Biocatalyst for synthesizing pregabalin intermediates and method for synthesizing same

Abstract

The present disclosure provides an engineered polypeptide that can catalyze the asymmetric hydrolysis of 3-isobutylglutarimide to produce (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid, which has high stereoselectivity, high catalytic activity, excellent process stability and thermal stability, can tolerate high product concentrations, and has high industrial application value. [C13] JPEG2025501363000043.jpg43170

Description

[Technical field]

[0001] The present invention relates to a biocatalyst and a process for the preparation of a pregabalin intermediate using said biocatalyst. [Background technology]

[0002] Pregabalin is a chiral small molecule compound with the chemical name S-(+)-3-isobutyl γ-aminobutyric acid. Pregabalin is related to endogenous inhibitory neurotransmitters and has antiepileptic activity, so it is commonly used as a chiral drug to treat epilepsy and neuralgia. This original drug is produced by Pfizer in the United States, approved by the European Union in July 2004 as a treatment for some epileptic seizures, and approved for sale by the US FDA in 2005. Its unique research route is shown in Figure 1.

[0003] One of the most important indicators in the production of pregabalin products is chiral purity. The synthesis methods of pregabalin and its intermediates in existing patents and literature are mainly divided into three categories: chemical / enzymatic resolution, asymmetric synthesis and chiral source synthesis, of which the first two are commonly used. In the resolution route, the ee value of the product is relatively low, and the compound of a different configuration needs to be racemized and reused, resulting in a very low yield of the final qualified product. For example, CN102102114B discloses a technology for preparing pregabalin intermediates by resolution of lipase to finally synthesize pregabalin. The conversion rate of the resolution step is about 40-45%, and the overall yield is only about 30%, and the route is shown in Figure 2.

[0004] On the other hand, asymmetric synthesis methods that introduce chirality into the reaction have a high utilization rate of raw materials and can be carried out by chiral catalysts or enzymes. However, chemical asymmetric synthesis methods require the use of expensive chiral catalysts, and the processes are often complicated and tedious. For example, the original research route developed by Pfizer in the United States requires nine steps. Although the synthesis process of patent CN105753726B is only four steps, it requires the use of chiral thiourea ammonium salt as a catalyst, there are processes such as hydrogenation, and the reaction conditions are harsh, and the route is shown in Figure 3.

[0005] Therefore, there is an urgent need to find a more suitable method for producing pregabalin. CN111944856A discloses a new synthetic route for pregabalin intermediates, which uses hydantoinase to asymmetrically hydrolyze 3-isobutylglutarimide to prepare pregabalin intermediate (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid with high chiral purity (see Figure 4). In this reaction step, (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid with ee ≥ 99% can be obtained, the steps of resolution and reracemization are avoided, the reaction path is shortened, the utilization rate of raw materials is improved, the cost is effectively reduced, and it is environmentally friendly. However, the hydantoinase disclosed in CN111944856A has a not sufficiently ideal catalytic performance, the enzyme usage is high, and the space-time yield of the product that can be achieved is low.

[0006] In response to these shortcomings, the present invention discloses a series of engineered hydantoinase polypeptides developed by directed evolution techniques, which significantly reduce the enzyme usage, simplify and streamline the enzymatic reaction and downstream processing, and improve the space-time yield. Summary of the Invention

[0007] 1. Overview The present invention provides an engineered polypeptide with high stereoselectivity, high catalytic activity, excellent process stability and thermal stability, and can withstand high product concentrations, which can catalyze the asymmetric hydrolysis of 3-isobutylglutarimide to produce (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid. The present invention also provides a gene for the engineered polypeptide, a recombinant expression vector containing the gene, an engineered strain, and an efficient method for preparing the engineered polypeptide, as well as a reaction process for preparing (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid using the engineered polypeptide.

[0008] As a result of experimental research, the present inventors have discovered a wild-type hydantoinase (GenBank: KF268426.1) derived from Pseudomonas fluorescens. Its amino acid sequence is shown in SEQ ID NO:2. Compared with the hydantoinase disclosed in CN111944856A, SEQ ID NO:2 has a better activity of catalyzing the production of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid by hydrolysis of 3-isobutylglutarimide. Among wild-type hydantoinases, SEQ ID NO:2 is an enzyme having a better activity for the reaction shown in FIG. 5, but is still far from industrial use and various performance improvements are required. Research on this wild-type hydantoinase was reported in Appl Biochem Biotechnol (2016) 179:1-15, and the results of the research showed that when wild-type hydantoinase catalyzes the hydrolysis reaction of substituted hydantoins, the optimal pH is 8.5 to 9.5, and when pH is < 7.5, the activity is significantly reduced, the thermal stability is also reduced, and the half-lives at 50°C, 55°C, and 60°C are 2.23h, 1.44h, and 0.78h, respectively, which is unfavorable for the production and storage of enzyme preparations. Research by the present inventors has revealed that in the absence of a catalyst, 3-isobutylglutarimide spontaneously hydrolyzes to form racemic 3-(carbamoylmethyl)-5-methylhexanoic acid. The rate of spontaneous hydrolysis is strongly correlated with pH, ​​and when pH>8.5, the rate of spontaneous hydrolysis becomes significant, and the racemic product produced contains the undesired chiral isomer (S)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid, which affects the chiral purity (i.e., ee value) of the final product. Therefore, spontaneous hydrolysis of 3-isobutylglutarimide is avoided as much as possible by the present invention. Since spontaneous hydrolysis of 3-isobutylglutarimide is hardly detected at pH≦7.0, the reaction shown in FIG. 5 must be carried out at pH≦7.0.

[0009] In addition to the need to improve the activity, thermostability and stability of SEQ ID NO:2 in catalyzing the reaction shown in Figure 5 at pH ≤ 7.0, the inventors have found through research that when the product concentration in the enzyme reaction exceeds a certain level, the activity of SEQ ID NO:2 is significantly inhibited, limiting further improvement of the space-time yield, so that the inhibition of the product of SEQ ID NO:2 is overcome (in other words, the tolerance of SEQ ID NO:2 to high product concentrations is improved), which is also a property of SEQ ID NO:2 that needs to be improved. The inventors have used directed evolution techniques combined with computer-aided design and screening to engineer SEQ ID NO:2 to obtain a series of engineered polypeptides with high stereoselectivity, high catalytic activity, excellent thermostability and reaction pH stability, and the ability to tolerate high product concentrations. These engineered polypeptides include amino acid sequences that have one or more residue differences compared to the reference sequence of SEQ ID NO:2, where these residue differences occur at amino acid positions that affect several different functional properties of the enzyme, such as catalytic activity, stereoselectivity, substrate and / or product tolerance, thermostability, reaction process stability (including pH variation range, ionic strength, solvent tolerance, etc.), recombinant expression efficiency, other properties that affect the enzyme preparation and catalytic performance, and various combinations of these properties.

[0010] In some embodiments, the engineered polypeptide comprises an amino acid sequence having at least 90% sequence identity to the polypeptide of SEQ ID NO:2, with one or more residue differences at residue positions selected from 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 compared to SEQ ID NO:2. The differences in amino acid residues compared in NO:2 were A8G, A39P, G46A, L51V, L51I, M62L, Q63E, L64I, L64T, L64S, L64A, F66Y, F66L, M67W, M67Y, M67F, A71T, A71S, E73D, I95V, I95L, I95M, N97G, and N 97D, N97L, N97Q, A113T, F152Y, F152M, F152L, I159L, I159F, I159Y, L189I, L189V, L189M, A199V, G201H, Q215A, Q215P, S254Q, S254L, S254N, S254G, S254F, K255F, K2 55Y, K255H, K255N, Q257W, V263T, L264C, A265P, G266Q, H267Y, M288C, F292L, F32 0S, F320L, R329A, R329L, R329Y, P336M, P336L, P336Q, N337P, A340P, F462R, K467D , P474W, A476P, R479Q, R479L, R479P, or in addition to these differences, insertions or deletions 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.

[0011] As provided herein, in some embodiments, the disclosed amino acid differences may be used alone or in various combinations to obtain engineered polypeptides with improved enzymatic properties. In some embodiments, the engineered polypeptide comprises an amino acid sequence having at least 90% sequence identity with a reference sequence SEQ ID NO:2 and having at least one residue difference 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.

[0012] More specifically, in some embodiments, the improved engineered polypeptides based on SEQ ID NO:2 include those 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, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 4, 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, 228, 230, These include polypeptides consisting of the amino acid sequences represented by 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.

[0013] In some embodiments, the improved engineered polypeptide is selected from the group consisting of SEQ ID NOs: 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, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 230, 231, 232, 233, 234, 235, 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, 288, 2 , 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, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 2 50, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, and the like.

[0014] The identity between two amino acid sequences or two nucleotide sequences can be obtained by algorithms commonly used in the field, and can be calculated using NCBI Blastp and Blastn software according to default parameters, or the Clustal W algorithm (Nucleic Acid Research, 22 (22): 4673-4680, 1994) can be employed. 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%.

[0015] In another aspect, the invention provides polynucleotide sequences encoding engineered polypeptides. In some embodiments, the polynucleotide may be part of an expression vector having one or more regulatory sequences for expressing the engineered polypeptide. In some embodiments, the polynucleotide is selected from the group consisting of 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, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 220, 221, 222, 223, 225, 227, 229, 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, 3, 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, 159, 161, 1 63, 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, 2 31, 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.

[0016] As known to those of skill in the art, due to the degeneracy of nucleotide codons, the sequences of 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, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 15 4, 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, 1 64, 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, 230, 23 The polynucleotide sequences encoding the amino acid sequences of 2, 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 set forth in 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, 4 7, 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, 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, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285. The nucleic acid sequence of the hydantoinase gene of the present invention is represented by SEQ ID NO: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, 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, 23 4, 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 or any other nucleic acid sequence encoding the amino acid sequence shown in

[0017] In another aspect, the disclosure provides a polynucleotide encoding an engineered polypeptide or an expression vector capable of expressing the engineered polypeptide, and a host cell. In some embodiments, the host cell may be a bacterial host cell, such as E. coli. The host cell may be used to express and isolate the engineered polypeptides described herein, or may be used directly in a reaction to convert a substrate to a product.

