclosure provides an engineered polypeptide capable of catalyzing asymmetric hydrolysis of 3-isobutyl glutarimide to generate (R)-(−)-3-(carbamoylmethyl)-5-methylhexanoic acid, which has high stereoselectivity, high catalytic activity, good process stability and thermal stability, and tolerance to high product concentration, and has good industrial application value.

Description

Technical Field

[0001] This invention relates to a biocatalyst and a method for preparing pregabalin intermediates using the biocatalyst. Background Technology

[0002] Pregabalin is a chiral small molecule drug with the chemical name S-(+)-3-isobutylγ-aminobutyric acid (SABA). It is associated with endogenous inhibitory neurotransmitters and possesses antiepileptic activity, thus it is commonly used as a chiral drug for treating epilepsy and neuropathic pain. The original drug was manufactured by Pfizer in the United States. In July 2004, the European Union approved it for the treatment of partial seizures, and in 2005, it was approved for marketing by the US FDA. Its original research route is as follows... Figure 1 As shown.

[0003] One of the most important indicators for producing pregabalin is chiral purity. Existing patents and literature mainly classify pregabalin and its intermediate synthesis methods into three categories: chemical / enzymatic resolution, asymmetric synthesis, and chiral source synthesis, with the first two being more commonly used. In resolution routes, the ee value of the product is relatively low, and it requires racemizing and reusing a compound with another configuration, resulting in a very low yield of the final qualified product. For example, CN102102114B discloses a technique for preparing pregabalin intermediates using lipase resolution and ultimately synthesizing pregabalin. The conversion rate of the resolution step is around 40-45%, while the overall yield is only about 30%. The route is as follows: Figure 2 As shown.

[0004] In contrast, asymmetric synthesis methods that introduce chirality into the reaction have higher raw material utilization rates and can be accomplished using chiral catalysts or enzymes. However, chemical asymmetric synthesis methods require expensive chiral catalysts, and the processes are often complex and cumbersome; for example, the original route developed by Pfizer requires nine steps. Patent CN105753726B describes a synthesis process with only four steps, but it still requires chiral thiourea ammonium salt as a catalyst and involves hydrogenation processes, with harsh reaction conditions, and the route is as follows. Figure 3 As shown.

[0005] Therefore, we urgently need to find a more suitable method to produce pregabalin. CN111944856A discloses a novel route for synthesizing a pregabalin intermediate, namely, using hydantoinase to asymmetricly hydrolyze 3-isobutylglutarimide to prepare the highly chiral and pure pregabalin intermediate (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid (e.g.) Figure 4As shown in the figure, this reaction step can yield (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid with ee ≥ 99%, avoiding the steps of resolution and reracemization, shortening the reaction path, improving the utilization rate of raw materials, effectively reducing costs, and being environmentally friendly. However, the catalytic performance of the hydantoin enzyme disclosed in CN111944856A is not ideal, the enzyme dosage is too high, and the space-time yield of the product is low.

[0006] To address these shortcomings, this invention discloses a series of engineered hydantoin peptides developed using directed evolution technology, which greatly reduces enzyme usage, makes the enzymatic reaction and post-processing simple and efficient, and improves space-time yield. Summary of the Invention

[0007] 1. Overview

[0008] This invention provides an engineered peptide with high stereoselectivity, high catalytic activity, good process stability and thermal stability, and tolerance to high product concentrations. It can be used to catalyze the asymmetric hydrolysis of 3-isobutylglutarimide to generate (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid. The invention also provides the gene for the engineered peptide, a recombinant expression vector containing this gene, an engineered bacterial strain, an efficient method for preparing the engineered peptide, and a reaction process for preparing (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid using the engineered peptide.

[0009] Through experimental research, the inventors discovered a wild-type hydantoinase derived from *Pseudomonas fluorescens* (GenBank: KF268426.1), with its amino acid sequence shown in SEQ ID NO: 2. Compared to the hydantoinase disclosed in CN111944856A, SEQ ID NO: 2 exhibits superior activity in catalyzing the hydrolysis of 3-isobutylglutarimide to (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid. Although SEQ ID NO: 2 is the wild-type hydantoinase... Figure 5The enzyme exhibits superior activity in the reaction shown, but it is still far from industrial application, and its performance in many aspects needs to be improved. Research on this wild-type hydantoin enzyme was reported in Appl Biochem Biotechnol (2016) 179:1–15. The results showed that the optimal pH for this wild-type hydantoin enzyme in catalyzing the hydrolysis of substituted hydantoins was between 8.5 and 9.5; its activity decreased significantly at pH < 7.5. Its thermal stability was also poor, with half-lives of 2.23 h, 1.44 h, and 0.78 h at 50 °C, 55 °C, and 60 °C, respectively, which is detrimental to the production and storage of enzyme preparations. The inventors discovered that, in the absence of any catalyst, 3-isobutylglutarimide spontaneously hydrolyzes to form racemic 3-(carbamoylmethyl)-5-methylhexanoic acid. The rate of this spontaneous hydrolysis is strongly pH-dependent; at pH > 8.5, the rate is significant. The resulting racemic product contains the undesirable chiral isomer (S)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid, which affects the chiral purity (i.e., ee value) of the final product. Therefore, the spontaneous hydrolysis of 3-isobutylglutarimide is something this invention strives to avoid. Furthermore, the spontaneous hydrolysis of 3-isobutylglutarimide is almost undetectable at pH ≤ 7.0. Figure 5 The reaction shown must be carried out at pH ≤ 7.0.

[0010] In addition to SEQ ID NO: 2 for catalysis Figure 5 Besides improving the activity, thermal stability, and stability at pH ≤ 7.0 of the reaction, the inventors discovered during their research that the activity of SEQ ID NO: 2 was severely inhibited when the product concentration in the enzymatic reaction accumulated to a certain level, limiting further improvements in space-time yield. Therefore, overcoming the product inhibition of SEQ ID NO: 2 (or, in other words, improving the tolerance of SEQ ID NO: 2 to high product concentrations) is also a property that SEQ ID NO: 2 needs to improve. Using directed evolution technology, combined with computer-aided design and screening, the inventors engineered SEQ ID NO: 2 to obtain a series of engineered peptides with high stereoselectivity, high catalytic activity, good thermal stability and reaction pH stability, and tolerance to high product concentrations. These engineered peptides include amino acid sequences that differ from the reference sequence of SEQ ID NO: 2 by one or more residues. These residue differences occur at amino acid positions that affect multiple different functional properties of the enzyme, including catalytic activity, stereoselectivity, substrate and / or product tolerance, thermal stability, reaction process stability (including pH fluctuation range, ionic strength, solvent tolerance, etc.), recombinant expression effect, and other properties that affect the preparation and catalytic performance of the enzyme, as well as various combinations of these properties.

[0011] In some embodiments, the engineered polypeptide may comprise an amino acid sequence having at least 90% sequence identity with the polypeptide of SEQ ID NO: 2 and differing from SEQ ID NO: 2 at one or more residue positions selected from the following: X8, X39, X46, X51, X62, X63, X64, X66, X67, X71, X73, X95, X97, X113, X152, X159, X189, X199, X201, X215, X254, X255, X257, X263, X264, X265, X266, X267, X288, X292, X320, X329, X336, X337, X340, X462, X467, X474, X476, X479. In some embodiments, compared with SEQ ID NO: 2, the polypeptide may contain amino acids that are different at one or more residue positions selected from the following: X8, X39, X46, X51, X62, X63, X64, X66, X67, X74, X476, X479. The amino acid residue differences compared to NO:2 are selected from the following: A8G, A39P, G46A, L51V, L51I, M62L, Q63E, L64I, L64T, L64S, L64A, F66Y, F66L, M67W, M67Y, M67F, A71T, A71S, E73D, I95V, I95L, I95M, N97 G, N97D, N97L, N97Q, A113T, F152Y, F152M, F152L, I159L, I159F, I159Y, L189I, L 189V, L189M, A199V, G201H, Q215A, Q215P, S254Q, S254L, S254N, S254G, S254F, K 255F, K255Y, K255H, K255N, Q257W, V263T, L264C, A265P, G266Q, H267Y, M288C, F292L, F320S, F320L, R329A, R329L, R329Y, P336M, P336L, P336Q, N337P, A340P, F462R, K467D, P474W, A476P, R479Q, R479L, R479P; or, based on these differences, the inclusion or deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24, 25 or more amino acid residues.

[0012] As provided herein, in some embodiments, the disclosed amino acid differences may be used alone or in various combinations to produce engineered peptides with improved enzyme properties. In some embodiments, the engineered peptide comprises an amino acid sequence having at least 90% sequence identity with the reference sequence SEQ ID NO:2 and a difference of at least one residue at residue position X64 compared to SEQ ID NO:2. In some embodiments, the amino acid residue at residue position X64 is selected from I, T, S, and A.

[0013] More specifically, in some implementations, the engineered peptides improved based on SEQ ID NO: 2 include those corresponding to SEQ ID No: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 1 60, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 22 The polypeptide composed of the amino acid sequences shown in 6, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286.

[0014] In some implementations, the improved engineered peptides include those with SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 1 72, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 24 4. The reference sequences of 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, and 286 have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more amino acid sequences that are identical to the reference sequences.

[0015] The identity between two amino acid sequences or two nucleotide sequences can be obtained using algorithms commonly used in this field. These can be calculated using NCBI Blastp and Blastn software with default parameters, or using the Clustal W algorithm (Nucleic Acid Research, 22(22):4673-4680, 1994). For example, using the Clustal W algorithm, the amino acid sequence identity between SEQ ID NO: 2 and SEQ ID NO: 184 is 97.9%.

[0016] In another aspect, the present invention provides a polynucleotide sequence encoding an engineered polypeptide. In some embodiments, the polynucleotide may be a portion of an expression vector having one or more control sequences for expressing the engineered polypeptide. In some embodiments, the polynucleotide may include the sequences corresponding to SEQ ID Nos: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 9 1, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 15 9, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 22 The polynucleotide sequences shown are 5, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, and 285.

[0017] As those skilled in the art will know, due to the degeneracy of nucleotide codons, the SEQ ID Nos encoded are: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92. 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 16 2, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 2 The polynucleotide sequences of amino acid sequences 30, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, and 286 are not limited to SEQ ID.No: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 ,47,49,51,53,55,57,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,8 9, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 1 57, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 22 1, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285. The nucleic acid sequence of the hydantoin gene of this invention can also be SEQ ID in the coding sequence listing.No: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 4 8, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 22 Any other nucleic acid sequence other than the amino acid sequences shown in 8, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286.

[0018] On the other hand, this disclosure provides a polynucleotide encoding an engineered polypeptide or an expression vector and host cell capable of expressing the engineered polypeptide. In some embodiments, the host cell may be a bacterial host cell, such as *Escherichia coli*. The host cell can be used to express and isolate the engineered polypeptide described herein, or optionally directly for reacting to transform a substrate into a product.

