Hydantoinase and application thereof in hydrolysis or preparation of chiral monomeric compound

Abstract

The invention relates to hydantoinase and application thereof in hydrolysis or preparation of chiral monomeric compounds. Compared with an amino acid sequence as shown in SEQ ID NO: 1, the amino acid sequence of the hydantoin enzyme is different in one or more positions selected from the 63 site, the 64 site, the 70 site, the 92 site, the 94 site, the 123 site, the 318 site, the 319 site and the 345 site. The invention also relates to a nucleic acid molecule, a nucleic acid construct, a recombinant vector and a host cell encoding the hydantoin enzyme, and an enzyme preparation containing the hydantoin enzyme. The invention also relates to a method for hydrolyzing or preparing a chiral monomeric compound by using the hydantoinase. The hydantoinase provided by the invention has better selectivity on substrate configuration and higher hydrolase activity, can catalyze various racemate hydantoin analogues, and selectively hydrolyzes R configuration with high reaction efficiency to obtain a compound chiral monomer with S configuration.

Description

Technical Field

[0001] The invention belongs to the field of biochemistry, and in particular relates to hydantoinase and its application in hydrolysis or preparation of chiral monomer compounds. Background Art

[0002] Hydantoinase, also known as hydantoinase, is a class of amide hydrolases that catalyze the cleavage of cyclic amide bonds of hydantoin, 5'-monosubstituted hydantoin and its derivatives. It is widely present in various organisms such as microorganisms, plants, and animals. However, hydantoinase with industrial application prospects mainly comes from microorganisms. Hydantoinase is classified as cyclic amidase (EC3.5.2), and is divided into four categories according to the different substrates it acts on: dihydropyrimidinase (EC3.5.2.2); allantoinase (EC3.5.2.5); carboxymethylhydantoinase (EC3.5.2.4); N-methylhydantoinase (EC3.5.2.14). In addition, there are some unnamed hydantoinase, including amidase with known metabolic pathways, carboxyethylhydantoinase and some hydantoinase whose metabolic mechanisms are not yet clear. According to the specificity of the substrate it acts on or the optical activity of the product, hydantoinase can also be divided into L-type, D-type or non-selective hydantoinase. At present, the industrial application of hydantoinase is mainly used to produce D-type and L-type amino acids.

[0003] (S)-3-aminopiperidine-2,6-dione can be used as a synthetic intermediate for compounds with medical value. Currently, it is mainly prepared by chemical methods, which require layering, filtration, drying, fractionation and other operations. The process is complicated and the steps are cumbersome. From the perspective of economy and environmental protection, it is not suitable for industrial large-scale production. Summary of the invention

[0004] In order to solve the defects of the prior art that the selectivity of hydantoinase for substrate configuration is not good enough and its hydrolase activity is not high enough, and the prior art still lacks a method for preparing chiral monomer compounds using bioenzyme catalysis, the present invention takes a different approach and uses hydantoinase to catalyze racemic hydantoin analogs to selectively hydrolyze one of the configurations (R configuration) to obtain a chiral monomer compound of the other configuration (S configuration), thereby providing a hydantoinase and its use in hydrolyzing or preparing chiral monomer compounds.

[0005] The first aspect of the present invention provides a hydantoinase, the amino acid sequence of which is different from the amino acid sequence shown in SEQ ID NO: 1 at one or more positions selected from: position 63, position 64, position 70, position 92, position 94, position 123, position 318, position 319 and position 345.

[0006] In one or more embodiments, the difference is selected from one or more of the following:

[0007] M63A, M63S or M63V;

[0008] P64I;

[0009] T70I;

[0010] F92Y or F92M;

[0011] L94Y;

[0012] H123M;

[0013] S318I;

[0014] F319L; and

[0015] R345Q.

[0016] In one or more embodiments, the amino acid sequence of the hydantoinase is shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9.

[0017] The second aspect of the present invention provides a nucleic acid molecule, the polynucleotide sequence of which is selected from:

[0018] (1) a polynucleotide sequence encoding the hydantoinase according to any embodiment of the present invention; or

[0019] (2) A complementary sequence to the polynucleotide sequence described in (1).

[0020] In one or more embodiments, the polynucleotide sequence described in (1) is shown as SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12.

[0021] The third aspect of the present invention provides a nucleic acid construct comprising the nucleic acid molecule according to any embodiment of the present invention.

[0022] In one or more embodiments, the nucleic acid construct is a gene expression cassette.

[0023] The fourth aspect of the present invention provides a recombinant vector comprising the nucleic acid molecule according to any embodiment of the present invention or the nucleic acid construct according to any embodiment of the present invention.