[0018] In some embodiments, the engineered polypeptide in the form of a whole cell, a crude extract, an isolated polypeptide, or a purified polypeptide can be used alone or in immobilized form (e.g., immobilized on a resin). [ka] [ka]

[0019] The disclosure also provides a method of converting a compound of formula A1 to a chiral compound of formula A2 using an engineered polypeptide disclosed herein, where the chiral compound of formula A2 is in excess of the corresponding enantiomer, the method comprising contacting a compound of formula A1 with an engineered polypeptide under reaction conditions suitable for converting A1 to A2, the engineered polypeptide being an engineered polypeptide described herein. In some embodiments, the engineered polypeptide has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:2 and is capable of converting a compound of formula A1 to a compound of formula A2.

[0020] In some embodiments, the compound of formula A2 is produced in at least 97%, 98%, 99% or more enantiomeric excess.

[0021] Specific embodiments of engineered polypeptides for use in the methods are further described in the Detailed Description of the Invention. An engineered polypeptide that may be used in the above methods may comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO:2, with one or more residue differences compared to SEQ ID NO:2 at residue positions selected from 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. The amino acid residue differences compared to NO:2 are A8G, A39P, G46A, L51V, L51I, M62L, Q63E, L64I, L64T, L64S, L64A, F66Y, F66L, M67W, M67Y, M67F, A71T, A71S, E73D, I95V, I95L, I95M, N97G, and N 97D, N97L, N97Q, A113T, F152Y, F152M, F152L, I159L, I159F, I159Y, L189I, L189V , L189M, A199V, G201H, Q215A, Q215P, S254Q, S254L, S254N, S254G, S254F, K255F, K 255Y, K255H, K255N, Q257W, V263T, L264C, A265P, G266Q, H267Y, M288C, F292L, F3 20S, F320L, R329A, R329L, R329Y, P336M, P336L, P336Q, N337P, A340P, F462R, K467 D, P474W, A476P, R479Q, R479L, R479P, or in addition to these differences, insertions or deletions 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.

[0022] In some embodiments, the engineered polypeptides that may be used in the above methods are selected from 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, 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 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, 286.

[0023] Any of the methods for using engineering polypeptides for formula A2 compounds disclosed herein can be carried out under a range of suitable reaction conditions, including but not limited to, pH, temperature, buffer, solvent system, substrate loading, polypeptide loading, pressure, and reaction time ranges. For example, in some embodiments, preparation of formula A2 compounds can be carried out, where suitable reaction conditions include: (a) substrate loading of about 1 g / L to 400 g / L compound A1, (b) engineering polypeptide loading of about 0.1 g / L to 50 g / L, (d) pH of about 6.0 to about 8.5, and (d) temperature of about 10° C. to 60° C.

[0024] In some embodiments, the engineered polypeptide is capable of converting substrate compound A1 to compound A2 under suitable reaction conditions with at least about a 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold or greater increase in activity compared to the reference polypeptide of SEQ ID NO:2. In some embodiments, the engineered polypeptide is capable of converting substrate compound A1 to compound A2 under suitable reaction conditions within about 48 hours, about 36 hours, about 24 hours or less of reaction time with at least about 5 g / Lh. -1 , 10g / Lh -1 , 15g / Lh -1 , 20g / Lh -1 or more.

[0025] 2 Details 2.1 Definition For the purposes of this disclosure, unless expressly defined otherwise, technical and scientific terms used herein have the meanings that are commonly understood by those of ordinary skill in the art.

[0026] "Protein," "polypeptide," and "peptide" are used interchangeably herein to mean a polymer of at least two amino acids covalently joined by amide bonds, regardless of length or post-translational modification (e.g., glycosylation, phosphorylation, lipidation, myristoylation, ubiquitination, etc.). Included within this definition are D- and L-amino acids, and mixtures of D- and L-amino acids.

[0027] The terms "engineered hydantoinase," "engineered hydantoinase polypeptide," "improved hydantoinase polypeptide," and "engineered polypeptide" are used interchangeably herein.

[0028] "Polynucleotide" and "nucleic acid" are used interchangeably herein.

[0029] "Coding sequence" refers to a nucleic acid portion (eg, a gene) that codes for the amino acid sequence of a protein.

[0030] "Naturally-occurring" or "wild-type" refers to a form found in nature. For example, a naturally-occurring or wild-type polypeptide or polynucleotide sequence is a sequence that occurs in an organism, isolatable from a source in nature, and has not been intentionally modified by human manipulation.

[0031] "Recombinant" or "engineered" or "non-naturally occurring," when used to refer to, for example, a cell, nucleic acid, or polypeptide, refers to material that is altered in a way that does not occur in nature, or that is the same but produced or obtained from synthetic material and / or by manipulation using recombinant techniques, or material that corresponds to the native or native form of such material.

[0032] "Sequence identity" and "homology" are used interchangeably herein to refer to a comparison between polynucleotides or polypeptides ("sequence identity" and "homology" are usually expressed as a percentage) and are determined by comparing two best aligned sequences in a comparison window, where a portion of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions (i.e., gaps) for best alignment of the two sequences compared to the reference sequence. The percentage is obtained by determining the number of positions in the two sequences where the same nucleic acid base or amino acid residue occurs to obtain the number of matching positions, and dividing this number of matching positions by the total number of positions in the comparison window, multiplying by 100 to obtain the percentage of sequence identity. Alternatively, the percentage may be obtained by determining the number of positions where the same nucleic acid base or amino acid residue occurs in both sequences, or the number of positions where the nucleic acid base or amino acid residue aligns with a gap to obtain the number of matching positions, and dividing this number of matching positions by the total number of positions in the comparison window, multiplied by 100 to obtain the percentage of sequence identity. Those skilled in the art will recognize that there are many established algorithms that can be used to align two sequences. Sequences for comparison can be best aligned using, for example, the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, the similarity search method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444, computer implementations of these algorithms (GAP, BESTFIT, FASTA, or TFASTA in the GCCG Wisconsin package), or by visual inspection (see generally, Current Protocols in Molecular Biology, eds. FM Ausubel et al., Current Protocols, a joint venture of Greene Publishing Associates Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)).Examples of suitable algorithms for determining percentage of sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms described in Altschul et al., 1990, J. Mol. Biol. 215:403-410, and Altschul et al., 1977, Nucleic Acids Res. 3389-3402, respectively. The software used to perform BLAST analysis is publicly available from the National Center for Biotechnology Information website. This algorithm involves identifying high-scoring sequence pairs (HSPs) by first identifying short words of length W in the query sequence that match or meet some positive threshold score T when compared to words of the same length in the database sequences. T is called the neighborhood word score threshold (Altschuletal et al., supra). These initial neighborhood word hits act as seeds to initiate searches to find longer HSPs that contain them. The word hits are then extended in both directions along each sequence to the point where the cumulative alignment score cannot be increased. For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for matching residue pairs, always >0) and N (penalty score for mismatching residues, always <0). For amino acid sequences, a score matrix is ​​used to calculate the cumulative score. Extension of the word hit string in each direction is terminated when the accumulation of one or more negative scoring residue alignments that would reduce the cumulative alignment score by an amount X from the maximum achieved causes the cumulative score to fall below 0 or when either end of the sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses default values ​​of a word length (W) of 11, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.For amino acid sequences, the BLASTP program uses as default values ​​a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, 1989, Proc Natl Acad Sci USA 89:10915). Exemplary sequence alignments and determination of percent sequence identity can be performed using the BESTFIT or GAP programs in the GCG Wisconsin software package (Accelrys, Madison WI) using the default parameters provided.

[0033] A "reference sequence" refers to a defined sequence used as a basis for sequence comparison. A reference sequence may 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 may each (1) contain sequences that are similar between the two sequences (i.e., a portion of the complete sequence) and (2) further contain sequences that differ between the two sequences, a comparison of sequences between two (or more) polynucleotides or polypeptides is 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, but may include engineered or modified sequences. For example, a "reference sequence based on SEQ ID NO:2 with a threonine at the residue corresponding to X64" refers to a reference sequence in which the residue corresponding to X64 in SEQ ID NO:2 (leucine) has been changed to threonine.

[0034] A "comparison window" refers to a conceptual segment of at least about 20 contiguous nucleotide positions or amino acid residues over which a sequence can be compared to a reference sequence of at least 20 contiguous nucleotides or amino acid residues, where the portion of the sequence within the comparison window may contain no more than 20% additions or deletions (i.e., gaps) compared to the reference sequence (no additions or deletions) for optimal alignment of the two sequences. The comparison window can be longer than 20 contiguous residues, and optionally includes a window of 30, 40, 50, 100, or more.

[0035] In the case of numbers used for a specified amino acid sequence or polynucleotide sequence, "corresponding to," "referring to," or "against" refers to the number of the specified reference sequence residue when the specified amino acid sequence or polynucleotide sequence is compared to the reference sequence. In other words, the residue numbers or residue positions of a given sequence are assigned based on the reference sequence, not the actual numerical positions of the residues in the given amino acid or polynucleotide sequence. For example, a given amino acid sequence, such as the amino acid sequence of an engineered hydantoinase, may be aligned with a reference sequence by introducing gaps to optimize the residue matches between the two sequences. In these cases, although gaps exist, the number of residues of a given amino acid or polynucleotide sequence is formulated relative to the reference sequence to which it is aligned.

[0036] An "amino acid difference" or "residue difference" refers to an amino acid residue difference at a position in a polypeptide sequence relative to an amino acid residue at the corresponding position in a reference sequence. The position of the amino acid difference is generally referred to herein as "Xn," where n refers to the corresponding position in the reference sequence that is the basis of the residue difference. For example, "residue difference at position X64 compared to SEQ ID NO:2" refers to an amino acid residue difference at a polypeptide position corresponding to position 64 of SEQ ID NO:2. Thus, if a reference polypeptide of SEQ ID NO:2 has a leucine at position 64, then "residue difference at position X64 compared to SEQ ID NO:2" refers to an amino acid substitution of any residue other than leucine at a polypeptide position corresponding to position 64 of SEQ ID NO:2. In most examples herein, a particular amino acid residue difference at a position is designated as "XnY," where "Xn" refers to the corresponding position as above, and "Y" is the one-letter code for the amino acid that appears in the engineered polypeptide (i.e., the residue that differs from that of the reference polypeptide). In some examples (e.g., Table 1), the disclosure also provides specific amino acid differences, represented by the conventional designation "AnB," where A is the one-letter designation for the residue in the reference sequence, "n" is the number of the residue position in the reference sequence, and B is the one-letter designation for the residue substitution in the sequence of the engineered polypeptide. In some examples, the polypeptides of the disclosure may include one or more amino acid residue differences relative to the reference sequence, represented by a list of the specific positions where there is a residue difference compared to the reference sequence.