[0019] In some implementations, engineered peptides in the form of whole cells, crude extracts, isolated peptides, or purified peptides can be used alone or in an immobilized form (e.g., immobilized on resin).

[0020]

[0021] This disclosure also provides a method for converting a compound of structural formula A1 into a chiral compound of structural formula A2 using an engineered polypeptide disclosed herein, wherein the chiral compound of structural formula A2 is in excess compared to the corresponding enantiomer, the method comprising contacting a compound of structural formula A1 with an engineered polypeptide under reaction conditions suitable for converting A1 to A2, wherein the engineered polypeptide is the engineered polypeptide described herein. In some embodiments, the engineered polypeptide has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with SEQ ID NO: 2 and is capable of converting a compound of structural formula A1 into a compound of structural formula A2.

[0022] In some embodiments, the compound of structure A2 is produced in an enantiomeric excess of at least 97%, 98%, or 99% or greater.

[0023] Specific implementation schemes for the engineered peptides used in this method are further provided in the details. Engineered peptides that can be used in the above methods may include amino acid sequences selected from those having at least 90% sequence identity with SEQ ID NO: 2 and differing from SEQ ID NO: 2 by one or more residue positions selected from the following: X8, X39, X46, X51, X62, X63, X64, X66, X67, X71, X73, X95, X97, X113, X152, X159, X189, X199, X201, X215, X254, X255, X257, X263, X264, X265, X266, X267, X288, X292, X320, X329, X336, X337, X340, X462, X467, X474, X476, X479. In some embodiments, compared with SEQ ID NO: 2, the amino acid sequence is... The amino acid residue differences compared to NO:2 are selected from the following: A8G, A39P, G46A, L51V, L51I, M62L, Q63E, L64I, L64T, L64S, L64A, F66Y, F66L, M67W, M67Y, M67F, A71T, A71S, E73D, I95V, I95L, I95M, N97 G, N97D, N97L, N97Q, A113T, F152Y, F152M, F152L, I159L, I159F, I159Y, L189I, L 189V, L189M, A199V, G201H, Q215A, Q215P, S254Q, S254L, S254N, S254G, S254F, K 255F, K255Y, K255H, K255N, Q257W, V263T, L264C, A265P, G266Q, H267Y, M288C, F292L, F320S, F320L, R329A, R329L, R329Y, P336M, P336L, P336Q, N337P, A340P, F462R, K467D, P474W, A476P, R479Q, R479L, R479P; or, based on these differences, the inclusion or deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24, 25 or more amino acid residues.

[0024] In some implementations, the engineered peptides that can be used in the above methods may include those selected from the corresponding SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90. 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 22 The amino acid sequences of 4, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, and 286.

[0025] Any of the methods disclosed herein for using engineered peptides in compounds of formula A2 can be performed under a range of suitable reaction conditions, including but not limited to ranges of pH, temperature, buffer solution, solvent system, substrate loading, peptide loading, pressure, and reaction time. For example, in some embodiments, the preparation of compound A2 can be performed, wherein suitable reaction conditions include: (a) a substrate loading of about 1 g / L to 400 g / L of compound A1; (b) a loading of about 0.1 g / L to 50 g / L of engineered peptide; (d) a pH of about 6.0 to about 8.5; and (e) a temperature of about 10°C to 60°C.

[0026] In some embodiments, the engineered peptide is capable of converting substrate compound A1 to compound A2 under appropriate reaction conditions, exhibiting an activity at least about 2, 3, 4, 5, 10, 15, 20, or more times greater than that of the reference peptide of SEQ ID NO:2. In some embodiments, the engineered peptide is capable of converting compound A1 to compound A2 under appropriate reaction conditions within a reaction time of about 48 hours, about 36 hours, about 24 hours, or less, exhibiting an activity of at least about 5 g / L h. -1 10g / L h -1 15g / L h -1 20g / L h -1 Or a higher spacetime productivity.

[0027] 2. Detailed Explanation

[0028] 2.1 Definition

[0029] With respect to this disclosure, unless otherwise expressly defined, the technical and scientific terms used herein shall have the meanings commonly understood by one of ordinary skill in the art.

[0030] The terms “protein,” “polypeptide,” and “peptide” are used interchangeably herein to refer to a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modifications (e.g., glycosylation, phosphorylation, lipidation, myristylation, ubiquitination, etc.). This definition includes D-amino acids and L-amino acids, as well as mixtures of D-amino acids and L-amino acids.

[0031] The terms “engineered hydantoin,” “engineered hydantoin peptide,” “improved hydantoin peptide,” and “engineered peptide” are used interchangeably in this article.

[0032] The terms “polynucleotide” and “nucleic acid” are used interchangeably in this article.

[0033] "Coding sequence" refers to the nucleic acid portion (e.g., a gene) of the amino acid sequence that encodes a protein.

[0034] "Naturally occurring" or "wild-type" refers to a form found in nature. For example, naturally occurring or wild-type polypeptide or polynucleotide sequences are sequences that exist in organisms, can be isolated from natural sources, and have not been intentionally modified by artificial means.

[0035] When used to refer to, for example, cells, nucleic acids, or polypeptides, “recombinant,” “engineered,” or “non-naturally occurring” means a material or a material corresponding to the natural or inherent form of that material that has been altered in a way that would not exist in nature, or is the same as it but produced or obtained from synthetic materials and / or through the use of recombinant techniques.

[0036] "Sequence identity" and "homology" are used interchangeably herein to refer to comparisons between polynucleotides or peptides ("sequence identity" and "homology" are typically expressed as percentages) and are determined by comparing two best-aligned sequences in a comparison window, where the portion of the polynucleotide or peptide sequence in the comparison window compared to a reference sequence may include additions or deletions (i.e., vacancies) for optimal alignment of the two sequences. The percentage can be calculated by determining the number of positions in both sequences where the same nucleic acid base or amino acid residue appears to generate the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the sequence identity percentage. Alternatively, the percentage can be calculated by determining the number of positions in both sequences where the same nucleic acid base or amino acid residue is present, or the number of positions where the nucleic acid base or amino acid residue is aligned with a vacancy to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the sequence identity percentage. Those skilled in the art will recognize that many algorithms exist for establishing the alignment of two sequences. The best sequence alignment for comparison can be achieved, for example, by the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, by the similarity search method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444, by computer implementations of these algorithms (GAP, BESTFIT, FASTA, or TFASTA in the GGWisconsin software package) or by visual inspection (see generally Current Protocols in Molecular Biology, eds. FMAusubel et al., Current Protocols, a joint venture between Greene Publishing Associates Inc. and John Wiley & Sons, Inc., (Supplement 1995) (Ausubel)). Examples of suitable algorithms for determining sequence identity and sequence similarity percentages are the BLAST and BLAST2.0 algorithms, described in Altschul et al., 1990, J.Mol.Biol.215:403-410 and Altschul et al., 1977, Nucleic Acids Res.3389-3402, respectively. Software for performing BLAST analyses is publicly available through the website of the National Center for Biotechnology Information (NCBI).The algorithm first identifies 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 compared to words of the same length in a database sequence. T is called the neighbor word scoring threshold (Altschul et al., as described above). These initial neighbor word hits act as seeds to initiate a search for longer HSPs containing them. Word hits are then extended along each sequence in both directions until the cumulative alignment score can no longer increase. For nucleotide sequences, the cumulative score is calculated using parameters M (reward score for matching residue pairs; always > 0) and N (penalty score for mismatched residues; always < 0). For amino acid sequences, a score matrix is ​​used to calculate the cumulative score. Extension of the word-matching string in each direction is terminated when: the cumulative alignment score decreases by an amount X from its maximum value; the cumulative score reaches 0 or below due to the accumulation of one or more negative-scoring residues; or either sequence end 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 a word length (W) of 11, an expected value (E) of 10, M = 5, N = -4, and a comparison of the two strands as default values. For amino acid sequences, the BLASTP program uses the following as default values: a word length (W) of 3, an expected value (E) of 10, and a BLOSUM62 score matrix (see Henikoff and Henikoff, 1989, Proc Natl Acad Sci USA 89: 10915). Exemplary determinations of sequence alignment and sequence identity percentage can be performed using the BESTFIT or GAP programs in the GCG Wisconsin software package (Accelrys, Madison WI) with the provided default parameters.

[0037] A “reference sequence” is a defined sequence used as the basis for sequence comparison. A reference sequence can be a subset of a larger sequence, such as a fragment of a full-length gene or polypeptide sequence. Generally, a reference sequence is at least 20 nucleotides or amino acid residues long, at least 25 residues long, at least 50 residues long, or the full length of a nucleic acid or polypeptide. Because two polynucleotides or polypeptides can each (1) include sequences similar between the two sequences (i.e., a portion of the complete sequence) and (2) can further include sequences different between the two sequences, sequence comparisons between two (or more) polynucleotides or polypeptides are typically performed by comparing the sequences of the two polynucleotides or polypeptides within a “comparison window” to identify and compare local regions of sequence similarity. In some embodiments, a “reference sequence” is not intended to be limited to wild-type sequences and can include engineered or altered sequences. For example, “a reference sequence based on SEQ ID NO:2 having threonine at the residue corresponding to X64” refers to a reference sequence in which the corresponding residue (which is leucine) at X64 in SEQ ID NO:2 has been altered to threonine.

[0038] A “comparison window” refers to a conceptual segment of at least about 20 adjacent nucleotide positions or amino acid residues, wherein the sequence can be compared with a reference sequence of at least 20 adjacent nucleotides or amino acids, and wherein a portion of the sequence in the comparison window may include 20% or less of additions or deletions (i.e., vacancies) compared to the reference sequence (which does not include additions or deletions) for optimal alignment of the two sequences. The comparison window may be longer than 20 adjacent residues and may optionally include windows of 30, 40, 50, 100 or longer.

[0039] In the context of numbering a specified amino acid or polynucleotide sequence, "corresponding to," "referring to," or "relative to" refers to the number of reference sequence residues specified when the specified amino acid or polynucleotide sequence is compared to a reference sequence. In other words, the residue numbering or position of a given sequence is assigned based on the reference sequence, not the actual numerical position of residues within the given amino acid or polynucleotide sequence. For example, a given amino acid sequence, such as the amino acid sequence of an engineered hydantoin, can be aligned to a reference sequence by introducing vacancies to optimize residue matching between the two sequences. In these cases, although vacancies exist, the residue numbering in the given amino acid or polynucleotide sequence is assigned relative to the reference sequence with which it has been aligned.