[0024] In one or more embodiments, the recombinant vector is a recombinant cloning vector or a recombinant expression vector.

[0025] A fifth aspect of the present invention provides a host cell, which contains the nucleic acid molecule described in any embodiment of the present invention, the nucleic acid construct described in any embodiment of the present invention or the recombinant vector described in any embodiment of the present invention, and / or expresses the hydantoinase described in any embodiment of the present invention.

[0026] In one or more embodiments, the host cell is selected from the group consisting of E. coli cells, insect cells, yeast cells, and mammalian cells.

[0027] A sixth aspect of the present invention provides an enzyme preparation, wherein the enzyme preparation contains the hydantoinase according to any embodiment of the present invention.

[0028] A seventh aspect of the present invention provides a method for hydrolyzing a chiral monomer compound represented by the following formula (iii) or preparing a chiral monomer compound represented by the following formula (ii):

[0029]

[0030] The method comprises using a hydantoinase from Geobacillus kaustophilus or a hydantoinase having at least 95% sequence identity with its amino acid sequence or a hydantoinase according to any embodiment of the present invention or an enzyme preparation according to any embodiment of the present invention to catalyze the hydrolysis of a chiral monomer compound represented by formula (iii) in a substrate;

[0031] When the method is to prepare a chiral monomer compound represented by formula (ii), the substrate is a mixture of chiral monomer compounds represented by formula (ii) and formula (iii), for example, the substrate is a racemate of a compound represented by formula (i) below; after catalysis, a chiral monomer compound represented by formula (ii) is obtained;

[0032]

[0033] Wherein, the R group in formula (i), formula (ii) or formula (iii) is halogen, unsubstituted or substituted by one or more R 2 Substituted phenyl, amino, Among them, R 1 C 1-6 The alkyl group is either unsubstituted or substituted with one or more R 1-1 Substituted phenyl; each R 1-1 are independently halogen, unsubstituted or substituted with one or more hydroxyl groups. 3-6 cycloalkyl, 3-6 membered heterocycloalkyl which is unsubstituted or substituted by one or more hydroxyl groups, or Each R 2 are independently halogen, unsubstituted or substituted with one or more hydroxyl groups. 3-6The cycloalkyl radical is a 3-6 membered heterocycloalkyl radical which is unsubstituted or substituted by one or more hydroxyl groups; the heteroatom of the 3-6 membered heterocycloalkyl radical is N, O or S, and the number of the heteroatom is 1.

[0034] In one or more embodiments, the method further comprises adding a cosolvent and / or a coenzyme; and / or, the R group in formula (i), formula (ii) or formula (iii) is:

[0035] -NH2 or Br.

[0036] In one or more embodiments, the R group in formula (i), formula (ii), or formula (iii) is -NH2.

[0037] In one or more embodiments, the compound of formula (i) is

[0038] In one or more embodiments, the amino acid sequence of the hydantoinase from Geobacillus alkalophilus is shown in SEQ ID NO: 1, or is the hydantoinase with NCBI accession number BAD75708.

[0039] In one or more embodiments, the method has one or more features selected from the group consisting of:

[0040] The co-solvent is selected from: dimethyl sulfoxide, alcohol solvent, toluene, or a combination thereof;

[0041] The reaction system contains metal ions: zinc ions, manganese ions or cobalt ions, preferably zinc ions;

[0042] The reaction system uses Tris-HCl buffer with a concentration of 10-200 mM, preferably 50-100 mM;

[0043] The pH of the reaction system is 6-10, preferably 6.5-7.5;

[0044] The reaction temperature is 10°C to 50°C, preferably 20°C to 45°C, more preferably 25°C to 32°C;

[0045] The reaction time is 0.1 to 96 hours, preferably 12 to 25 hours;

[0046] The mass of hydantoinase in the reaction system is 0.5-100 times, preferably 1-4 times, the mass of the substrate; and,

[0047] The mass of the metal ions in the reaction system is 0.01-0.5% of the mass of the substrate, preferably 0.05-0.2%.

[0048] In one or more embodiments, in the method, the cosolvent is dimethyl sulfoxide, the metal ion used is zinc ion, the hydantoinase is from alkalophilic Bacillus, preferably the hydantoinase or its enzyme preparation having an amino acid sequence as shown in SEQ ID NO: 1, or the hydantoinase is the hydantoinase or its enzyme preparation as described in any embodiment of the present invention.