[0037] "Deletion" refers to the modification of a polypeptide by removing one or more amino acids from a reference polypeptide. Deletions may include removal of one or more, two or more amino acids, five or more, ten or more, fifteen or more, or twenty or more amino acids, up to 10% of the total number of amino acids that make up the reference enzyme, or up to 20% of the total number of amino acids that make up the reference enzyme, while retaining the enzymatic activity of the engineered hydantoinase and / or retaining the improved properties of the engineered hydantoinase. Deletions may include internal and / or terminal portions of the polypeptide. In various embodiments, deletions may include contiguous segments or may be discontinuous.

[0038] "Insertion" refers to the modification of a polypeptide by the addition of one or more amino acids from a reference polypeptide. In some embodiments, the improved engineered hydantoinase comprises one or more amino acid insertions into a naturally occurring hydantoinase polypeptide and one or more amino acid insertions into other improved hydantoinase polypeptides. The insertions can be made internally in the polypeptide or at the carboxy- or amino-terminus. As used herein, insertions include fusion proteins known in the art. The insertions can be contiguous segments of amino acids or can be separated by one or more amino acids in a naturally occurring polypeptide.

[0039] As used herein, a "fragment" refers to a polypeptide having an amino-terminal and / or carboxy-terminal deletion, but where the amino acid sequence retained is the same as at corresponding positions within the sequence. Fragments may be at least 10 amino acids long, at least 20 amino acids long, at least 50 amino acids long or more, and up to 70%, 80%, 90%, 95%, 98%, and 99% of the full-length hydantoinase polypeptide.

[0040] An "isolated polypeptide" refers to a polypeptide that is substantially isolated from other materials that are naturally associated with it, such as proteins, lipids, and polynucleotides. The term includes a polypeptide that has been removed or purified from its naturally occurring environment or expression system (e.g., a host cell or in vitro synthesis). The improved hydantoinase polypeptide may be present intracellularly, in a cell culture medium, or prepared in various forms, such as a lysate or isolated preparation. Thus, in some embodiments, the improved hydantoinase polypeptide may be an isolated polypeptide.

[0041] A "chiral center" refers to a carbon atom that connects four different groups.

[0042] "Stereoselectivity" refers to the preferential formation of one stereoisomer over another or isomers in a chemical or enzymatic reaction. Stereoselectivity may be partial, with one stereoisomer being formed in preference to the others, or complete, with only one stereoisomer being formed. Stereoselectivity is also called enantioselectivity when the stereoisomers are enantiomers, and the excess of one enantiomer in a mixture of two enantiomers (usually reported as a percentage) is usually reported as enantiomeric excess (ee) where appropriate. Stereoselectivity is referred to as diastereoselectivity when the stereoisomers are diastereoisomers, i.e., the excess (usually reported as a percentage) of one diastereomer in a mixture of two diastereomers, usually reported as "diastereoisomeric excess" (abbreviated as de) as appropriate. This ratio (typically a percentage) is arbitrarily reported in the art as the enantiomeric excess (ee), which is usually calculated from {major enantiomer concentration - minor enantiomer concentration} / {major enantiomer concentration + minor enantiomer concentration}.

[0043] "Stereoisomers," "stereoisomeric forms," ​​and similar expressions are used interchangeably herein and refer to all isomers whose molecules differ only in the orientation of their atoms in space. This includes enantiomers and isomers of compounds with multiple chiral centers that are not mirror images of one another (i.e., "diastereomers").

[0044] "Improved enzymatic properties" refers to a hydantoinase polypeptide that exhibits any improved enzymatic properties compared to a reference hydantoinase, such as a wild-type hydantoinase or another improved engineered hydantoinase. Enzymatic properties for which improvement is desirable include, but are not limited to, enzymatic activity (which can be expressed as a percentage of substrate conversion), thermal stability, solution stability, pH activity profile, resistance to inhibitors (e.g., sediment or product inhibition), and stereoselectivity.

[0045] "Conversion" refers to the enzymatic conversion of a substrate to a corresponding product. "Conversion percentage" or "conversion rate" refers to the percentage of a substrate in a reaction system that is converted to a product under specified reaction conditions and within a specified reaction time. Thus, the "enzyme activity" or "activity" of a hydantoinase polypeptide can be expressed as the "conversion percentage" of a substrate to a product. The conversion rate is generally calculated as {molar concentration of product} / {molar concentration of substrate+molar concentration of product} by taking a sample and measuring the product concentration and substrate concentration in the reaction system.

[0046] "Thermostable" refers to a hydantoinase polypeptide that retains activity similar to the wild-type enzyme after exposure to elevated temperatures (eg, 72°C or higher) for a period of time (eg, 2.5 hours or more).

[0047] "Solvent stability" or "solvent resistance" refers to the ability of a hydantoinase polypeptide to maintain activity similar to that of a wild-type enzyme after exposure to various concentrations (e.g., 5-99%) of solvents (e.g., 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).

[0048] "Suitable reaction conditions" refers to conditions in a biocatalytic reaction solution (e.g., ranges of enzyme loading, substrate loading, temperature, pH, buffers, co-solvents, etc.) under which a hydantoinase polypeptide according to the present disclosure can convert a substrate into a desired product compound. Exemplary "suitable reaction conditions" are provided in the present disclosure and validated by the Examples.

[0049] "Hydrocarbyl" refers to a linear or branched chain hydrocarbyl group. The symbol "C" followed by a subscripted number designates the number of carbon atoms that may be included in a particular group. For example, "C1-C8" refers to a linear or branched chain hydrocarbyl group having 1 to 8 carbon atoms. The hydrocarbyl group may be optionally substituted by one or more substituents. "Aryl" refers to a monovalent aromatic hydrocarbyl having from 6 to about 20 carbon atoms. "Heteroaryl" and "heteroaromatic" refer to an aryl group in which one or more carbon atoms of the parent aromatic ring system are replaced by a heteroatom (O, N, or S). "Substituted," when used to modify a designated group or radical, means that one or more hydrogen atoms of the designated group or radical have each been replaced independently of one another. As used herein, a "compound" refers to any compound encompassed by the structural formula and / or chemical name shown for the compounds disclosed herein. A compound may be represented by its chemical structure and / or chemical name. In the event that the chemical structure conflicts with the chemical name, the chemical structure determines the compound. Unless otherwise stated or indicated, the chemical structures depicted herein encompass all possible isomeric forms of the depicted compounds.

[0050] Replaced by the same or different substituents. "Substituted hydrocarbyl, aryl, or heteroaryl" refers to a hydrocarbyl, aryl, or heteroaryl group in which one or more hydrogen atoms have been replaced by another substituent. "Optional" or "optionally" means that the described event or circumstance may or may not occur. For example, "optionally substituted aryl" refers to an aryl group that may be substituted or unsubstituted, and this description includes both substituted and unsubstituted aryl groups.

[0051] 2.2 Engineered Polypeptides The engineered polypeptide disclosed in the present invention is derived from a wild-type hydantoinase that has been modified through an innovative directed evolution process and undergoes mutations such as substitution, insertion or deletion of a specific number of amino acid residues. For a description of directed evolution technology, see "Directed Evolution: Bringing New Chemistry to Life" by 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. As a result of detection by the present inventors, the wild-type hydantoinase corresponding to SEQ ID NO:2 still has low activity against A1, is highly affected by pH, has particularly low resistance to high concentration product A2, and is poorly thermostable. These defects make SEQ ID NO:2 useless for industrial applications and therefore need to be engineered by enzyme directed evolution techniques.

[0052] The protein corresponding to SEQ ID NO:2 does not have a disclosed 3D structure, and the inventors use Yasara software to construct its 3D structural model, and then, in combination with bioinformatics technology, design site-directed saturation mutation libraries or multi-point combinatorial mutation libraries targeting multiple residues, and then screen them at various stages of development using the screening reaction conditions shown in Table 1.1, Table 2.1, 2.2, and Table 3.1-3.4. The method for constructing the mutation library can use site-directed mutation PCR (shown in Example 2) or multi-site mutation PCR, which are common in the art (see "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.).

[0053] In order to develop an enzyme catalyst with excellent properties for the reaction shown in Figure 5, the present invention carried out stepwise directed evolution of SEQ ID NO:2, and designed different high-throughput screening reaction conditions according to different properties of the enzyme to be improved. The first step is mainly to improve the activity of the enzyme. The designed high-throughput screening reaction conditions are shown in Table 1.1 or Example 8. Table 1 lists some exemplary engineering polypeptides obtained in the first step and their screening reaction results.

[0054] JPEG2025501363000004.jpg141170

[0055] JPEG2025501363000005.jpg98170

[0056] In practical industrial applications, the simpler the reaction system, the better, generally no co-solvent such as DMSO is used, and the substrate loading should be as high as possible. In order to detect the catalytic effect of the engineering polypeptides shown in Table 1 under conditions relevant to industrial applications and compare it with the wild-type SEQ ID NO:2, the present invention used the following reaction conditions to detect the exemplary engineering polypeptides obtained in the first stage: the loading amount of substrate A1 is 10g / L, the loading amount of wet cells expressing the engineering polypeptide is 50g / L, 0.1M PBS pH 7.0, 30°C. The reaction process was as described in Example 12. The results are shown in Table 1.2.

[0057] JPEG2025501363000006.jpg42170

[0058] In the second stage, not only the activity of the enzyme is improved, but also the pH stability is improved. The designed high-throughput screening reaction conditions are shown in Tables 2.1 and 2.2. Table 2 lists some exemplary engineered polypeptides obtained in the second stage and their screening reaction results.