[0040] "Amino acid difference" or "residue difference" refers to the difference of an amino acid residue at a position in a polypeptide sequence relative to the corresponding amino acid residue at a position in a reference sequence. The position of the amino acid difference is generally referred to as "Xn" in this document, where n indicates the corresponding position in the reference sequence upon which the residue difference is based. For example, "residue difference at position X64 compared to SEQ ID NO:2" refers to the difference of an amino acid residue at position 64 of the polypeptide corresponding to SEQ ID NO:2. Therefore, if the reference polypeptide of SEQ ID NO:2 has a leucine residue at position 64, then "residue difference at position X64 compared to SEQ ID NO:2" refers to the substitution of any amino acid residue other than leucine at position 64 of the polypeptide corresponding to SEQ ID NO:2. In most instances herein, a specific amino acid residue difference at a position is denoted as "XnY", where "Xn" refers to the corresponding position as described above, and "Y" is a one-letter identifier of the amino acid found in the engineered polypeptide (i.e., a different residue from the reference polypeptide). In some instances (e.g., in Table 1), this disclosure also provides specific amino acid differences represented by the conventional symbol “AnB”, where A is a one-letter identifier of a residue in the reference sequence, “n” is the residue position number in the reference sequence, and B is a single-letter identifier of residue substitution in the sequence of the engineered polypeptide. In some instances, the polypeptide of this disclosure may contain one or more amino acid residue differences relative to the reference sequence, represented by a list of specific positions where residue differences exist relative to the reference sequence.

[0041] "Deficiency" refers to a modification of a peptide by removing one or more amino acids from a reference peptide. Deficiency may include the removal of one or more, two or more, five or more, ten or more, fifteen or more, or twenty or more amino acids, up to 10% or up to 20% of the total number of amino acids constituting the reference enzyme, while retaining the enzymatic activity and / or improved properties of the engineered hydantoin. Deficiency may involve internal portions and / or terminal portions of the peptide. In various embodiments, the deficiency may include continuous segments or may be discontinuous.

[0042] "Insertion" refers to the modification of a polypeptide by adding one or more amino acids from a reference polypeptide. In some embodiments, improved engineered hydantoins include one or more amino acid insertions into a naturally occurring hydantoin polypeptide, and one or more amino acid insertions into other improved hydantoin polypeptides. Insertions can be made within the polypeptide or at the carboxyl or amino terminus. As used herein, insertions include fusion proteins known in the art. Insertions can be continuous segments of amino acids or separated by one or more amino acids in a naturally occurring polypeptide.

[0043] As used herein, "fragment" refers to a polypeptide having an amino-terminal and / or carboxyl-terminal deletion, but retaining an amino acid sequence that is identical to the corresponding position in the sequence. Fragments can be at least 10 amino acids long, at least 20 amino acids long, at least 50 amino acids long or longer, and up to 70%, 80%, 90%, 95%, 98%, and 99% of the full-length hydantoin polypeptide.

[0044] "Isolated polypeptide" refers to a polypeptide that is substantially separated from its natural companion substances such as proteins, lipids, and polynucleotides. This term includes polypeptides that have been removed or purified from their natural environment or expression system (e.g., host cells or in vitro synthesis). Modified hydantoin polypeptides can be present in cells, in cell culture media, or prepared in various forms, such as lysates or isolated preparations. Thus, in some embodiments, modified hydantoin polypeptides can be isolated polypeptides.

[0045] A "chiral center" refers to a carbon atom that connects four different groups.

[0046] "Stereoselectivity" refers to the preferential formation of one stereoisomer relative to one or more other isomers in a chemical or enzymatic reaction. Stereoselectivity can be partial, where one stereoisomer is formed more preferentially than another; or it can be complete, where only one stereoisomer is formed. When the stereoisomers are enantiomers, stereoselectivity is called enantioselectivity, and the excess fraction (usually reported as a percentage) of one enantiomer in a mixture of two enantiomers is optionally reported as "enantiomeric excess" (EE). When the stereoisomers are diastereomers, stereoselectivity is referred to as diastereoselectivity, which is the excess fraction (usually reported as a percentage) of one diastereomer in a mixture of two diastereomers, and is typically reported as “diastereoisomeric excess” (de). In the art, this fraction (typically a percentage) is typically reported as enantiomeric excess (ee) calculated from the following formula: {major enantiomer concentration – minor enantiomer concentration} / {major enantiomer concentration + minor enantiomer concentration}.

[0047] The terms “stereoisomer,” “stereoisomeric form,” and similar expressions are used interchangeably in this article and refer to all isomers that differ only in the spatial orientation of their atoms. This includes enantiomers and isomers of compounds that have more than one chiral center and are not mirror images of each other (i.e., “diastereomers”).

[0048] "Improved enzyme properties" refers to an improved hydantoin polypeptide that exhibits any enzyme properties compared to a reference hydantoin, such as a wild-type hydantoin or another improved engineered hydantoin. Desired improved enzyme properties include, but are not limited to, enzyme activity (which can be expressed as a percentage of substrate conversion), thermal stability, solution stability, pH activity characteristics, tolerance to inhibitors (e.g., substrate or product inhibition), and stereoselectivity.

[0049] "Conversion" refers to the enzymatic conversion of a substrate into a corresponding product. "Conversion percentage" or "conversion rate" refers to the percentage of substrate in a reaction system that is converted into the product under specified reaction conditions and within a specified reaction time. Therefore, the "enzyme activity" or "activity" of a hydantoin peptide can be expressed as the "conversion percentage" from substrate to product. The conversion rate is generally calculated by sampling and determining the concentrations of the product and substrate in the reaction system: {product molar concentration} / {substrate molar concentration + product molar concentration}.

[0050] "Thermostable" means that the hydantoin peptide maintains similar activity to the wild-type enzyme after exposure to elevated temperatures (e.g., 72°C or higher) for a period of time (e.g., 2.5 hours or more).

[0051] "Solvent stable" or "solvent tolerant" means that the hydantoin peptide maintains similar activity to the wild-type enzyme after exposure to different concentrations (e.g., 5-99%) of solvents (methanol, ethanol, isopropanol, dimethyl sulfoxide (DMSO), tetrahydrofuran, 2-methyltetrahydrofuran, acetone, toluene, butyl acetate, methyl tert-butyl ether, etc.) for a period of time (e.g., 0.5-24 hours).

[0052] "Suitable reaction conditions" refer to those conditions in the biocatalytic reaction solution (e.g., ranges of enzyme loading, substrate loading, temperature, pH, buffer solution, co-solvent, etc.) under which the hydantoin of this disclosure can convert the substrate into the desired product compound. Exemplary "suitable reaction conditions" are provided in this disclosure and illustrated by examples.

[0053] "Hydrocarbon group" refers to a straight-chain or branched hydrocarbon group. The number following the symbol "C" specifies the number of carbon atoms that a particular group may contain. For example, "C1-C8" refers to a straight-chain or branched hydrocarbon group having 1 to 8 carbon atoms. The hydrocarbon group may optionally be substituted by one or more substituent groups. "Aryl" refers to a monovalent aromatic hydrocarbon group having 6 to 20 carbon atoms. "Heteroaryl" and "heteroaromatic" refer to aryl groups in which one or more carbon atoms of the parent aromatic ring system are replaced by heteroatoms (O, N, or S). "Substituted" when used to modify a specified group or free radical means that one or more hydrogen atoms of the specified group or free radical are each independently substituted. As used herein, "compound" refers to any compound whose structural formula and / or chemical name are indicated accompanying the compounds disclosed herein. A compound may be identified by its chemical structure and / or chemical name. When there is a conflict between the chemical structure and the chemical name, the chemical structure determines the identity of the compound. Unless otherwise specifically described or indicated, the chemical structures described herein cover all possible isomers of the described compounds.

[0054] Replaced by the same or different substituents. "Substituted hydrocarbon, aryl, or heteroaryl" refers to a hydrocarbon, aryl, or heteroaryl group in which one or more hydrogen atoms are replaced by another substituent. "Optional" or "optionally" means that the described event or situation may or may not occur; for example, "optionally substituted aryl" refers to an aryl group that may be substituted or may not be substituted, and this description includes both substituted and unsubstituted aryl groups.

[0055] 2.2 Engineered Peptides

[0056] The engineered polypeptide disclosed in this invention is derived from a wild-type hydantoinase through a creative directed evolution process, involving mutations such as substitutions, insertions, or deletions of a certain number of amino acid residues. For a description of directed evolution technology, please refer to "Directed Evolution: Bringing New Chemistry to Life," Frances H. Arnold, Angewandte Chemie, November 28, 2017. Frances H. Arnold was awarded the 2018 Nobel Prize in Chemistry for his pioneering contributions to enzyme directed evolution technology. This wild-type hydantoinase is derived from *Pseudomonas fluorescens*, and its amino acid sequence is shown in SEQ ID NO: 2. According to the inventors' testing, the wild-type hydantoinase corresponding to SEQ ID NO: 2 still exhibits poor activity towards A1, is highly affected by pH, and shows particularly poor tolerance to high concentrations of product A2, as well as poor thermal stability. These defects are detrimental to industrial applications, necessitating engineered improvements to SEQ ID NO: 2 through enzyme directed evolution technology.

[0057] The protein corresponding to SEQ ID NO: 2 does not have a publicly disclosed 3D structure. The inventors constructed its 3D structural model using Yasara software and then, combining bioinformatics techniques, designed site-directed saturation mutant libraries or multi-site combined mutant libraries targeting multiple residues. These libraries were then screened at different development stages using the screening reaction conditions shown in Tables 1.1, 2.1, 2.2, and 3.1-3.4. The method for constructing mutant libraries can employ site-directed mutagenesis PCR (as shown in Example 2) or multi-site mutagenesis PCR (refer to "Mutagenesis and Synthesis of Novel Recombinant Genes Using PCR," Chapter 32, in PCR Primer, 2nd edition (eds. Dieffenbach and Dveksler). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2003.).

[0058] In order to develop for Figure 5 The high-performance enzyme catalyst shown in the diagram was used in this invention for the directed evolution of SEQ ID NO: 2 in stages. Different high-throughput screening reaction conditions were designed for different characteristics of the enzyme to be enhanced. The first stage mainly focused on enhancing enzyme activity, and the designed high-throughput screening reaction conditions are shown in Table 1.1 or Example 8. Table 1 lists some exemplary engineered peptides obtained in the first stage and their screening reaction results.

[0059] Table 1. Exemplary engineered peptides obtained in the first stage of directed evolution.

[0060]

[0061]

[0062] Table 1.1

[0063]

[0064]

[0065] In practical industrial applications, the simpler the reaction system, the better. Generally, co-solvents such as DMSO are not used, and the substrate loading should be as high as possible. To test the catalytic effect of the engineered peptides shown in Table 1 under industrial application-related conditions and to compare them with the wild-type SEQ ID NO: 2, the present invention used the following reaction conditions to test the exemplary engineered peptides obtained in the first stage: substrate A1 loading of 10 g / L, wet bacterial cells expressing the engineered peptide directly using a loading of 50 g / L, 0.1 M PBS pH 7.0, 30°C. The reaction process is as described in Example 12. The results are shown in Table 1.2.