[0049] In an eighth aspect, the present invention provides use of a hydantoinase or an enzyme preparation thereof from alkalophilic Geobacillus and / or a hydantoinase or an enzyme preparation thereof according to any embodiment of the present invention in hydrolyzing a chiral monomer compound represented by the following formula (iii) or preparing a chiral monomer compound represented by the following formula (ii);

[0050]

[0051] Wherein, the R group in formula (ii) or formula (iii) is halogen, unsubstituted or substituted with one or more R 2 Substituted phenyl, amino, Among them, R 1 C 1-6 The alkyl group is either unsubstituted or substituted with one or more R 1-1 Substituted phenyl; each R 1-1 are independently halogen, unsubstituted or substituted with one or more hydroxyl groups. 3-6 cycloalkyl, 3-6 membered heterocycloalkyl which is unsubstituted or substituted by one or more hydroxyl groups, or Each R 2 are independently halogen, unsubstituted or substituted with one or more hydroxyl groups. 3-6 A cycloalkyl group, a 3-6-membered heterocycloalkyl group which is unsubstituted or substituted by one or more hydroxyl groups; the heteroatom of the 3-6-membered heterocycloalkyl group is N, O or S, and the number of the heteroatom is 1;

[0052] In one or more embodiments, the amino acid sequence of the hydantoinase from Geobacillus alkaliphilus is as shown in SEQ ID NO: 1, or is the hydantoinase with NCBI accession number BAD75708; and / or, the R group in formula (ii) or formula (iii) is:

[0053] -NH2 or Br;

[0054] In one or more embodiments, the R group in formula (ii) or formula (iii) is -NH2.

[0055] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0056] The reagents and raw materials used in the present invention are commercially available.

[0057] The positive and progressive effects of the present invention are:

[0058] The hydantoinase provided by the present invention has better selectivity for substrate configuration and higher hydrolase activity, and can catalyze a variety of racemic hydantoin analogs to selectively hydrolyze one of the configurations (R configuration) with high reaction efficiency to obtain a chiral monomer of a compound with another configuration (S configuration).

[0059] Compared with traditional chemical synthesis methods, bio-enzyme catalysis has the advantages of high reaction efficiency, good stereoselectivity, mild reaction conditions, low energy consumption, and environmental friendliness. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 This is the SFC spectrum of (S)-3-aminopiperidine-2,6-dione after the reaction according to the method described in Example 1. The peak at t=1.513 is the target compound (S)-3-aminopiperidine-2,6-dione.

[0061] Figure 2 This is the SFC spectrum of (S)-3-aminopiperidine-2,6-dione after the reaction according to the method described in Example 2. The peak at t=1.508 is the target compound (S)-3-aminopiperidine-2,6-dione.

[0062] Figure 3 This is the SFC spectrum of (S)-3-aminopiperidine-2,6-dione after the reaction according to the method described in Example 3. The peak at t=1.544 is the target compound (S)-3-aminopiperidine-2,6-dione.

[0063] Figure 4 This is the SFC spectrum of (S)-3-aminopiperidine-2,6-dione after the reaction according to the method described in Example 4. The peak at t=1.508 is the target compound (S)-3-aminopiperidine-2,6-dione.

[0064] Figure 5 This is the SFC spectrum of (S)-3-aminopiperidine-2,6-dione after the reaction according to the method described in Example 5. The peak at t=1.514 is the target compound (S)-3-aminopiperidine-2,6-dione.

[0065] Figure 6 This is the SFC spectrum of (S)-3-bromopiperidine-2,6-dione after the reaction according to the method described in Example 6. The peak at t=1.575 is the target compound (S)-3-bromopiperidine-2,6-dione.

[0066] Figure 7 This is the SFC spectrum of (S)-3-bromopiperidine-2,6-dione after the reaction according to the method described in Example 7. The peak at t=1.587 is the target compound (S)-3-bromopiperidine-2,6-dione.

[0067] Figure 8 This is the SFC spectrum of (S)-3-aminopiperidine-2,6-dione after the reaction according to the method described in Example 8. The peak at t=1.466 is the target compound (S)-3-aminopiperidine-2,6-dione.

[0068] Fig. 9 This is the SFC spectrum of (S)-tert-butyl 2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)oxy)acetate after the reaction described in Example 9. The peak at t=1.250 is the target compound (S)-tert-butyl 2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl)oxy)acetate. DETAILED DESCRIPTION

[0069] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a preferred technical solution.