[0059] JPEG2025501363000007.jpg241170JPEG2025501363000008.jpg203170

[0060] JPEG2025501363000009.jpg81170

[0061] JPEG2025501363000010.jpg90170

[0062] The exemplary engineering polypeptide obtained in the second step was detected using the following reaction conditions: the loading amount of substrate A1 was 10g / L, the loading amount of wet bacterial cells expressing the engineering polypeptide was 6g / L, 0.1M PBS pH 7.0, 30°C. The reaction process was as described in Example 13. The results are shown in Table 2.3.

[0063] JPEG2025501363000011.jpg70170

[0064] In the third stage, based on the evolution of enzyme activity and pH stability, resistance to high product concentrations and improved thermostability are also added. 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 polypeptides obtained in the third stage and their screening reaction results.

[0065] JPEG2025501363000012.jpg144170JPEG2025501363000013.jpg249170JPEG2025501363000014.jpg250170JPEG2025501363000015.jpg84170

[0066] JPEG2025501363000016.jpg89170

[0067] JPEG2025501363000017.jpg72170

[0068] JPEG2025501363000018.jpg80170

[0069] JPEG2025501363000019.jpg80170

[0070] JPEG2025501363000020.jpg251170JPEG2025501363000021.jpg32170

[0071] The exemplary engineering polypeptide obtained in the third step was detected using the following reaction conditions: the loading amount of substrate A1 was 10g / L, the loading amount of wet bacterial cells expressing the engineering polypeptide was 1g / L, 0.1M PBS pH 7.0, 40°C. The reaction process was as described in Example 14. The results are shown in Table 3.6.

[0072] JPEG2025501363000022.jpg72170

[0073] Based on the properties of the exemplary polypeptides listed in Tables 1, 2, and 3, improved enzymatic activity (i.e., converting compound A1 to compound A2) is associated with residue differences at residue positions 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, etc. In some embodiments, SEQ ID NO: The amino acid residue differences compared to NO:2 are 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, Q215 A, 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.

[0074] Based on the properties of the exemplary polypeptides listed in Tables 2 and 3.5, improved pH stability of the enzyme is associated with residue differences at residue positions 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, etc. In some embodiments, SEQ ID NO: The amino acid residue differences compared to NO:2 are 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, G201 H, Q215A, Q215P, S254Q, S254L, S254N, S254G, S254F, K255F, K255Y, K255H, K255N, Q257W, V263T, L264C, A265P, G266Q, H267Y, M288C, F292L, R329A, R329L, R329Y, N337P, A340P, F462R, K467D, P474W, A476P.

[0075] Based on the properties of the exemplary polypeptides listed in Table 3.5, improved product tolerance and / or thermostability of the enzyme is associated with residue differences at residue positions X39, X51, X64, X66, X71, X97, X113, X159, X189, X199, X215, X255, X257, X337, X340, etc. In some embodiments, the amino acid residue differences compared to SEQ ID NO:2 are selected from A39P, L51I, L64T, F66Y, A71T, N97L, N97Q, A113T, I159L, I159Y, I159F, L189V, L189I, L189M, A199V, Q215A, Q215P, K255H, K255N, Q257W, N337P, A340P.

[0076] As will be apparent to one of skill in the art, the aforementioned residue positions, and the specific amino acid residues at each residue position, may be used alone or in various combinations to synthesize hydantoinase polypeptides having desired improved properties, including enzymatic activity, stereoselectivity, stability, and the like.

[0077] In light of the guidance provided herein, it can further be envisioned that any of the exemplary engineered polypeptides having even sequence identifiers of SEQ ID NOs:4-286 can be used to synthesize starting amino acid sequences of other engineered polypeptides, for example, by adding various amino acid differences from the residue positions set out in Table 1, Table 2, Table 3. Further refinements are possible by including amino acid differences at positions that were unchanged in the first three stages of evolution.

[0078] Thus, in some embodiments, an engineered polypeptide capable of converting compound A1 to compound A2 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a reference sequence having an even-numbered sequence identifier selected from SEQ ID NOs: 4-286, and is selected from SEQ ID and having one or more residue differences compared to NO:2 at residue positions selected from 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.

[0079] In some embodiments, the engineered polypeptide capable of converting compound A1 to compound A2 under suitable reaction conditions has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a reference sequence having an even-numbered sequence identifier selected from SEQ ID NOs: 4 to 286, inclusive, and has SEQ ID Compared with NO:2, 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, The amino acid sequence comprises one or more residue differences selected from 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.

[0080] In addition to the residue positions specified above, any of the engineered polypeptides disclosed herein include residue differences relative to the reference polypeptide sequence of SEQ ID NO:2 at other residue positions, i.e., residue positions other than 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 may provide additional variants to the amino acid sequence without altering the ability of the polypeptide to convert compound A1 to compound A2, particularly with respect to improved enzymatic activity, improved pH stability, improved enzyme product tolerance, and improved enzymatic activity. Thus, in some embodiments, in addition to the amino acid residue differences in any one of the engineered polypeptides selected from the polypeptides having even sequence identifiers SEQ ID NO:4-286, the sequence may 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.

[0081] 2.3 Polynucleotides, control sequences, expression vectors, and host cells useful for preparing engineered polypeptides In another aspect, the present disclosure provides a polynucleotide encoding an engineered polypeptide having hydantoinase activity as described herein. The polynucleotide can be operably linked to one or more heterologous regulatory sequences that control gene expression to generate a recombinant polynucleotide capable of expressing the polypeptide. An expression construct comprising a heterologous polynucleotide encoding an engineered hydantoinase can be introduced into a suitable host cell to express the corresponding engineered polypeptide.

[0082] As will be apparent to one of skill in the art, the availability of protein sequences and knowledge of the codons corresponding to various amino acids provides a description of all polynucleotides that can code for a target protein sequence. The degeneracy of the genetic code, in which the same amino acid is coded for by alternative or synonymous codons, allows for the generation of a very large number of nucleic acids, all of which code for the improved hydantoinase polypeptides disclosed herein. Thus, after determining a particular amino acid sequence, one of skill in the art can generate any number of different nucleic acids by modifying the sequence of only one or more codons, without altering the amino acid sequence of the protein. In this regard, the present disclosure specifically contemplates all possible modifications of polynucleotides that may be prepared by selecting combinations based on possible codon choices, and for any polypeptide herein, includes the amino acid sequences of exemplary engineered polypeptides provided in Table 1, Table 2, Table 3, and any polypeptide disclosed as a sequence having an even-numbered sequence identifier of SEQ ID NO:4-286 in the Sequence Listing, which is incorporated herein by reference, all of which modifications are considered to be specifically disclosed.

[0083] In various embodiments, codons are preferably selected to be compatible with the host cell in which the protein is produced, for example, bacterially preferred codons are used to express genes in bacteria, yeast preferred codons are used to express genes in yeast, and mammalian preferred codons are used to express genes in mammalian cells.

[0084] In some embodiments, the polynucleotide encodes a hydantoinase polypeptide comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence having an even-numbered sequence identifier selected from SEQ ID NOs:4-286, wherein the polypeptide has one or more of the improved properties described herein, such as hydantoinase activity, improved activity compared to the polypeptide of SEQ ID NO:2, or the ability to convert compound A1 to product compound A2.

[0085] In some embodiments, the polynucleotide encodes an engineered polypeptide comprising an amino acid sequence having the above percentage identity compared to SEQ ID NO:2 and having one or more amino acid residue differences. In some embodiments, the disclosure provides engineered polypeptides with hydantoinase activity comprising at least 90% sequence identity to a reference sequence of SEQ ID NO:2 and having a combination of residue differences at positions selected from 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.

[0086] In some embodiments, the polynucleotide encoding the engineered polypeptide is selected from the group consisting of 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, 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, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285.

[0087] In some embodiments, the polynucleotide encodes a polypeptide as 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%, 99% or more sequence identity at the nucleotide level to a reference polynucleotide encoding an engineered hydantoinase. In some embodiments, the reference polynucleotide sequence is represented by 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, 4 7, 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 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, 285.

[0088] An isolated polynucleotide encoding an engineered polypeptide can be manipulated in a variety of ways to provide for expression of the polypeptide, including further sequence modification by codon optimization to improve expression, insertion into appropriate expression elements with or without additional control sequences, and transformation into a host cell suitable for expression and production of the polypeptide.

[0089] Depending on the expression vector, manipulation of the isolated polynucleotide may be desirable or necessary prior to its insertion into the vector. Techniques for modifying polynucleotides 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, Ausubel. F. (ed.), Greene Pub. Associates, 1998, updated in 2010.

[0090] In another aspect, the present disclosure also relates to a recombinant expression vector comprising a polynucleotide encoding an engineered polypeptide or variant thereof, and one or more expression control regions, such as a promoter terminator and an origin of replication, depending on the type of host into which it is to be introduced. Alternatively, the nucleic acid sequence of the present disclosure can be expressed by inserting the nucleic acid sequence or a nucleic acid construct comprising the sequence into an appropriate expression vector. In producing an expression vector, the coding sequence is placed in the vector such that the coding sequence is operably linked to the appropriate control sequence for expression.

[0091] The recombinant expression vector may be any vector (e.g., a plasmid or a virus) that can be conveniently used in recombinant DNA steps and that can result in the expression of a polynucleotide sequence. The choice of vector usually depends on the compatibility of the vector with the host cell into which it is introduced. The vector may be a linear or closed circular plasmid. The expression vector may be an autonomously replicating vector, i.e. a vector that exists as an extrachromosomal entity whose replication is independent of chromosomal replication, such as a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means to ensure self-replication. Alternatively, the vector may be one that, when introduced into a host cell, is integrated into the genome and replicates together with the chromosome into which it is integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the host cell genome may be used.

[0092] Many expression vectors useful for embodiments of the present disclosure are commercially available. An exemplary expression vector can be prepared by operably linking a polynucleotide encoding an improved hydantoinase polypeptide to the plasmid pACYC-Duet-1 (Novagen).