[0066] Table 1.2 Catalytic effect of the first-stage mutant enzyme under reaction conditions relevant to industrial applications

[0067]

[0068] The second stage, while enhancing enzyme activity, also incorporated the evolution of pH stability. The designed high-throughput screening reaction conditions are shown in Tables 2.1 and 2.2. Table 2 lists some exemplary engineered peptides obtained in the second stage and their screening reaction results.

[0069] Table 2. Exemplary engineered peptides obtained in the second stage of directed evolution.

[0070]

[0071]

[0072]

[0073]

[0074] Table 2.1

[0075]

[0076] Table 2.2

[0077]

[0078]

[0079] The exemplary engineered peptide obtained in the second stage was tested using the following reaction conditions: substrate A1 loading of 10 g / L, wet bacterial cells expressing the engineered peptide directly using a loading of 6 g / L, 0.1 M PBS, pH 7.0, and 30°C. The reaction procedure was as described in Example 13. The results are shown in Table 2.3.

[0080] Table 2.3 Catalytic effect of the second-stage mutant enzyme under reaction conditions relevant to industrial applications

[0081]

[0082] The third stage, building upon the evolution of enzyme activity and pH stability, further enhanced the tolerance and thermal stability of high-concentration products. The designed high-throughput screening reaction conditions are shown in Tables 3.1, 3.2, 3.3, and 3.4. Tables 3 and 3.5 list some exemplary engineered peptides obtained in the third stage and their screening reaction results.

[0083] Table 3. Exemplary engineered peptides obtained in the third stage of directed evolution.

[0084]

[0085]

[0086] Table 3.1

[0087]

[0088]

[0089] Table 3.2

[0090]

[0091] Table 3.3

[0092]

[0093] Table 3.4

[0094]

[0095] Table 3.5

[0096]

[0097]

[0098] The exemplary engineered peptide obtained in the third stage was tested using the following reaction conditions: substrate A1 loading of 10 g / L, direct use of wet bacterial cells expressing the engineered peptide at a loading of 1 g / L, 0.1 M PBS, pH 7.0, and 40 °C. The reaction procedure was as described in Example 14. The results are shown in Table 3.6.

[0099] Table 3.6 Catalytic effect of the third-stage mutant enzyme under reaction conditions relevant to industrial applications

[0100]

[0101]

[0102] Based on the characteristics of the exemplary peptides listed in Tables 1, 2, and 3, the increase in enzyme activity (i.e., the conversion of compound A1 to compound A2) is associated with residue differences at the following residue positions, as well as others: X8, X39, X46, X51, X62, X63, X64, X66, X67, X71, X73, X95, X97, X152, X159, X189, X199, X201, X215, X254, X255, X257, X263, X264, X265, X266, X267, X288, X292, X320, X329, X336, X337, X340, X462, X467, X474, X476, X479. In some embodiments, with SEQ ID The amino acid residue differences compared to NO:2 are selected from the following: A8G, A39P, G46A, L51V, L51I, M62L, Q63E, L64I, L64T, L64S, L64A, F66Y, F66L, M67W, M67Y, M67F, A71T, A71S, E73D, I95V, I95L, I95M, N97G, N97D, N97L, N97Q, F152Y, F152M, F152L, I159L, I159F, I159Y, L189I, L189V, L189M, A199V, G201H, Q 215A, Q215P, S254Q, S254L, S254N, S254G, S254F, K255F, K255Y, K255H, K255N, Q257W, V263T, L264C, A265P, G266Q, H267Y, M288C, F292L, F320S, F320L, R329A, R329L, R329Y, P336M, P336L, P336Q, N337P, A340P, F462R, K467D, P474W, A476P, R479Q, R479L, R479P.

[0103] Based on the properties of the exemplary peptides listed in Tables 2 and 3.5, the increase in enzyme pH stability is associated with residue differences at the following residue positions, as well as others: X8, X39, X46, X51, X64, X66, X67, X71, X73, X95, X97, X113, X152, X159, X189, X201, X215, X254, X255, X257, X263, X264, X265, X266, X267, X288, X292, X329, X337, X340, X462, X467, X474, X476. In some embodiments, with SEQ ID The amino acid residue differences compared to NO:2 are selected from the following: A8G, A39P, G46A, L51V, L51I, L64T, L64I, L64S, F66Y, M67F, M67Y, M67W, A71T, A71S, E73D, I95V, I95L, I95M, N97L, N97Q, A113T, F152Y, F152M, F152L, I159Y, I159F, I159L, L189I, L189V, G 201H, Q215A, Q215P, S254Q, S254L, S254N, S254G, S254F, K255F, K255Y, K255H, K255N, Q257W, V263T, L26 4C, A265P, G266Q, H267Y, M288C, F292L, R329A, R329L, R329Y, N337P, A340P, F462R, K467D, P474W, A476P.

[0104] Based on the characteristics of the exemplary peptides listed in Table 3.5, the increase in enzyme product tolerance and / or thermal stability is associated with residue differences at the following residue positions, as well as others: X39, X51, X64, X66, X71, X97, X113, X159, X189, X199, X215, X255, X257, X337, X340. In some embodiments, the amino acid residue differences compared to SEQ ID NO: 2 are selected from the following: A39P, L51I, L64T, F66Y, A71T, N97L, N97Q, A113T, I159L, I159Y, I159F, L189V, L189I, L189M, A199V, Q215A, Q215P, K255H, K255N, Q257W, N337P, A340P.

[0105] As will be apparent to those skilled in the art, the aforementioned residue positions and specific amino acid residues at each residue position can be used alone or in various combinations to synthesize hydantoin peptides with desired improved properties, including enzyme activity, stereoselectivity, stability, and others.

[0106] Based on the guidance provided herein, it is further conceivable that any exemplary engineered polypeptide having an even-numbered sequence identifier as specified in SEQ ID NO:4–286 can be used as a starting amino acid sequence for the synthesis of other engineered polypeptides, for example, by adding various amino acid differences from residue positions described in Tables 1, 2, and 3. Further improvements can be achieved by including amino acid differences at positions that have remained unchanged during the first three stages of evolution.

[0107] Therefore, in some embodiments, it is possible to engineer a polypeptide that converts compound A1 into compound A2, comprising having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with a reference sequence selected from even-numbered sequence identifiers in SEQ ID NO:4-286, and with SEQ ID NO:4-286. NO:2 differs from one or more residues at the following positions: X8, X39, X46, X51, X62, X63, X64, X66, X67, X71, X73, X95, X97, X113, X152, X159, X189, X199, X201, X215, X254, X255, X257, X263, X264, X265, X266, X267, X288, X292, X320, X329, X336, X337, X340, X462, X467, X474, X476, X479.

[0108] In some embodiments, engineered polypeptides capable of converting compound A1 to compound A2 under appropriate reaction conditions include those having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with a reference sequence selected from even-numbered sequence identifiers in SEQ ID NO:4-286, and with SEQ ID NO:4-286. NO:2 differs from the amino acid sequence selected from one or more residues below: A8G, A39P, G46A, L51V, L51I, M62L, Q63E, L64I, L64T, L64S, L64A, F66Y, F66L, M67W, M67Y, M67F, A71T, A71S, E73D, I95V, I95L, I95M, N97G, N97D, N97L, N97Q, A113T, F152Y, F152M, F152L, I159L, I159F, I159Y, L189I, L189V, L189M, A199V. G201H, Q215A, Q215P, S254Q, S254L, S254N, S254G, S254F, K255F, K255Y, K255H, K255N, Q257W, V263T, L264C, A265P, G266Q, H267Y, M28 8C, F292L, F320S, F320L, R329A, R329L, R329Y, P336M, P336L, P336Q, N337P, A340P, F462R, K467D, P474W, A476P, R479Q, R479L, R479P.

[0109] In addition to the residue positions specified above, any engineered polypeptide disclosed herein may also include residue differences relative to the reference polypeptide sequence of SEQ ID NO:2 at other residue positions, i.e., residue positions other than the following residue positions: X8, X39, X46, X51, X62, X63, X64, X66, X67, X71, X73, X95, X97, X113, X152, X159, X189, X199, X201, X215, X254, X255, X257, X263, X264, X265, X266, X267, X288, X292, X320, X329, X336, X337, X340, X462, X467, X474, X476, X479. Residue differences at these other residue positions can provide additional variants in the amino acid sequence without altering the peptide's ability to convert compound A1 to compound A2, particularly regarding increases in enzyme activity, pH stability, enzyme product tolerance, and thermal stability. Therefore, in some embodiments, in addition to amino acid residue differences from any of the engineered peptides selected from peptides having even-numbered sequence identifiers in SEQ ID NO:4-286, the sequence may also include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 residue differences at other amino acid residue positions compared to SEQ ID NO:2.

[0110] 2.3 It can be used to prepare polynucleotides, control sequences, expression vectors, and host cells for engineered peptides.

[0111] On the other hand, this disclosure provides polynucleotides encoding engineered polypeptides with hydantoin activity as described herein. The polynucleotides can be operatively linked to one or more heterologous regulatory sequences controlling gene expression to produce recombinant polynucleotides capable of expressing the polypeptides. Expression constructs containing heterologous polynucleotides encoding engineered hydantoin can be introduced into suitable host cells to express the corresponding engineered polypeptides.

[0112] As will be apparent to those skilled in the art, the availability of protein sequences and knowledge of codons corresponding to a variety of amino acids provide an indication of all polynucleotides capable of encoding the target protein sequence. The degeneracy of the genetic code, where the same amino acid is encoded by alternative or synonymous codons, allows for the generation of a vast number of nucleic acids, all of which encode the modified hydantoin polypeptides disclosed herein. Therefore, once a specific amino acid sequence is determined, those skilled in the art can generate any number of different nucleic acids by modifying only the sequence of one or more codons without altering the amino acid sequence of the protein. In this regard, this disclosure is particularly conceived of individual and every possible variation of the polynucleotides that can be prepared by selecting combinations based on possible codon selections, and all such variations are considered particularly disclosed for any polypeptide disclosed herein, including the amino acid sequences of exemplary engineered polypeptides provided in Tables 1, 2, and 3, and any polypeptides disclosed by reference as even-numbered sequence identifiers in the sequence listing incorporated herein by reference.

[0113] In various embodiments, the codons are preferably selected to suit the host cell in which the protein is produced. For example, preferred codons for bacteria are used to express genes in bacteria; preferred codons for yeast are used for expression in yeast; and preferred codons for mammals are used for expression in mammalian cells.

[0114] In some embodiments, the polynucleotide encodes a hydantoin polypeptide comprising an amino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence-identical to a reference sequence selected from even-numbered sequence identifiers in SEQ ID NO:4-286, wherein the polypeptide has one or more of hydantoin activity and the improved properties described herein, such as the ability to convert compound A1 to product compound A2 with increased activity compared to the polypeptide of SEQ ID NO:2.