[0093] In another aspect, the present disclosure provides a host cell comprising a polynucleotide encoding an improved hydantoinase polypeptide of the present disclosure, said polynucleotide being operably linked to one or more control sequences for expression of the hydantoinase in the host cell. Host cells for expressing the polypeptides encoded by the expression vectors of the present disclosure are well known in the art and include, but are not limited to, bacterial cells such as E. coli, Arthrobacter sp. KNK168, Streptomyces sp., 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 E. coli BL21(DE3). The host cells may be wild-type or engineered cells that have been subjected to genome editing, such as knocking out the wild-type hydantoinase gene contained in the genome of the host cells. Suitable culture media and growth conditions for the host cells are well known in the art.

[0094] Polynucleotides for expressing hydantoinase can be introduced into cells by various methods known in the art, including electroporation, bioparticle bombardment, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion. Various methods for introducing polynucleotides into cells will be apparent to those skilled in the art.

[0095] 2.4 Methods for Producing Engineered Polypeptides If the sequence of an engineered polypeptide is known, a polynucleotide encoding the polypeptide can be prepared by standard solid-phase methods according to known synthesis methods. In some embodiments, fragments of up to about 100 bases can be synthesized separately and then ligated (e.g., by enzymatic or chemical ligation or polymerase-mediated methods) to form any desired contiguous sequence. For example, the polynucleotides and oligonucleotides of the present disclosure are typically implemented in the classical phosphoramidite method, e.g., as described in Beaucage et al., 1981, TetLett 22:1859-69, or the method described in Matthes et al., 1984, EMBOJ.3:801-05, e.g., automated synthesis methods. According to the phosphoramidite method, oligonucleotides are synthesized, e.g., in an automated DNA synthesizer, purified, annealed, ligated, and cloned into an appropriate vector. Essentially any nucleic acid can also be obtained from any of a variety of commercial sources.

[0096] In some embodiments, the disclosure also provides a method for preparing or making an engineered polypeptide, the method comprising culturing a host cell capable of expressing a polynucleotide encoding the engineered polypeptide under culture conditions suitable for expression of the polypeptide. In some embodiments, the method for preparing a polypeptide further comprises isolating the polypeptide. The engineered polypeptide may be expressed in a suitable cell and isolated (or recovered) from the host cell and / or culture medium using any one or more of the well-known techniques for protein purification. The techniques for protein purification include, among others, lysozyme treatment, sonication, filtration, salting out, heat treatment, ultracentrifugation, chromatography and chromatography.

[0097] 2.5 Method of using engineered hydantoinase and preparation of compounds using same The present disclosure also provides a method for preparing a compound of structural formula (I) using the improved engineered hydantoinase polypeptides described herein, comprising: said compound of formula (I) has the stereochemical configuration as shown at the chiral centre marked with an *, said compound of formula (I) being in excess of the corresponding enantiomer; The method provides a preparation method comprising the step of contacting a hydantoinase derivative having structural formula (II) as a substrate with an engineered hydantoinase polypeptide. [ka] (where: n=0 or 1, R1 and R2 are each independently selected from H, optionally substituted or unsubstituted aryl or heteroaryl, optionally substituted or unsubstituted linear or branched C1-C4 alkyl, optionally substituted or unsubstituted linear or branched C1-C4 alkenyl, optionally substituted or unsubstituted cycloalkyl, -OR', -NH2, -NR'R', -SR', -COR', or -C(O)R'; Each R' is independently selected from -H or (C1-C4) hydrocarbyl. [ka] (In the structural formula (II), the definitions of n, R1, and R2 are the same as those in the structural formula (I).)

[0098] In another aspect, the present disclosure also provides a method for preparing a compound of structural formula (III) using the improved engineered hydantoinase polypeptides described herein, comprising: said compound of formula (III) having the stereochemical configuration as shown at the chiral center marked with an *, said compound of formula (III) being in excess of the corresponding enantiomer; The method provides a preparation method comprising the step of contacting an imide derivative having structural formula (IV) as a substrate with an engineered hydantoinase polypeptide. [ka] (n=0 or 1, R1 and R2 are each independently selected from H, optionally substituted or unsubstituted linear or branched C1-C4 alkyl, or optionally substituted or unsubstituted C6H6; When n=0, R1 and R2 may together form a ring structure group, which may be selected from monocyclic or polycyclic, optionally substituted or unsubstituted aryl, or monocyclic or polycyclic, optionally substituted or unsubstituted heteroaryl. [ka] (In the structural formula (IV), the definitions of n, R1, and R2 are the same as those in the structural formula (III).)

[0099] In another aspect, the improved engineered polypeptides described herein convert DL-p-hydroxyphenylhydantoin to N-carbamoyl-Dp-hydroxyphenylglycine, which is further converted to Dp-hydroxyphenylglycine by the action of hydrochloric acid. [ka] [ka] [ka]

[0100] In another aspect, the improved engineered polypeptides described herein can convert A1 to A2. In some embodiments, the engineered polypeptides are useful in methods for preparing enantiomeric excess of formula A2 compounds. [ka]

[0101] In these embodiments, the method comprises contacting a compound of formula A1 with an engineered polypeptide disclosed herein under suitable reaction conditions. [ka]

[0102] In some embodiments of the above method, the compound of formula A2 is produced in at least 97%, 98%, 99% or more enantiomeric excess.

[0103] Specific embodiments of engineered hydantoinase polypeptides for use in the methods are provided further in the details of the invention. Improved engineered polypeptides that can be used in the above methods include those of 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, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 120, 122, 124, 126, 128, 129, 130, 131, 132, 133, 134, 135, 136, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 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, 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,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, 5 4, 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, 1 80, 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, 25 Also included are amino acid sequences that have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any of the reference amino acid sequences selected from the following sequences: 6, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286.

[0104] As described herein and verified in the Examples, the present disclosure contemplates suitable reaction conditions that can be used in the methods herein, including, but not limited to, ranges of pH, temperature, buffers, solvent systems, substrate loading, polypeptide loading, and reaction time. Additional suitable reaction conditions for carrying out the methods of biocatalytically converting substrate compounds to product compounds using engineered hydantoinase polypeptides described herein can be readily optimized by routine experimentation, including, but not limited to, contacting the engineered polypeptide with the substrate compound under experimental reaction conditions of concentration, pH, temperature, solvent conditions, and detecting the product compound using, for example, the methods described in the Examples provided herein.

[0105] As described above, the engineered polypeptides having hydantoinase activity used in the methods of the present disclosure are generally selected from the group consisting of 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, 1 80, 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, 2 56, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286.

[0106] The substrate compound in the reaction mixture may vary, taking into consideration, for example, the amount of the desired product compound, the effect of substrate concentration on enzyme activity, the stability of the enzyme under reaction conditions, and the percentage of substrate to product conversion. In some embodiments of the method, suitable reaction conditions include a loading of substrate A1 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 more. Although the substrate loading values ​​provided herein are based on the molecular weight of compound A1, it is also contemplated that equimolar amounts of various hydrates and salts of compound A1 may be used in the method.

[0107] In reaction embodiments, reaction conditions may include a suitable pH. As noted above, the desired pH or desired pH range can be maintained by using an acid or base, a suitable buffer, or a combination of a buffer and the addition of 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.

[0108] In embodiments of the methods herein, suitable temperatures can be used for the reaction conditions, for example, taking into account increased reaction rates at higher temperatures, enzyme activity over sufficiently long reaction times. Thus, in some embodiments, suitable reaction conditions include temperatures of about 10°C to about 60°C, about 25°C to about 50°C, about 25°C to about 40°C, or about 25°C to about 30°C. In some embodiments, suitable reaction temperatures include temperatures of 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 enzyme reaction can be maintained at a specific temperature throughout the reaction. In some embodiments, the temperature during the enzyme reaction can be adjusted to the temperature profile during the reaction.

[0109] The method using the engineered hydantoinase is usually carried out in water or a solvent. Suitable solvents include aqueous buffers, organic solvents, and / or co-solvent systems, and the co-solvent system usually includes 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, the method using the engineered polypeptide is usually carried out in an aqueous co-solvent system including an organic solvent (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.), an ionic liquid (e.g., 1-ethyl 4-methylimidazole tetrafluoroborate, 1-butyl-3-methylimidazole tetrafluoroborate, 1-butyl-3-methylimidazole hexafluorophosphate, etc.). The organic solvent component in the aqueous co-solvent system can be miscible with the aqueous component to provide a single liquid phase, or partially miscible or immiscible with the aqueous component to provide two liquid phases. Carbon dioxide generated during the hydrolysis reaction can cause foam formation, so an antifoaming agent can be added as appropriate. Exemplary aqueous co-solvent systems include water and one or more organic solvents. Typically, the organic solvent component of the aqueous co-solvent system is selected so as not to completely inactivate the hydantoinase. A suitable co-solvent system can be readily identified by measuring the enzymatic activity of a particular engineered hydantoinase using an enzymatic activity assay as described herein with a defined substrate of interest in a candidate solvent system.

[0110] Suitable reaction conditions can include a combination of reaction parameters that provide for the biocatalytic conversion of the substrate compound to the corresponding product compound. Thus, in some embodiments of the method, the combination of reaction parameters includes: (a) a substrate A1 loading of about 1 g / L to 400 g / L, (b) an engineering polypeptide concentration of about 0.1 g / L to 50 g / L, (c) a pH of about 6.0 to 8.5, and (d) a temperature of about 10° C. to 60° C.

[0111] In some embodiments, the method comprises contacting 10 g / L or more of A1 substrate with an engineered polypeptide described herein at reaction conditions of about 30° C. to about 50° C. and pH 6.0 to 8.0, wherein 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 more. In some embodiments, the hydantoinase polypeptide capable of carrying out the above reaction comprises an amino acid sequence corresponding to the even-numbered sequence identifiers of SEQ ID NOs: 4 to 286.

[0112] Exemplary reaction conditions include those provided in Examples 12-22.

[0113] In carrying out the enzymatic reactions described herein, the engineering polypeptide may be added to the reaction mixture in the form of partially purified or purified enzyme, heat-treated enzyme solution, whole cells transformed with a gene encoding the enzyme, and / or cell extracts of such cells, and / or lysates. Whole cells transformed with a gene encoding the engineering polypeptide, or cell extracts thereof, lysates thereof, and isolated enzymes may be used in various forms, such as solid (e.g., freeze-dried, spray-dried, etc.) or semi-solid (e.g., crude pastes such as wet cells). Cell extracts or cell lysates may be partially purified by precipitation (e.g., treatments such as ammonium sulfate, polyethyleneimine, heat treatment, etc.), followed by a desalting procedure (e.g., ultrafiltration, dialysis, etc.) before lyophilization. Any enzyme product may be stabilized by cross-linking or immobilization to a solid phase material (e.g., a resin) using known cross-linking agents, such as glutaraldehyde.

[0114] In some embodiments of the enzymatic reactions described herein, the reactions are carried out under suitable reaction conditions as described herein, and the engineering polypeptide is immobilized on a solid support. Solid supports that can be used to immobilize the engineering polypeptide for carrying out the enzymatic reaction include, but are not limited to, microspheres or resins that include epoxy-functionalized polymethacrylate, aminoepoxy-functionalized polymethacrylate, octadecyl-functionalized styrene / DVB copolymer, or octadecyl-functionalized polymethacrylate. Exemplary solid supports include, but are not limited to, chitosan beads, EupergitC, and SEPABEAD (Mitsubishi), including different types of SEPABEAD:EC-EP, EC-HFA / S, EXA252, EXE119, and EXE120.

[0115] In some embodiments, where the engineered polypeptide can be expressed as a secreted polypeptide, medium containing the secreted polypeptide can be used in the methods herein.

[0116] In some embodiments, solid reactants (e.g., enzymes, salts, etc.) may be applied to the reaction in a variety of forms, such as powders (e.g., lyophilized, spray dried, etc.), solutions, emulsions, suspensions, etc. Reactants can be readily lyophilized or spray dried using methods and equipment well known to those of skill in the art. For example, a small volume of protein solution is frozen at -80°C and added to a pre-cooled lyophilization chamber, followed by vacuum.

[0117] In some embodiments, there are several options for the order or manner in which the reactants are added: The reactants can be added simultaneously to the solvent (e.g., a single-phase solvent, a two-phase aqueous co-solvent system, etc.), or some reactants can be added first and other reactants added in a flow stream or in interval batches. Various features and embodiments of the present disclosure are illustrated in the following representative examples, which are intended to be illustrative and not limiting. [Brief description of the drawings]

[0118] [Figure 1] FIG. 1 shows the unique research route of pregabalin. [Diagram 2] A lipase-splitting route for the synthesis of pregabalin and its intermediates. [Diagram 3] Asymmetric chemical synthesis of pregabalin and its intermediates. [Figure 4] Asymmetric hydrolysis of 3-isobutylglutarimide by hydantoinase to prepare pregabalin intermediate. [Diagram 5] The hydrolysis of 3-isobutylglutarimide catalyzed by hydantoinase to produce (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid. [Figure 6] Protein electropherogram analysis. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0119] The present invention will be further described below with reference to examples, but the present invention is not limited thereto. In the following examples, experimental methods for which no specific conditions are specified are usually performed under normal conditions or conditions proposed by the manufacturer.

[0120] Example 1: Gene cloning and expression vector construction The gene sequence of wild-type hydantoinase derived from Pseudomonas fluorescens was found from NCBI (GenBank: KF268426.1), synthesized by common techniques in this field, and cloned into expression vector pACYC-Duet-1 (Novagen). The recombinant expression plasmid was transformed into competent cells of E. coli BL21 (DE3). The transformation conditions were 42°C, 90 seconds of heat shock. The transformation solution was applied onto an LB plate containing chloramphenicol, and inverted cultured at 37°C overnight to obtain recombinant transformants.

[0121] Example 2: Construction of a hydantoinase mutation library All commercially available reagents used herein are preferably Quikchange kits (supplier: Agilent). The sequence of the mutation primers was designed according to the kit's instructions.

[0122] PCR system: 10×buffer 2.5μL, dNTP mix 1μL, primer Oligomix 2μL (5μM), plasmid template 2.5μL (50ng / μl), high fidelity enzyme 1μL, ddH2O 16μL

[0123] PCR amplification steps: (1) 95°C, 1 min pre-denaturation, (2) 95°C, 1 min denaturation, (3) 55°C, 1 min annealing, (4) 65°C, 6 min extension. Repeat steps (2) to (4) 29 times. (5) Continue extension at 65°C for 5 min and cool to 4°C. Add 2 μl DpnI (Kit) to the PCR product and enzymatically digest at 37°C for 2 h. The product was transformed into E. coli BL21 (DE3) electrocompetent cells, plated on LB plates containing chloramphenicol, and inverted at 37°C overnight to obtain library colonies.

[0124] Example 3: Expression of mutant enzyme library and preparation of enzyme solution for screening Colonies of the mutant enzyme library were picked from the agar plate, inoculated into a 96-well plate containing LB medium containing chloramphenicol, and cultured overnight in a shaker at 30°C. 600 When the OD of the medium reached 2-3, 20 μL was taken from the 96-well plate and inoculated into a 96-well deep well plate (400 μL of TB medium per well) containing TB medium containing chloramphenicol, and cultured in a shaker at 30°C. 600 When the affinity of the enzyme reached 0.6-0.8, IPTG was added as an inducer to a final concentration of 1 mM, and the mixture was placed on a shaker at 30°C to allow expression overnight (18-20 h). After expression was completed, the deep-well plate containing the bacterial solution was centrifuged, and the supernatant of the bacterial solution was removed to obtain wet bacterial cells. A cell lysis solution (1 g / L lysozyme, 0.5 g / L PMBS, dissolved in PBS buffer, pH 7) was added to the wet bacterial cells, and the mixture was shaken for 1 h to disrupt the cells, obtaining a lysate. The lysate was centrifuged, and the supernatant was transferred to a new deep-well plate to obtain an enzyme solution that can be used in screening reactions.

[0125] Example 4: Expression of Engineered Polypeptides A single microbial colony containing E. coli BL21(DE3) carrying the target engineered polypeptide expression plasmid was inoculated into a 250 mL Erlenmeyer flask containing 50 mL of LB medium (containing 30 μg / mL chloramphenicol) and grown overnight in a shaker at 30 °C on a rocking table. The OD of the culture was 600 When the OD of the medium reached 2, it was inoculated into 250 mL of TB medium in a 1000 mL Erlenmeyer flask at 5% (v / v) inoculum and cultured in a shaker at 30°C. 600 When the β-amino acid concentration reached 0.6, IPTG was added to a final concentration of 1 mM to induce the expression of hydantoinase. After culturing for 20 h, the culture was centrifuged (8000 rpm, 10 min), and the supernatant was discarded and the cells were collected to obtain wet cells. The wet cells can be used directly to prepare the enzyme solution or frozen and stored at -20°C until use. The wet cells were resuspended in PBS buffer, disrupted by ultrasonication in an ice bath, and centrifuged to recover the supernatant, thereby obtaining an enzyme solution containing the engineering polypeptide.

[0126] Example 5: Quantification of hydantoinase polypeptides in enzyme solution samples According to the method of Example 4, the enzyme solution of SEQ ID NO:2 was prepared and diluted 100 times (sample 1) and 200 times (sample 2), and electrophoretic analysis was performed together with different concentrations of BCA protein standard samples (Easy II Protein Quantitative Kit, brand: Transgen). Using computer software, grayscale analysis of protein bands on the electrophoretic chart was performed, and the calibration curve of BCA bands (samples 3 to 7 in Figure 6) grayscale values ​​and BCA concentration was obtained. The concentration of hydantoinase polypeptide in the enzyme solution sample was obtained by substituting the grayscale values ​​of the target bands of hydantoinase (shown by the dotted arrow in Figure 6) into the equation of the calibration curve. JPEG2025501363000032.jpg31170

[0127] Example 6: High-Throughput Conversion Analysis Method for Liquid Phase Orifice Plates: HPLC analysis method: the chromatography column is Gemini C18 250mm*4.6mm*5um, the mobile phase used is 70% 0.4% HCLO4:30% ACN, the flow rate is 1mL / min, the column oven temperature is 40℃, the detection wavelength is 210nm, the solvent is 50% ACN, the injection volume is 10μL, the retention time of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid is 5.030min, and the retention time of 3-isobutylglutarimide is 11.188min.

[0128] Example 7: Chiral Analysis Method Sample derivatization process: 1 mL of the reaction solution was taken, and potassium carbonate and 2-bromoacetophenone were weighed out in a ratio of product:potassium carbonate:2-bromoacetophenone (mass ratio) = 5:3:1. 1 mL of acetonitrile was mixed with 1 mL of the reaction solution, and the mixture was shaken at 1500 rpm for 15 min. 3 mL of ethyl acetate was added and the mixture was shaken at 1500 rpm for 15 min. The ethyl acetate layer was centrifuged and freeze-dried, dissolved in 50% ACN, and detected by HPLC.

[0129] HPLC analysis method: the chromatography column is CHIRALPAK AD-RH 4.6*150mm*5um, the mobile phase used is 50% water (adjusted to pH2.50 with phosphoric acid):50%ACN, the flow rate is 0.5ml / min, the column oven temperature is 30℃, the detection wavelength is 210nm, the injection volume is 10ul, the retention time of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid is 15.2min, and the retention time of (S)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid is 13.2min.

[0130] 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]}

[0131] Example 8: Screening reactions for catalytic activity in the first stage of directed evolution Referring to the method of Example 3, an enzyme solution at pH 7.0 was prepared and immediately used in the screening reaction.

[0132] In a 96-well plate, the enzyme solution and the substrate mother solution (substrate A1 dissolved in DMSO) were mixed, and the final concentrations of each component in the reaction system were 2 g / L substrate, 10% DMSO, 10 g / L enzyme, and 0.05 M PBS, and the well plate was placed on a shaker at 250 rpm and 30° C. and shaken for 22 hours. After the reaction was completed, 200 μL of pure acetonitrile was added per well to quench the reaction, and the plate was placed on a plate shaker and shaken for 30 min (800 rpm), then centrifuged (4000 rpm, 10 min). The supernatant after centrifugation was collected and subjected to HPLC analysis according to the method of Example 6 to calculate the conversion rate of A1 to A2, and the ee value of the product A2 was detected according to the method of Example 7.