[0115] In some embodiments, the polynucleotide encodes an engineered polypeptide comprising an amino acid sequence having the percentage of identity described above compared to SEQ ID NO:2 and having one or more amino acid residue differences. In some embodiments, this disclosure provides engineered polypeptides having hydantoin activity, said engineered polypeptide comprising a combination of at least 90% sequence identity with a reference sequence of SEQ ID NO:2 and residue differences selected from the following positions: X8, X39, X46, X51, X62, X63, X64, X66, X67, X71, X73, X95, X97, X113, X152, X159, X189, X199, X201, X215, X254, X255, X257, X263, X264, X265, X266, X267, X288, X292, X320, X329, X336, X337, X340, X462, X467, X474, X476, X479.

[0116] In some embodiments, the polynucleotide encoding the engineered polypeptide comprises a subset selected from SEQ ID No: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 15 7, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 2 55, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285 sequences.

[0117] In some embodiments, the polynucleotide encodes the polypeptide described herein, but has about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity at the nucleotide level with a reference polynucleotide encoding an engineered hydantoinase. In some embodiments, the reference polynucleotide sequence is selected from SEQ ID NO. No: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 15 7, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 2 55, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285 sequences.

[0118] Isolated polynucleotides encoding engineered peptides can be manipulated in a variety of ways to provide peptide expression, including further sequence modification through codon optimization to improve expression, insertion into expression elements with or without additional control sequences, and conversion into host cells suitable for expression and peptide production.

[0119] Depending on the expression vector, manipulation of the isolated polynucleotides prior to insertion into the vector may be desired or necessary. Techniques for modifying polynucleotide and nucleic acid sequences using recombinant DNA methods are well known in the art. Guidance is provided in: Sambrook et al., 2001, *Molecular Cloning: A Laboratory Manual*, 3rd edition, Cold Spring Harbor Laboratory Press; and *Current Protocols in Molecular Biology*, edited by Ausubel F., Greene Pub. Associates, 1998, updated 2010.

[0120] In another aspect, this disclosure also relates to recombinant expression vectors, which, depending on the type of host they will be introduced into, include a polynucleotide encoding an engineered polypeptide or a variant thereof, and one or more expression regulatory regions, such as promoters and terminators, origins of replication, etc. Optionally, the nucleic acid sequence of this disclosure can be expressed by inserting the nucleic acid sequence or a nucleic acid construct including the sequence into a suitable expression vector. In generating the expression vector, the coding sequence is located in the vector such that the coding sequence is operatively linked to a suitable control sequence for expression.

[0121] Recombinant expression vectors can be any vector (e.g., plasmids or viruses) that can be readily used in recombinant DNA steps and deliver the expression of polynucleotide sequences. The choice of vector will generally depend on its compatibility with the host cell to which it will be introduced. Vectors can be linear or closed circular plasmids. Expression vectors can be self-replicating vectors, i.e., vectors that exist as extrachromosomal entities and whose replication is independent of chromosomal replication, such as plasmids, extrachromosomal elements, microchromosomes, or artificial chromosomes. Vectors can contain any tools used to ensure self-replication. Optionally, a vector can be one that integrates into the genome upon introduction into the host cell and replicates along with the chromosome into which it is integrated. Furthermore, a single vector or plasmid, or two or more vectors or plasmids containing the total DNA to be introduced into the host cell's genome, can be used.

[0122] Many expression vectors useful for embodiments of this disclosure are commercially available. Exemplary expression vectors can be prepared by operatively linking a polynucleotide encoding an improved hydantoin polypeptide to the plasmid pACYC-Duet-1 (Novagen).

[0123] On the other hand, this disclosure provides a host cell comprising a polynucleotide encoding an improved hydantoin polypeptide of this disclosure, said polynucleotide being operatively linked to one or more control sequences in the host cell for the expression of the hydantoin. Host cells for expressing the polypeptide encoded by the expression vector of this disclosure are well known in the art and include, but are not limited to, bacterial cells such as *Escherichia coli*, *Arthrobacter* species KNK168, *Streptomyces*, and *Salmonella typhimurium* cells; fungal cells such as yeast cells (e.g., *Saccharomyces cerevisiae* or *Pichia pastoris*); 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. An exemplary host cell is *Escherichia coli* BL21(DE3). The host cells described above can be wild-type or genome-edited engineered cells, such as those with the wild-type hydantoin gene knocked out from the host cell genome. Suitable culture media and growth conditions for these host cells are well known in the art.

[0124] Polynucleotides for expressing hydantoin can be introduced into cells by a variety of methods known in the art. These techniques include, among others, electroporation, bioparticle bombardment, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion. The different methods for introducing polynucleotides into cells will be apparent to those skilled in the art.

[0125] 2.4 Methods for generating engineered peptides

[0126] When the sequence of the engineered polypeptide is known, the polynucleotide encoding the polypeptide can be prepared using standard solid-phase methods according to known synthetic methods. In some embodiments, fragments of up to about 100 bases can be synthesized individually and then ligated (e.g., by enzymatic or chemical ligation methods or polymerase-mediated methods) to form any desired continuous sequence. For example, the polynucleotides and oligonucleotides of this disclosure can be prepared by chemical synthesis using, for example, the classic phosphoramidite method described in Beaucage et al., 1981, Tet Lett 22: 1859-69, or the method described in Mattes et al., 1984, EMBO J. 3: 801-05, as typically practiced in automated synthetic methods. According to the phosphoramidite method, the oligonucleotides are synthesized, purified, annealed, ligated, and cloned into suitable vectors, for example, in an automated DNA synthesizer. Furthermore, virtually any nucleic acid can be obtained from any of a variety of commercial sources.

[0127] In some embodiments, this disclosure also provides methods for preparing or manufacturing engineered peptides, wherein the method includes culturing host cells capable of expressing polynucleotides encoding the engineered peptide under culture conditions suitable for peptide expression. In some embodiments, the method for preparing the peptide further includes isolating the peptide. The engineered peptide can be expressed in suitable cells and isolated (or recovered) from the host cells and / or culture medium using any one or more well-known techniques for protein purification, including, among others, lysozyme treatment, sonication, filtration, salting out, heat treatment, ultracentrifugation, chromatography, and chromatography.

[0128] 2.5 Methods using engineered hydantoin and compounds prepared from it

[0129] This disclosure also provides a method for preparing compounds of structural formula (I) using the improved engineered hydantoin peptides described herein:

[0130]

[0131] The compound of structural formula (I) has the stereochemical configuration shown at the chiral center marked with *; the compound of structural formula (I) shown is in excess compared to the corresponding enantiomer, wherein

[0132] n = 0 or 1;

[0133] R1 and R2 are independently selected from H, optionally substituted or unsubstituted aryl or heteroaryl, straight or branched and optionally substituted or unsubstituted C1-C4 alkyl, straight or branched and optionally substituted or unsubstituted C1-C4 alkenyl, optionally substituted or unsubstituted cycloalkyl, -OR', -NH2 or -NR'R', -SR', -CO2R' or -C(O)R';

[0134] Each R' is independently selected from -H or (C1-C4) hydrocarbon groups;

[0135] The method includes using a hydantoin derivative having structural formula (II) as a substrate.

[0136]

[0137] When in contact with engineered hydantoin polypeptide, the definitions of n, R1, and R2 in structural formula (II) are the same as those in structural formula (I).

[0138] On the other hand, this disclosure also provides a method for preparing compounds of structural formula (III) using the improved engineered hydantoin peptides described herein:

[0139]

[0140] The compound of structural formula (III) has the stereochemical configuration shown at the chiral center marked with *; the compound of structural formula (III) shown is in excess compared to the corresponding enantiomer, wherein

[0141] n = 0 or 1;

[0142] R1 and R2 are independently selected from H, straight-chain or branched and optionally substituted or unsubstituted C1-C4 alkyl or optionally substituted or unsubstituted C6H6.

[0143] When n = 0, R1 and R2 can also form a ring structure group together. This group is selected from monocyclic or polycyclic, optionally substituted or unsubstituted aryl or monocyclic or polycyclic, optionally substituted or unsubstituted heteroaryl.

[0144] The method includes using an imide derivative having structural formula (IV) as a substrate.

[0145]

[0146] When in contact with engineered hydantoin polypeptides, the definitions of n, R1, and R2 in structural formula (IV) are the same as those in structural formula (III).

[0147] On the other hand, the improved engineered peptide described in this article can convert DL-p-hydroxyphenylhydantoin into N-carbamoyl-D-p-hydroxyphenylglycine, which can then be further converted into D-p-hydroxyphenylglycine under the action of hydrochloric acid.

[0148]

[0149] On the other hand, the improved engineered peptide described herein can convert A1 to A2. In some embodiments, the engineered peptide can be used in methods for preparing enantiomeric excess of a compound of formula A2:

[0150]

[0151] In these embodiments, the method includes, under suitable reaction conditions, reacting the compound represented by structural formula A1:

[0152]

[0153] Steps for contacting the engineered peptides disclosed herein.

[0154] In some embodiments of the above methods, the compound of formula A2 is produced in an enantiomeric excess of at least 97%, 98%, 99% or greater.

[0155] Specific implementation schemes of engineered hydantoin peptides for use in this method are further provided in the details. Improved engineered peptides that can be used in the above methods may include those selected from the corresponding SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90. 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 22 The amino acid sequence of 4, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286.This also includes those selected from SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 17 4, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 2 An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of the reference amino acid sequences 48, 250, 252, 254, 256, 258, 260, 262, 264, 266.

[0156] As described herein and illustrated in the examples, this disclosure envisions a range of suitable reaction conditions that can be used in the methods described herein, including, but not limited to, ranges of pH, temperature, buffer solution, solvent system, substrate loading, peptide loading, and reaction time. Further suitable reaction conditions for performing the method of biocatalytically converting a substrate compound into a product compound using the engineered hydantoin peptides described herein can be readily optimized through routine experiments, including but not limited to contacting the engineered peptide with the substrate compound under experimental reaction conditions of concentration, pH, temperature, and solvent, and detecting the product compound, for example, using the methods described in the examples provided herein.

[0157] As described above, the engineered polypeptides with hydantoin activity used in the methods of this disclosure typically comprise a subset of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 17 4, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 2 The amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of the reference amino acid sequences 48, 250, 252, 254, 256, 258, 260, 262, 264, 266.

[0158] The substrate compound in the reaction mixture can vary, taking into account factors such as the desired amount of product compound, the effect of substrate concentration on enzyme activity, enzyme stability under reaction conditions, and the percentage conversion of substrate to product. In some embodiments of the method, suitable reaction conditions include substrate loadings of at least about 1 g / L, at least about 5 g / L, at least about 10 g / L, at least about 15 g / L, at least about 20 g / L, at least about 30 g / L, at least about 50 g / L, at least about 75 g / L, at least about 100 g / L, at least about 150 g / L, at least about 200 g / L, or even greater. While the substrate loading values ​​provided herein are based on the molecular weight of compound A1, it is also contemplated that equal molar amounts of various hydrates and salts of compound A1 may be used in the method.