[0133] Example 9: Screening reactions for pH stability in the second stage of directed evolution Referring to the method of Example 3, an enzyme solution of pH 6.3 was prepared and shaken at room temperature (20°C to 25°C) for 23 hours, after which PBS buffer was added to adjust the pH of the enzyme solution to 7.0, and a screening reaction was carried out. In a 96-well plate, the pretreated enzyme solution and the substrate mother solution (substrate A1 dissolved in DMSO) were mixed, and the final concentrations of each component in the reaction system were 2 g / L substrate, 10% DMSO, 3 g / L enzyme, and 0.05 M PBS. The well plate was placed on a shaker at 250 rpm and 30° C. and shaken for 22 hours. After the reaction was completed, 200 μL of pure acetonitrile was added per well to quench the reaction, and the plate was placed on a plate shaker and shaken for 30 min (800 rpm), then centrifuged (4000 rpm, 10 min). The supernatant after centrifugation was collected and subjected to HPLC analysis according to the method of Example 6 to calculate the conversion rate of A1 to A2, and the ee value of the product A2 was detected according to the method of Example 7.

[0134] Example 10: Screening reactions for product resistance in the third stage of directed evolution Referring to the method of Example 3, an enzyme solution at pH 7.0 was prepared and immediately thereafter a screening reaction was carried out. In a 96-well plate, the enzyme solution, the substrate mother solution (substrate A1 dissolved in DMSO), and the product mother solution (product A2 dissolved in PBS buffer) were mixed, and the final concentrations of each component in the reaction system were 2 g / L substrate, 50 g / L product, 10% DMSO, 0.3 g / L enzyme, and 0.05 M PBS, and the well plate was placed on a shaker at 250 rpm and 30 ° C. and shaken for 22 hours. After the reaction was completed, 200 μL of pure acetonitrile was added per well to quench the reaction, and the plate was placed on a plate shaker and shaken for 30 min (800 rpm), then centrifuged (4000 rpm, 10 min), and the supernatant after centrifugation was collected and HPLC analysis was performed according to the method of Example 6 to calculate the conversion rate from A1 to A2.

[0135] Example 11: Screening reactions for thermostability in the third stage of directed evolution An enzyme solution at pH 7.0 was prepared according to the method of Example 3, and after shaking at 50° C. for 23 hours, a screening reaction was carried out.

[0136] In a 96-well plate, the enzyme solution and the substrate mother solution (substrate A1 dissolved in DMSO) were mixed, and the final concentrations of each component in the reaction system were 2 g / L substrate A, 10% DMSO, 0.3 g / L enzyme, and 0.05 M PBS, and the well plate was placed on a shaker at 250 rpm and 30° C. and shaken for 22 hours. After the reaction was completed, 200 μL of pure acetonitrile was added per well to quench the reaction, and the plate was placed on a plate shaker and shaken for 30 min (800 rpm), then centrifuged (4000 rpm, 10 min), and the supernatant after centrifugation was collected and subjected to HPLC analysis according to the method of Example 6 to calculate the conversion rate from A1 to A2.

[0137] Example 12: Reaction method for measuring conversion rate by reacting 5 mL of shake flask bacteria from the first stage To a reaction flask with a total volume of 30 mL, 250 mg of wet cells expressing SEQ ID NO:8 were added, 50 mg of substrate A1 was added, and finally PBS buffer (0.1 M, pH 7.0) was added to make the total reaction volume 5.0 mL. The concentrations of each component in the reaction system were 50 g / L of wet cells and 10 g / L of substrate A1. A magnetic stirrer rotor was added to the reaction flask, and the reaction was started by placing it on a magnetic stirrer set at 400 rpm and 30°C. After reacting for 24 hours, 5 mL of acetonitrile was added to the flask and the reaction was quenched for 30 min. The inactivated solution was transferred to a 2 mL centrifuge tube and centrifuged (13000 rpm, 3 min). The supernatant after centrifugation was collected, 1) HPLC analysis was performed according to the method of Example 6 to calculate the conversion rate from A1 to A2, and 2) HPLC analysis was performed according to the method of Example 7 to calculate the ee value of A2.

[0138] Example 13: Reaction method for measuring conversion rate by reacting 5 mL of shake flask bacteria in the second stage To a reaction flask with a total volume of 30 mL, 30 mg of wet cells expressing SEQ ID NO:50 were added, 50 mg of substrate A1 was added, and finally PBS buffer (0.1 M, pH 7.0) was added to make the total reaction volume 5.0 mL. The concentrations of each component in the reaction system were 6 g / L of wet cells and 10 g / L of substrate A1. A magnetic stirrer rotor was added to the reaction flask, and the reaction was started by placing it on a magnetic stirrer set at 400 rpm and 30°C. After reacting for 24 hours, 5 mL of acetonitrile was added to the flask and the reaction was quenched for 30 min. The inactivated solution was transferred to a 2 mL centrifuge tube and centrifuged (13000 rpm, 3 min). The supernatant after centrifugation was collected, 1) HPLC analysis was performed according to the method of Example 6 to calculate the conversion rate from A1 to A2, and 2) HPLC analysis was performed according to the method of Example 7 to calculate the ee value of A2.

[0139] Example 14: Reaction method for measuring conversion rate by reacting 5 mL of shake flask bacteria from the third stage 5 mg of wet cells expressing SEQ ID NO:184 were added to a reaction flask with a total volume of 30 mL, 50 mg of substrate A1 was added, and finally PBS buffer (0.1 M, pH 7.0) was added to make the total volume of the reaction 5.0 mL. The concentrations of each component in the reaction system were 1 g / L of wet cells and 10 g / L of substrate A1. A magnetic stirrer rotor was added to the reaction flask, and the reaction was started by placing it on a magnetic stirrer set at 400 rpm and 40°C. After reacting for 24 hours, 5 mL of acetonitrile was added to the flask and the reaction was quenched for 30 min. The inactivated solution was transferred to a 2 mL centrifuge tube and centrifuged (13000 rpm, 3 min). The supernatant after centrifugation was collected, 1) HPLC analysis was performed according to the method of Example 6 to calculate the conversion rate from A1 to A2, and 2) HPLC analysis was performed according to the method of Example 7 to calculate the ee value of A2.

[0140] Example 15: Catalytic process of pregabalin intermediate synthesis by engineering hydantoinase polypeptide SEQ ID NO:10 In a 500mL reaction vessel, 100mL 0.05M PBS pH7.0 buffer was added, and 50mL of enzyme solution (SEQ ID NO:10) was further added. A water bath was started to control the temperature at 30°C, and the stirring speed was set to 200 rpm. Finally, 3g of substrate (3-isobutylglutarimide) was added to start the reaction. During the reaction, the pH was adjusted with ammonia water to control it to 7.0±0.2, and the reaction was terminated after 20h. Samples were taken and detected, and the conversion rate after 20h reaction was 71%.

[0141] After the reaction, the reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated to about 100 mL. Then, hydrochloric acid was added dropwise to adjust the pH of the concentrated solution to 3.0. After stirring for 30 min, the solution was filtered to obtain a wet crude product, which was then baked and weighed. Finally, about 2.2 g of crude product was obtained. ee%=99.8%.

[0142] Example 16: Catalytic process of pregabalin intermediate synthesis by engineering hydantoinase polypeptide SEQ ID NO:24 In a 500mL reaction vessel, 100mL 0.05M PBS pH7.0 buffer was added, and 50mL of enzyme solution (SEQ ID NO:24) was further added. A water bath was started to control the temperature at 30°C, and the stirring speed was set to 200 rpm. Finally, 3g of substrate (3-isobutylglutarimide) was added to start the reaction. During the reaction, the pH was adjusted with ammonia water to control it to 7.0±0.2, and the reaction was terminated after 20h. Samples were taken and detected, and the conversion rate after 20h reaction was 73%.

[0143] After the reaction, the reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated to about 100 mL. Hydrochloric acid was added dropwise to adjust the pH of the concentrated solution to 3.0, and the solution was stirred for 30 min. After filtration, the wet crude product was obtained, baked, and weighed. Finally, about 2.3 g of crude product was obtained. ee%=99.7%.

[0144] Example 17: Catalytic process of pregabalin intermediate synthesis by engineering hydantoinase polypeptide SEQ ID NO:52 In a 500mL reaction vessel, 140mL 0.05M PBS pH7.0 buffer was added, 10mL of enzyme solution (SEQ ID NO:52) was further added, a water bath was started to control the temperature at 35°C, the stirring speed was set to 200rpm, and finally, 10g of substrate (3-isobutylglutarimide) was added to start the reaction. During the reaction, the pH was adjusted with ammonia water to control it to 7.0±0.2, and the reaction was terminated after 20h. Samples were taken and detected, and the conversion rate after 20h reaction was 95%.

[0145] After the reaction, the reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated to about 100 mL. Then, hydrochloric acid was added dropwise to adjust the pH of the concentrated solution to 3.0. After stirring for 30 min, the solution was filtered to obtain a wet crude product, which was then baked and weighed. Finally, 10.1 g of crude product was obtained. ee%≧99.6%.

[0146] Example 18: Catalytic process of pregabalin intermediate synthesis by engineering hydantoinase polypeptide SEQ ID NO:162 In a 500mL reaction vessel, 140mL 0.05M PBS pH7.0 buffer was added, 10mL of enzyme solution (SEQ ID NO:162) was further added, a water bath was started to control the temperature at 35°C, the stirring speed was set to 200rpm, and finally, 10g of substrate (3-isobutylglutarimide) was added to start the reaction. During the reaction, the pH was adjusted with ammonia water to control it to 7.0±0.2, and the reaction was terminated after 20h. Samples were taken and detected, and the conversion rate after 20h reaction was 96%.

[0147] After the reaction, the reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated to about 100 mL. Then, hydrochloric acid was added dropwise to adjust the pH of the concentrated solution to 3.0. After stirring for 30 min, the solution was filtered to obtain a wet crude product, which was then baked and weighed. Finally, 10.4 g of crude product was obtained. ee%=99.5%.

[0148] Example 19: Catalytic process of pregabalin intermediate synthesis by engineering hydantoinase polypeptide SEQ ID NO:184 In a 500mL reaction vessel, 145mL 0.05M PBS pH7.0 buffer was added, and 5mL of enzyme solution (SEQ ID NO:184) was further added. A water bath was started to control the temperature at 45°C, the stirring speed was set to 200rpm, and finally 30g of substrate (3-isobutylglutarimide) was added to start the reaction. During the reaction, the pH was adjusted with ammonia water to control it to 7.0±0.2, and the reaction was terminated after 20h. Samples were taken and detected, and the conversion rate after 20h reaction was 98%.

[0149] After the reaction, the reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated to about 100 mL. Then, hydrochloric acid was added dropwise to adjust the pH of the concentrated solution to 3.0. After stirring for 30 min, the solution was filtered to obtain a wet crude product, which was then baked and weighed to obtain 32.1 g of crude product. ee%=99.7%.

[0150] Example 20: Catalytic process of pregabalin intermediate synthesis by engineering hydantoinase polypeptide SEQ ID NO:264 In a 500mL reaction vessel, 145mL 0.05M PBS pH7.0 buffer was added, 5mL of enzyme solution (SEQ ID NO:264) was further added, a water bath was started to control the temperature at 45°C, the stirring speed was set to 200rpm, and finally, 20g of substrate (3-isobutylglutarimide) was added to start the reaction. During the reaction, the pH was adjusted with ammonia water to control it to 7.0±0.2, and the reaction was terminated after 20h. Samples were taken and detected, and the conversion rate after 20h reaction was 72%.

[0151] After the reaction, the reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated to about 100 mL. Hydrochloric acid was added dropwise to adjust the pH of the concentrated solution to 3.0, and the solution was stirred for 30 min. After filtration, the wet crude product was obtained, baked, and weighed. Finally, 15.8 g of crude product was obtained. ee%=99.8%.

[0152] Example 21: Catalytic process of pregabalin intermediate synthesis by engineering hydantoinase polypeptide SEQ ID NO:286 In a 500mL reaction vessel, 145mL 0.05M PBS pH7.0 buffer was added, 5mL of enzyme solution (SEQ ID NO:286) was further added, a water bath was started to control the temperature at 45°C, the stirring speed was set to 200rpm, and finally, 36g of substrate (3-isobutylglutarimide) was added to start the reaction. During the reaction, the pH was adjusted with ammonia water to control it to 7.0±0.2, and the reaction was terminated after 20h. Samples were taken and detected, and the conversion rate after 20h reaction was 96%. After the reaction, the reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated to about 100 mL. Then, hydrochloric acid was added dropwise to adjust the pH of the concentrated solution to 3.0. After stirring for 30 min, the solution was filtered to obtain a wet crude product, which was then baked and weighed. Finally, 37.9 g of crude product was obtained. ee%=99.8%.

[0153] Example 22: Catalytic process of Dp-hydroxyphenylglycine synthesis by engineering hydantoinase polypeptide SEQ ID NO:214 The following is a typical process for detecting the conversion rate in a reaction volume of 5 mL. In a reaction flask with a total volume of 30 mL, 70 μL of the enzyme solution of SEQ ID NO: 214 was added, 50 mg of p-hydroxyphenylhydanto was added, and finally 5 mL of phosphate buffer (0.1 M, pH 7.5) was added to make the concentrations of each component of the reaction system 14 mL / L of the enzyme solution of SEQ ID NO: 214 and 10 g / L of p-hydroxyphenylhydanto. A magnetic stirrer rotor was added to the reaction flask, which was then placed on an IKA magnetic stirrer set at 400 rpm and 40°C to start the reaction. After reacting for 1 hour, 5 mL of acetonitrile was added to the flask to quench the reaction. Concentrated hydrochloric acid was added to the quenched reaction flask until the concentration reached 2 mmol / L, and then 27 mg of sodium bisulfite was added to the reaction flask, which was then placed on a magnetic stirrer at 50°C and stirred at 400 rpm to hydrolyze. After 3 hours, 5 mL of 0.1% glacial acetic acid was added to the reaction flask, the reaction solution was centrifuged (13,000 rpm, 3 min), the supernatant after centrifugation was collected, and the sample was analyzed by HPLC, resulting in a conversion rate of 42.3%. [ka]

[0154] It should be understood that, after reading the above content of the present invention, one skilled in the art can make various changes or modifications to the present invention, and that equivalent forms thereof are also included within the scope defined by the appended claims of this application.

Claims

1. 1. An engineered hydantoinase polypeptide, comprising:

1. An engineered hydantoinase polypeptide that catalyzes the asymmetric hydrolysis of 3-isobutylglutarimide to produce (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid, and has an ee value of at least 97%, said polypeptide having at least 90% sequence identity with reference sequence SEQ ID NO:2 and comprising an amino acid sequence having at least one residue difference at residue position X64 compared to SEQ ID NO:2, wherein the amino acid residue at residue position X64 is selected from I, T, S, and A.

2. 2. The polypeptide of claim 1, wherein the reaction conditions include a loading of 3-isobutylglutarimide of about 1 g / L to 400 g / L, a loading of engineering polypeptide of about 0.1 g / L to 50 g / L, a pH of 6.0 to 8.5, and a temperature of 10 to 60° C.

3. The amino acid sequences are 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, 224, 226, 228, 230, 232, 23 3. The polypeptide of claim 1 or 2, having an amino acid sequence represented by the following formula: 4, 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.

4. A polypeptide immobilized on a solid material by a method of chemical binding or physical adsorption, A polypeptide selected from the polypeptides according to any one of claims 1 to 3.

5. A polynucleotide encoding the polypeptide according to any one of claims 1 to 4.

6. The polynucleotide sequences are selected from the group consisting of SEQ ID NOs: 1, 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, 9 3, 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, 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, 2 6. The polynucleotide of claim 5, which is a polynucleotide sequence of: 33, 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.

7. An expression vector comprising the polynucleotide according to claims 5 to 6.

8. The expression vector of claim 7 , comprising a plasmid, cosmid, phage, or viral vector.

9. A host cell comprising an expression vector according to any one of claims 7 to 8, preferably Escherichia coli.

10. 10. A method for preparing a hydantoinase polypeptide, comprising culturing the host cell of claim 9 and obtaining the polypeptide from the culture.

11. A hydantoinase catalyst selected from the culture according to any one of claims 7 to 10, a host cell or culture solution containing a hydantoinase polypeptide obtained from the culture, or a processed product using these, wherein the product is an extract obtained from the host cell, an isolated product obtained by isolating or purifying the hydantoinase in the extract, or an immobilized product obtained by immobilizing the host cell and the extract thereof or an isolated product of the extract.

12. the compound of formula (I) shown has the stereochemical configuration shown at the chiral center marked with an *, the compound of formula (I) shown is in excess of the corresponding enantiomer, A method for preparing a compound of structural formula (I), comprising contacting a hydantoinase derivative having structural formula (II) as a substrate with an engineered hydantoinase polypeptide according to any one of claims 1 to 4. 【Chemistry 1】 (wherein n=0 or 1, R １ , and R ２ are each independently H, an optionally substituted or unsubstituted aryl or heteroaryl, an optionally substituted or unsubstituted linear or branched C １ ~C ４ Alkyl, optionally substituted or unsubstituted, linear or branched C １ ~C ４ Alkenyl, optionally substituted or unsubstituted cycloalkyl, -OR', -NH ２ , -NR'R', -SR', -CO ２ R', or -C(O)R'; Each R' is independently -H or C １ ~C ４ is selected from hydrocarbyl. 【Chemistry 2】 (In structural formula (II), n, R １ , and R ２ The definitions are the same as those in structural formula (I).

13. the compound of structural formula (III) has the stereochemical configuration as shown at the chiral center marked with an *, and the compound of structural formula (III) as shown is in excess over the corresponding enantiomer, A method for preparing a compound of formula (III), comprising contacting an imide derivative having formula (IV) as a substrate with an engineered hydantoinase polypeptide according to any one of claims 1 to 4. 【Chemistry 3】 (where: n=0 or 1; R １ , and R ２ each independently represents H, optionally substituted or unsubstituted straight or branched chain C １ ~C ４ Alkyl or optionally substituted or unsubstituted C ６ H ６ is selected from When n=0, R １ , and R ２ may together form a ring structure group, which is selected from monocyclic or polycyclic, optionally substituted or unsubstituted aryl, or monocyclic or polycyclic, optionally substituted or unsubstituted heteroaryl. 【Chemistry 4】 (In structural formula (IV), n, R １ , and R ２ The definition of is the same as in structural formula (III).

14. A method for producing a hydantoinase according to any one of claims 1 to 4, which converts a substrate DL-p-hydroxyphenylhydantoin into N-carbamoyl-D-p-hydroxyphenylglycine by the action of the hydantoinase according to any one of claims 1 to 4, A process for the preparation of the formula Dp-hydroxyphenylglycine, further converting N-carbamoyl-Dp-hydroxyphenylglycine into Dp-hydroxyphenylglycine by the action of hydrochloric acid. 【Chemistry 5】 【Chemistry 6】 【Chemistry 7】

15. A method for preparing the compound of formula A2, (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid, comprising contacting the compound of formula A1, 3-isobutylglutarimide, with an engineered hydantoinase polypeptide of any one of claims 1-4. 【Chemistry 8】 【Chemistry 9】

16. The method of any one of claims 12 to 15, wherein the product is produced in at least 97%, 98%, 99% or more enantiomeric excess.

17. The method according to any one of claims 12 to 15, wherein the reaction solvent includes water, methanol, ethanol, propanol, isopropanol, dimethylsulfoxide, dimethylformamide, isopropyl acetate, ethyl acetate, butyl acetate, 1-octanol, heptane, octane, methyl tert-butyl ether (MTBE), toluene, and the like.

18. The method according to any one of claims 12 to 17, wherein the temperature is from 10°C to 60°C.

19. The method according to any one of claims 12 to 18, wherein the pH is between pH 6.0 and pH 8.

5.

20. The method according to any one of claims 12 to 19, wherein the substrate loading is from 1 g / L to 400 g / L.

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