[0159] In embodiments of the reaction, reaction conditions may include a suitable pH. As described above, a desired pH or a desired pH range can be maintained by using an acid or base, a suitable buffer, or a combination of buffering and adding an acid or base. The pH of the reaction mixture can be controlled before and / or during the reaction process. In some embodiments, suitable reaction conditions include a solution pH of about 6 to about 8.5. In some embodiments, reaction conditions include a solution pH of about 6, 6.5, 7, 7.5, 8, or 8.5.

[0160] In embodiments of the methods described herein, suitable temperatures may be used as reaction conditions, taking into account, for example, the increase in reaction rate at higher temperatures and the enzyme activity for a sufficiently long reaction duration. Accordingly, in some embodiments, suitable reaction conditions include temperatures from about 10°C to about 60°C, from about 25°C to about 50°C, from about 25°C to about 40°C, or from about 25°C to about 30°C. In some embodiments, suitable reaction temperatures include temperatures from about 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, or 60°C. In some embodiments, the temperature during the enzymatic reaction may be maintained at a constant temperature throughout the reaction process. In some embodiments, the temperature during the enzymatic reaction may be adjusted as a temperature profile during the reaction process.

[0161] Methods using engineered hydantoin are typically carried out in water or solvents. Suitable solvents include aqueous buffer solutions, organic solvents, and / or co-solvent systems, with the co-solvent system typically comprising both an aqueous solvent and an organic solvent. The aqueous solution (water or aqueous co-solvent system) may be pH buffered or unbuffered. In some embodiments, methods using engineered peptides are typically carried out in an aqueous co-solvent system comprising: organic solvents (e.g., methanol, ethanol, propanol, isopropanol (IPA)), dimethyl sulfoxide (DMSO), dimethylformamide (DMF), isopropyl acetate, ethyl acetate, butyl acetate, 1-octanol, heptane, octane, methyl tert-butyl ether (MTBE), toluene, etc.), and ionic liquids (e.g., 1-ethyl-4-methylimidazole tetrafluoroborate, 1-butyl-3-methylimidazole tetrafluoroborate, 1-butyl-3-methylimidazole hexafluorophosphate, etc.). In an aqueous cosolvent system, the organic solvent component may be miscible with the aqueous component, providing a single liquid phase, or partially miscible or immiscible with the aqueous component, providing a two-phase system. Carbon dioxide generated during the hydrolysis reaction may cause foam formation; an antifoaming agent may be added as appropriate. An exemplary aqueous cosolvent system comprises water and one or more organic solvents. Typically, the organic solvent component of the aqueous cosolvent system is selected such that it does not completely inactivate the hydantoin. Suitable cosolvent systems can be readily identified by measuring the enzyme activity of a specific engineered hydantoin using a chosen substrate of interest in a candidate solvent system and employing enzyme activity assays such as those described herein.

[0162] Suitable reaction conditions may include a combination of reaction parameters that provide for the biocatalytic conversion of a substrate compound into its corresponding product compound. Accordingly, in some embodiments of the method, the combination of reaction parameters includes: (a) a loading of substrate A1 from about 1 g / L to 400 g / L; (b) an engineered peptide concentration from about 0.1 g / L to 50 g / L; (c) a pH from about 6.0 to 8.5; and (d) a temperature from about 10 °C to 60 °C.

[0163] In some embodiments, the method includes contacting a substrate of ≥10 g / L with the engineered peptide described herein at a temperature of about 30°C to about 50°C and a pH of 6.0 to 8.0, such that at least 70%, 80%, 90%, 95% or more of the substrate A1 is converted to product A2 within 24 hours, and product A2 is produced in an enantiomeric excess of at least 97%, 98%, 99% or greater. In some embodiments, the hydantoin peptide capable of performing the above reaction comprises an amino acid sequence corresponding to an even-numbered sequence identifier in SEQ ID NO:4-286.

[0164] Exemplary reaction conditions include those provided in Examples 12-22.

[0165] In carrying out the enzyme-catalyzed reactions described herein, engineered peptides may be added to the reaction mixture in the form of partially purified or purified enzymes, heat-treated enzyme solutions, complete cells transformed with a gene encoding the enzyme, and / or cell extracts and / or lysates of such cells. Complete cells transformed with a gene encoding the engineered peptide, or their cell extracts, lysates, and isolated enzymes may be used in a variety of different forms, including solids (e.g., lyophilized, spray-dried, etc.) or semi-solids (e.g., coarse pastes such as wet bacterial cells). Cell extracts or cell lysates may be partially purified by precipitation (e.g., ammonium sulfate, polyethyleneimine, heat treatment, or similar treatments), followed by a desalting process (e.g., ultrafiltration, dialysis, and similar processes) before lyophilization. Any enzyme product may be stabilized by cross-linking or immobilizing to a solid material (e.g., resin) using a known cross-linking agent such as glutaraldehyde.

[0166] In some embodiments of the enzyme-catalyzed reaction described herein, the reaction is carried out under suitable reaction conditions described herein, wherein the engineered peptide is immobilized onto a solid support. Solid supports that can be used to immobilize engineered peptides performing enzyme-catalyzed reactions include, but are not limited to, microspheres or resins comprising polymethacrylates having epoxy functional groups, polymethacrylates having amino epoxy functional groups, styrene / DVB copolymers having octadecyl functional groups, or polymethacrylates having octadecyl functional groups. Exemplary solid supports include, but are not limited to, chitosan beads, EupergitC, and SEPABEAD (Mitsubishi), including the following different types of SEPABEAD: EC-EP, EC-HFA / S, EXA252, EXE119, and EXE120.

[0167] In some embodiments, the engineered peptide can be expressed in the form of a secretory peptide, and a culture medium containing the secretory peptide can be used in the method described herein.

[0168] In some embodiments, solid reactants (e.g., enzymes, salts, etc.) can be provided to the reaction in various forms, including powders (e.g., lyophilized, spray-dried, etc.), solutions, emulsions, suspensions, etc. Reactants can be readily lyophilized or spray-dried using methods and instruments well known to those skilled in the art. For example, a protein solution can be frozen in small quantities to -80°C and then added to a pre-cooled lyophilization chamber, followed by vacuum drying.

[0169] In some implementations, there are multiple options for the order or manner in which the reactants are added. The reactants may be added to the solvent simultaneously (e.g., a single-phase solvent, a two-phase aqueous co-solvent system, etc.); or alternatively, some reactants may be added first, while other reactants may be added in a flow or in batches at intervals.

[0170] Different features and implementations of this disclosure are exemplified in the following representative embodiments, which are intended to be illustrative rather than limiting. Attached Figure Description

[0171] Figure 1 Roadmap for the original research of pregabalin

[0172] Figure 2 Synthesis of pregabalin and its intermediates via lipase resolution route

[0173] Figure 3 Chemical asymmetric synthesis of pregabalin and its intermediates

[0174] Figure 4 Pregabalin intermediate prepared by asymmetric hydrolysis of 3-isobutylglutarimide with hydantoin.

[0175] Figure 5 The hydrolysis of 3-isobutylglutarimide by hydantoin catalyzed by hydantoin catalyzed to produce (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid

[0176] Figure 6 For protein electrophoresis image analysis Detailed Implementation

[0177] The present invention is further illustrated below with examples, but the invention is not limited thereto. Experimental methods in the following examples, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer.

[0178] Example 1: Construction of gene cloning and expression vectors

[0179] The gene sequence of the wild-type hydantoin derived from *Pseudomonas fluorescens* was retrieved from NCBI (GenBank: KF268426.1), then synthesized using common techniques in the field and cloned into the expression vector pACYC-Duet-1 (Novagen). The recombinant expression plasmid was transformed into competent *E. coli* BL21(DE3) cells. The transformation conditions were 42°C with a 90-second heat shock. The transformation solution was plated on LB agar plates containing chloramphenicol and incubated overnight at 37°C inverted to obtain the recombinant transformant.

[0180] Example 2: Construction of a Heinz mutant library

[0181] All reagents used here are commercial, with the Quikchange kit (supplier: Agilent) being the preferred choice. The sequence design of the mutation primers was performed according to the kit instructions.

[0182] The PCR system consisted of: 2.5 μL of 10× buffer, 1 μL of dNTP mix, 2 μL (5 μM) of primer Oligomix, 2.5 μL (50 ng / μL) of plasmid template, 1 μL of high-fidelity enzyme, and 16 μL of ddH2O.

[0183] The PCR amplification steps were as follows: (1) 95℃ pre-denaturation for 1 min; (2) 95℃ denaturation for 1 min; (3) 55℃ annealing for 1 min; (4) 65℃ extension for 6 min; steps (2) to (4) were repeated 29 times; (5) 65℃ extension for another 5 min, then cooled to 4℃. 2 μl of DpnI (Kit) was added to the PCR product, and the mixture was digested at 37℃ for 2 h. The product was transformed into E. coli BL21 (DE3) electrocompetent cells and plated on LB agar plates containing chloramphenicol. The cells were incubated upside down at 37℃ overnight to obtain the library colonies.

[0184] Example 3: Preparation of enzyme solution for expression and screening of mutant enzyme library

[0185] Colonies from the mutant enzyme library were picked from agar plates and inoculated into 96-well plates containing chloramphenicol LB medium. The plates were then incubated overnight at 30°C on a shaker. When the OD of the culture medium... 600 When the OD of the culture medium reaches 2-3, take 20 μL from the 96-well plate and inoculate it into a 96-well deep-well plate containing chloramphenicol TB medium (400 μL TB medium per well), and incubate on a shaker at 30°C. 600 When the expression level reaches 0.6–0.8, add IPTG to a final concentration of 1 mM as an inducer and incubate overnight (18–20 h) at 30°C on a shaker. After expression, centrifuge the deep-well plate containing the bacterial culture, remove the supernatant, and obtain wet bacterial cells. Add cell lysis buffer (1 g / L lysozyme, 0.5 g / L PMBS, dissolved in PBS buffer, pH 7) to the wet bacterial cells and shake for 1 h to lyse the cells, obtaining the lysis buffer. Centrifuge the lysis buffer and transfer the supernatant to a new deep-well plate to obtain the enzyme solution suitable for screening reactions.

[0186] Example 4: Expression of engineered peptides

[0187] A single colony of *E. coli* BL21(DE3) containing the target engineered peptide expression plasmid was inoculated into a 250 mL Erlenmeyer flask containing 50 mL of LB medium (containing 30 μg / mL chloramphenicol) and incubated overnight at 30 °C with shaking. When the OD of the culture medium... 600 When the OD2 ratio reaches 2, inoculate 5% (v / v) into a 1000mL Erlenmeyer flask containing 250mL of TB culture medium and incubate at 30℃ with shaking. 600 When the concentration reached 0.6, IPTG was added to a final concentration of 1 mM to induce hydantoin expression. After culturing for 20 h, the culture medium was centrifuged (8000 rpm, 10 min), the supernatant was discarded, and the cells were collected to obtain wet bacterial cells. The wet bacterial cells can be used directly to prepare enzyme solutions, or they can be frozen and stored at -20°C until use.

[0188] The wet bacterial cells were resuspended in PBS buffer, sonicated in an ice bath, and the supernatant was collected by centrifugation to obtain an enzyme solution containing engineered peptides.

[0189] Example 5: Quantification of hydantoin peptides in enzyme solution samples

[0190] According to the method in Example 4, the enzyme solution of SEQ ID NO: 2 was prepared, diluted 100-fold (sample 1) and 200-fold (sample 2), and subjected to electrophoretic analysis together with BCA protein standard samples of different concentrations (Easy II Protein Quantitative Kit, brand: Transgen). The protein bands on the electrophoresis image were analyzed by grayscale analysis using computer software to obtain the BCA bands. Figure 6 The standard curve of gray value versus BCA concentration for samples 3-7. The target band of the hydantoin enzyme solution ( Figure 6 By substituting the gray value (indicated by the dashed arrow) into the equation of the standard curve, the concentration of hydantoin in the enzyme solution sample can be obtained.

[0191] Electrophoresis samples Enzyme solution 1 Enzyme solution 2 BCA BCA BCA BCA BCA Protein concentration (μg / mL) 42.9 23.9 100 50 25 12.5 6.25

[0192] Example 6: High-throughput conversion analysis method for liquid phase orifice plates:

[0193] HPLC analysis method: The chromatographic column was a Gemini C18 250mm*4.6mm*5um, the mobile phase was 70% 0.4% HClO4:30% ACN, the flow rate was 1mL / min, the column temperature was 40℃, the detection wavelength was 210nm, the solvent was 50% ACN, and the injection volume was 10uL. The retention time of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid was 5.030min, and the retention time of 3-isobutylglutarimide was 11.188min.

[0194] Example 7: Chiral Analysis Method

[0195] Sample derivatization process: Take 1 mL of reaction solution, weigh potassium carbonate and 2-bromoacetophenone in a ratio of product:potassium carbonate:2-bromoacetophenone (mass ratio) = 5:3:1, add 1 mL of acetonitrile and 1 mL of reaction solution, mix and shake at 1500 rpm for 15 min, add 3 mL of ethyl acetate, shake at 1500 rpm for 15 min, centrifuge, take the ethyl acetate layer, freeze dry, dissolve in 50% ACN and then detect by HPLC.

[0196] HPLC analysis method: The chromatographic column was CHIRALPAK AD-RH 4.6*150mm*5um, the mobile phase used was 50% water (pH adjusted to 2.50 with phosphoric acid): 50% ACN, the flow rate was 0.5ml / min, the column temperature was 30℃, the detection wavelength was 210nm, the injection volume was 10ul, the retention time of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid was 15.2min, and the retention time of (S)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid was 13.2min.

[0197] ee={[(R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid]–[(S)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid]} / {[(R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid]+[(S)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid]}.

[0198] Example 8: Screening reaction for catalytic activity in the first stage of directed evolution

[0199] Following the method in Example 3, an enzyme solution with pH 7.0 was prepared and immediately used for screening reactions.

[0200] In a 96-well plate, the enzyme solution and substrate stock solution (prepared by dissolving substrate A1 in DMSO) were mixed to achieve a final concentration of [substrate 2 g / L, DMSO 10%, enzyme 10 g / L, 0.05 M PBS]. The plate was then placed in a shaker at 250 rpm and 30°C for 22 hours. After the reaction, 200 μL of pure acetonitrile was added to each well to quench the reaction. The plate was then shaken on a plate shaker for 30 min (800 rpm) and centrifuged (4000 rpm, 10 min). The supernatant was collected and analyzed by HPLC according to the method in Example 6 to calculate the conversion rate from A1 to A2. The ee value of product A2 was determined according to the method in Example 7.

[0201] Example 9: Screening reaction for pH stability in the second stage of directed evolution

[0202] Following the method in Example 3, an enzyme solution with pH 6.3 was prepared and shaken at room temperature (20℃-25℃) for 23 hours. Then, PBS buffer was added to adjust the pH of the enzyme solution to 7.0 for screening reaction.

[0203] In a 96-well plate, the pretreated enzyme solution was mixed with the substrate stock solution (prepared by dissolving substrate A1 in DMSO) to achieve a final concentration of [substrate 2 g / L, DMSO 10%, enzyme 3 g / L, 0.05 M PBS]. The plate was then placed in a shaker at 250 rpm and 30°C for 22 hours. After the reaction, 200 μL of pure acetonitrile was added to each well to quench the reaction. The plate was then shaken on a plate shaker for 30 min (800 rpm) and centrifuged (4000 rpm, 10 min). The supernatant was collected and analyzed by HPLC according to the method in Example 6 to calculate the conversion rate from A1 to A2. The ee value of product A2 was determined according to the method in Example 7.

[0204] Example 10: Screening reaction for tolerance of products in the third stage of directed evolution

[0205] Following the method in Example 3, an enzyme solution with pH 7.0 was prepared and immediately subjected to a screening reaction.

[0206] In a 96-well plate, the enzyme solution was mixed with the substrate stock solution (prepared by dissolving substrate A1 in DMSO) and the product stock solution (dissolved by dissolving product A2 in PBS buffer) to achieve the following final concentrations: [substrate 2 g / L, product 50 g / L, DMSO 10%, enzyme 0.3 g / L, 0.05 M PBS]. The plate was then placed in a shaker at 250 rpm and 30°C for 22 hours. After the reaction, 200 μL of pure acetonitrile was added to each well to quench the reaction. The plate was then shaken on a plate shaker for 30 min (800 rpm) and centrifuged (4000 rpm, 10 min). The supernatant was collected and analyzed by HPLC according to the method in Example 6 to calculate the conversion rate from A1 to A2.

[0207] Example 11: Screening reaction for thermal stability in the third stage of directed evolution

[0208] Following the method in Example 3, an enzyme solution with pH 7.0 was prepared and shaken at 50°C for 23 hours before a screening reaction was performed.

[0209] In a 96-well plate, the enzyme solution was mixed with the substrate stock solution (prepared by dissolving substrate A1 in DMSO) to achieve the following final concentrations: [substrate A1 2 g / L, DMSO 10%, enzyme 0.3 g / L, 0.05 M PBS]. The plate was then placed in a shaker at 250 rpm and 30°C for 22 hours. After the reaction, 200 μL of pure acetonitrile was added to each well to quench the reaction. The plate was then shaken on a plate shaker for 30 min (800 rpm) and centrifuged (4000 rpm, 10 min). The supernatant was collected and analyzed by HPLC according to the method in Example 6 to calculate the conversion rate of A1 to A2.

[0210] Example 12: Reaction method for determining conversion rate in a 5mL shake-flask culture during the first stage.

[0211] In a 30 mL reaction flask, 250 mg of wet bacterial cells expressing SEQ ID NO: 8 and 50 mg of substrate A1 were added. Finally, PBS buffer (0.1 M, pH 7.0) was added to bring the total reaction volume to 5.0 mL. The concentrations of each component in the reaction system were [wet bacterial cells 50 g / L, substrate A1 10 g / L]. A magnetic stir bar was added to the reaction flask, and the flask was placed on a magnetic stirrer set to 400 rpm and 30 °C to start the reaction. After 24 hours of reaction, 5 mL of acetonitrile was added to quench the reaction for 30 min. The inactivation solution was transferred to a 2 mL centrifuge tube and centrifuged (13000 rpm, 3 min). The supernatant was collected and analyzed by HPLC according to 1) the method of Example 6 to calculate the conversion rate of A1 to A2; and 2) the method of Example 7 to calculate the ee value of A2.

[0212] Example 13: Reaction method for determining conversion rate in a 5mL shake-flask culture during the second stage.

[0213] In a 30 mL reaction flask, 30 mg of wet bacterial cells expressing SEQ ID NO: 50 and 50 mg of substrate A1 were added. Finally, PBS buffer (0.1 M, pH 7.0) was added to bring the total reaction volume to 5.0 mL. The concentrations of each component in the reaction system were [wet bacterial cells 6 g / L, substrate A1 10 g / L]. A magnetic stir bar was added to the reaction flask, and the flask was placed on a magnetic stirrer set to 400 rpm and 30 °C to begin the reaction. After 24 hours of reaction, 5 mL of acetonitrile was added to quench the reaction for 30 min. The inactivation solution was transferred to a 2 mL centrifuge tube and centrifuged (13000 rpm, 3 min). The supernatant was collected and analyzed by HPLC according to 1) the method of Example 6 to calculate the conversion rate of A1 to A2; and 2) the method of Example 7 to calculate the ee value of A2.

[0214] Example 14: Reaction method for determining conversion rate in a 5mL shake-flask bacterial reaction during the third stage.

[0215] In a 30 mL reaction flask, 5 mg of wet bacterial cells expressing SEQ ID NO: 184 and 50 mg of substrate A1 were added. Finally, PBS buffer (0.1 M, pH 7.0) was added to bring the total reaction volume to 5.0 mL. The concentrations of each component in the reaction system were [1 g / L wet bacterial cells, 10 g / L substrate A1]. A magnetic stir bar was added to the reaction flask, and the flask was placed on a magnetic stirrer set to 400 rpm and 40 °C to begin the reaction. After 24 hours of reaction, 5 mL of acetonitrile was added to quench the reaction for 30 min. The inactivation solution was transferred to a 2 mL centrifuge tube and centrifuged (13000 rpm, 3 min). The supernatant was collected and analyzed by HPLC according to 1) the method in Example 6 to calculate the conversion rate of A1 to A2; and 2) the method in Example 7 to calculate the ee value of A2.

[0216] Example 15: Process for the engineered synthesis of pregabalin intermediates catalyzed by hydantoin polypeptide SEQ ID No: 10

[0217] In a 500 mL reaction vessel, 100 mL of 0.05 M PBS pH 7.0 buffer was added, followed by 50 mL of enzyme solution (SEQ ID No: 10). The water bath was turned on and the temperature was maintained at 30 °C, with stirring at 200 rpm. Finally, 3 g of substrate (3-isobutylglutarimide) was added to initiate the reaction. During the reaction, the pH was adjusted with ammonia water to maintain it at 7.0 ± 0.2. The reaction was stopped after 20 h. Samples were taken and the conversion rate after 20 h was measured to be 71%.

[0218] After the reaction was completed, the reaction solution was filtered with diatomaceous earth as an aid. The filtrate was concentrated to about 100 mL, and hydrochloric acid was added dropwise to adjust the pH of the concentrate to 3.0. After stirring for 30 min, the solution was filtered to obtain a wet crude product. After drying, the product was weighed, and finally about 2.2 g of crude product was obtained, with ee% = 99.8%.

[0219] Example 16: Process for the engineered synthesis of pregabalin intermediates catalyzed by hydantoin polypeptide SEQ ID No: 24

[0220] In a 500 mL reaction vessel, 100 mL of 0.05 M PBS pH 7.0 buffer was added, followed by 50 mL of enzyme solution (SEQ ID No: 24). The water bath was turned on and the temperature was maintained at 30 °C, with stirring at 200 rpm. Finally, 3 g of substrate (3-isobutylglutarimide) was added to initiate the reaction. During the reaction, the pH was adjusted with ammonia water to maintain it at 7.0 ± 0.2. The reaction was stopped after 20 h. Samples taken after 20 h of reaction showed a conversion rate of 73%.

[0221] After the reaction was completed, the reaction solution was filtered with diatomaceous earth as an aid. The filtrate was concentrated to about 100 mL, and hydrochloric acid was added dropwise to adjust the pH of the concentrate to 3.0. After stirring for 30 min, the solution was filtered to obtain a wet crude product. After drying, the product was weighed, and finally about 2.3 g of crude product was obtained, with ee% = 99.7%.

[0222] Example 17: Process for the engineered synthesis of pregabalin intermediates catalyzed by hydantoin polypeptide SEQ ID No: 52

[0223] In a 500 mL reaction vessel, 140 mL of 0.05 M PBS pH 7.0 buffer was added, followed by 10 mL of enzyme solution (SEQ ID No: 52). The water bath was turned on and the temperature was maintained at 35 °C, with stirring at 200 rpm. Finally, 10 g of substrate (3-isobutylglutarimide) was added to initiate the reaction. During the reaction, the pH was adjusted with ammonia water to maintain it at 7.0 ± 0.2. The reaction was stopped after 20 h. Samples taken after 20 h showed a conversion rate of 95%.

[0224] After the reaction was completed, the reaction solution was filtered with diatomaceous earth as an aid. The filtrate was concentrated to about 100 mL, and hydrochloric acid was added dropwise to adjust the pH of the concentrate to 3.0. After stirring for 30 min, the solution was filtered to obtain a wet crude product. After drying, the product was weighed to obtain 10.1 g of crude product with ee% ≥ 99.6%.

[0225] Example 18: Process for the engineered synthesis of pregabalin intermediates catalyzed by hydantoin polypeptide SEQ ID No: 162

[0226] In a 500 mL reaction vessel, 140 mL of 0.05 M PBS pH 7.0 buffer was added, followed by 10 mL of enzyme solution (SEQ ID No: 162). The water bath was turned on and the temperature was maintained at 35 °C, with stirring at 200 rpm. Finally, 10 g of substrate (3-isobutylglutarimide) was added to initiate the reaction. During the reaction, the pH was adjusted with ammonia water to maintain it at 7.0 ± 0.2. The reaction was stopped after 20 h. Samples taken after 20 h of reaction showed a conversion rate of 96%.

[0227] After the reaction was completed, the reaction solution was filtered with diatomaceous earth as an aid. The filtrate was concentrated to about 100 mL, and hydrochloric acid was added dropwise to adjust the pH of the concentrate to 3.0. After stirring for 30 min, the solution was filtered to obtain a wet crude product. After drying, the product was weighed, and finally 10.4 g of crude product was obtained, with ee% = 99.5%.

[0228] Example 19: Process for the engineered synthesis of pregabalin intermediates catalyzed by hydantoin polypeptide SEQ ID No: 184

[0229] In a 500 mL reaction vessel, 145 mL of 0.05 M PBS pH 7.0 buffer was added, followed by 5 mL of enzyme solution (SEQ ID No: 184). The water bath was turned on and the temperature was maintained at 45 °C, with stirring at 200 rpm. Finally, 30 g of substrate (3-isobutylglutarimide) was added to initiate the reaction. During the reaction, the pH was adjusted with ammonia water to maintain it at 7.0 ± 0.2. The reaction was stopped after 20 h. Samples were taken and the conversion rate after 20 h was measured to be 98%.

[0230] After the reaction was completed, the reaction solution was filtered with diatomaceous earth as an aid. The filtrate was concentrated to about 100 mL, and hydrochloric acid was added dropwise to adjust the pH of the concentrate to 3.0. After stirring for 30 min, the solution was filtered to obtain a wet crude product. After drying, the product was weighed, and finally 32.1 g of crude product was obtained, with ee% = 99.7%.

[0231] Example 20: Process for the engineered synthesis of pregabalin intermediates catalyzed by hydantoin polypeptide SEQ ID No: 264

[0232] In a 500 mL reaction vessel, 145 mL of 0.05 M PBS pH 7.0 buffer was added, followed by 5 mL of enzyme solution (SEQ ID No: 264). The water bath was turned on and the temperature was maintained at 45 °C, with stirring at 200 rpm. Finally, 20 g of substrate (3-isobutylglutarimide) was added to initiate the reaction. During the reaction, the pH was adjusted with ammonia water to maintain it at 7.0 ± 0.2. The reaction was stopped after 20 h. Samples taken after 20 h of reaction showed a conversion rate of 72%.

[0233] After the reaction was completed, the reaction solution was filtered with diatomaceous earth as an aid. The filtrate was concentrated to about 100 mL, and hydrochloric acid was added dropwise to adjust the pH of the concentrate to 3.0. After stirring for 30 min, the solution was filtered to obtain a wet crude product. After drying, the product was weighed, and finally 15.8 g of crude product was obtained, with ee% = 99.8%.

[0234] Example 21: Process for the engineered synthesis of pregabalin intermediates catalyzed by hydantoin polypeptide SEQ ID No: 286

[0235] In a 500 mL reaction vessel, 145 mL of 0.05 M PBS pH 7.0 buffer was added, followed by 5 mL of enzyme solution (SEQ ID No: 286). The water bath was turned on and the temperature was maintained at 45 °C, with stirring at 200 rpm. Finally, 36 g of substrate (3-isobutylglutarimide) was added to initiate the reaction. During the reaction, the pH was adjusted with ammonia water to maintain it at 7.0 ± 0.2. The reaction was stopped after 20 h. Samples were taken and the conversion rate after 20 h was measured to be 96%.

[0236] After the reaction was completed, the reaction solution was filtered with diatomaceous earth as an aid. The filtrate was concentrated to about 100 mL, and hydrochloric acid was added dropwise to adjust the pH of the concentrate to 3.0. After stirring for 30 min, the solution was filtered to obtain a wet crude product. After drying, the product was weighed, and finally 37.9 g of crude product was obtained, with ee% = 99.8%.

[0237] Example 22: Process for the engineered hydantoin polypeptide SEQ ID No: 214 to catalyze the synthesis of D-p-hydroxyphenylglycine.

[0238] The following is a representative procedure for detecting conversion using a 5 mL reaction volume. In a 30 mL reaction flask, 70 μL of the enzyme solution (SEQ ID NO:214), 50 mg of p-hydroxyphenylhydantoin, and finally 5 mL of phosphate buffer (0.1 M, pH 7.5) were added to achieve the following concentrations: [SEQ ID NO:214 enzyme solution 14 mL / L, p-hydroxyphenylhydantoin 10 g / L]. A magnetic stir bar was added to the flask, and the flask was placed on an IKA magnetic stirrer set to 400 rpm and 40 °C to begin the reaction. After 1 hour of reaction, 5 mL of acetonitrile was added to quench the reaction. Concentrated hydrochloric acid was added to the quenched flask to a concentration of 2 mmol / L, followed by 27 mg of sodium bisulfite. The flask was then placed on a magnetic stirrer at 50 °C and 400 rpm for hydrolysis. Three hours later, 5 mL of 0.1% glacial acetic acid was added to the reaction flask, and then the reaction solution was centrifuged (13000 rpm, 3 min). The supernatant after centrifugation was used to analyze the sample by HPLC, and the conversion rate was found to be 42.3%.

[0239]

[0240] It should be understood that after reading the above description of the present invention, those skilled in the art can make various alterations or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An engineered hydantoin polypeptide that catalyzes the asymmetric hydrolysis of 3-isobutylglutarimide to generate (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid, wherein the ee value is at least 97%; The amino acid sequence of the polypeptide is SEQ ID No: 8, 10, 12, 14, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172 The amino acid sequences shown are 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, and 286.

2. The reaction conditions for hydrolyzing the polypeptide according to claim 1 to generate (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid include: Loading of 3-isobutylglutarimide from 1 g / L to 400 g / L, loading of engineered hydantoin peptide from 0.1 g / L to 50 g / L, pH from 6.0 to 8.5, and reaction temperature from 10 to 60 °C.

3. A polypeptide that is immobilized on a solid material by means of chemical bonding or physical adsorption, wherein the polypeptide is selected from the polypeptides described in any one of claims 1-2.

4. A polynucleotide, said polynucleotide encoding a polypeptide according to any one of claims 1-3.

5. The polynucleotide of claim 4, wherein the polynucleotide sequence is the corresponding SEQ ID. No: 7, 9, 11, 13, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 14 3. 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 1 The polynucleotide sequences of 73, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285.

6. An expression vector comprising the polynucleotide of any one of claims 4-5.

7. The expression vector of claim 6, wherein the expression vector comprises a plasmid, a granule, a bacteriophage, or a viral vector.

8. A host cell comprising the expression vector according to any one of claims 6-7.

9. The host cell according to claim 8, wherein the host cell is Escherichia coli.

10. A method for preparing a hydantoin polypeptide, comprising the following steps: culturing the host cell as described in any one of claims 8-9, and obtaining the hydantoin polypeptide from the culture.

11. A hydantoin catalyst selected from the cultures of claim 10, obtained from host cells or culture media containing hydantoin polypeptides, or articles thereof; wherein, The product refers to an extract obtained from host cells, a separated product obtained by separating or purifying the hydantoin in the extract, or an immobilized product obtained by immobilizing host cells and their extracts or the separated products of the extracts.

12. A method for preparing D-p-hydroxyphenylglycine, the method comprising: a substrate DL-p-hydroxyphenylhydantoin Under the action of the hydantoin peptide according to any one of claims 1-3, it is converted into N-carbamoyl-D-p-hydroxyphenylglycine. N-Carbamoyl-D-p-hydroxyphenylglycine is further converted to D-p-hydroxyphenylglycine under the action of hydrochloric acid. .

13. A method for preparing compound (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid of formula A2, The method includes using compound A1, 3-isobutylglutarimide. The step of contacting the hydantoin peptide according to any one of claims 1-3.

14. The method of any one of claims 12-13, wherein the product is produced in excess of at least 97% enantiomers.

15. The method according to any one of claims 12-13, wherein the reaction solvent comprises one or more of water, methanol, ethanol, propanol, isopropanol, dimethyl sulfoxide, dimethylformamide, isopropyl acetate, ethyl acetate, butyl acetate, 1-octanol, heptane, octane, methyl tert-butyl ether, or toluene.

16. The method according to any one of claims 12-13, wherein the temperature of the reaction conditions is from 10°C to 60°C.

17. The method of any one of claims 12-13, wherein the pH of the reaction conditions is from 6.0 to 8.

5.

18. The method of any one of claims 12-13, wherein the substrate loading is from 1 g / L to 400 g / L.

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