[0070] The present invention has found that the hydantoinase of the present invention can hydrolyze the chiral monomer compound represented by the following formula (iii) or prepare the chiral monomer compound represented by the following formula (ii) with a high conversion rate and chirality:

[0071]

[0072] Wherein, the R group in formula (ii) or formula (iii) is halogen, unsubstituted or substituted with one or more R 2 Substituted phenyl, amino, Among them, R 1 C 1-6 The alkyl group is either unsubstituted or substituted with one or more R 1-1 Substituted phenyl; each R 1-1 are independently halogen, unsubstituted or substituted with one or more hydroxyl groups. 3-6 cycloalkyl, 3-6 membered heterocycloalkyl which is unsubstituted or substituted by one or more hydroxyl groups, or Each R 2 are independently halogen, unsubstituted or substituted with one or more hydroxyl groups. 3-6 The cycloalkyl radical is a 3-6 membered heterocycloalkyl radical which is unsubstituted or substituted by one or more hydroxyl groups; the heteroatom of the 3-6 membered heterocycloalkyl radical is N, O or S, and the number of the heteroatom is 1.

[0073] In a particularly preferred embodiment, the R group in formula (ii) or formula (iii) is:

[0074] -NH2 or Br.

[0075] The present invention finds that by mutating the wild-type hydantoinase, the obtained enzyme mutant can catalyze one configuration (R configuration) of a plurality of racemic hydantoin analogs with better selectivity for substrate configuration and higher hydrolase activity to obtain a chiral monomer of a compound with another configuration (S configuration).

[0076] In a particularly preferred embodiment, the target chiral compound of the present invention is (S)-3-aminopiperidine-2,6-dione represented by the following formula (iv):

[0077]

[0078] In the present invention, the hydantoinase is preferably a hydantoinase from Geobacillus kaustophilus, and exemplary hydantoinase from Geobacillus kaustophilus includes a hydantoinase having an amino acid sequence as shown in SEQ ID NO: 1. The hydantoinase of the present invention also includes a mutant of SEQ ID NO: 1, such as a hydantoinase having an amino acid sequence with the amino acid sequence as shown in SEQ ID NO: 1 having at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% sequence identity. The sequence identity can be calculated using software commonly used in the art, such as BLAST (from NCBI) with software default parameters.

[0079] In one or more embodiments, the mutant of hydantoinase of the present invention is a mutant derived from SEQ ID NO: 1 obtained by performing multiple amino acid mutations on the amino acid sequence shown in SEQ ID NO: 1 and retaining the hydantoinase activity possessed by SEQ ID NO: 1. The one or more amino acid mutations include amino acid mutations within 20, preferably within 15, more preferably within 10, more preferably within 8, more preferably within 5, more preferably within 3, such as substitution, insertion or deletion of amino acid residues. Preferably, the mutation is substitution.

[0080] In the present invention, exemplary hydantoinase mutants include, but are not limited to, hydantoinase with accession number BAD75708.

[0081] In some embodiments, the particularly preferred hydantoinase mutants of the present invention are mutants obtained by amino acid differences at one or more key positions of position 63, position 64, position 70, position 92, position 94, position 123, position 318, position 319 and position 345. Preferred mutations are substitution mutations.

[0082] In some embodiments, the substitution is a conservative substitution. In some embodiments, the wild-type M at position 63 is substituted with other non-polar amino acids, such as alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, proline, preferably substituted with alanine, valine, leucine, isoleucine; more preferably substituted with alanine. The wild-type T at position 64 is substituted with a non-polar amino acid, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, proline, preferably substituted with isoleucine, phenylalanine, tryptophan, proline; more preferably substituted with isoleucine. The wild-type F at position 92 is substituted with an uncharged polar amino acid, serine, threonine, tyrosine, asparagine, glutamine, cysteine ​​and glycine, preferably substituted with asparagine, tyrosine, cysteine, more preferably substituted with tyrosine. The wild-type L at position 94 is substituted with a non-polar amino acid, such as alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, methionine, or proline, preferably with an aromatic non-polar amino acid, such as phenylalanine or tryptophan; more preferably with phenylalanine. The wild-type H at position 123 is substituted with a non-polar amino acid, such as alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, proline, or methionine, preferably with methionine, isoleucine, phenylalanine, tryptophan, or proline; more preferably with methionine. The wild-type S at position 318 is substituted with an aliphatic non-polar amino acid, such as alanine, leucine, valine, proline, or isoleucine, preferably with isoleucine, leucine, or valine; more preferably with valine. The wild-type F at position 319 is substituted with a non-polar amino acid, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, proline, preferably leucine, phenylalanine, tryptophan, proline; more preferably leucine. The wild-type R at position 345 is substituted with an uncharged polar amino acid, serine, threonine, tyrosine, asparagine, glutamine, cysteine ​​and glycine, preferably asparagine, glutamine, cysteine; more preferably glutamine.

[0083] In a preferred embodiment, the substitution mutation is one or more of the following substitution mutations: M63A, M63S or M63V; P64I; T70I; F92Y or F92M; L94Y; H123M; S318I; F319L; and R345Q. In a further preferred embodiment, the amino acid sequence of the hydantoin mutant is as shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9.

[0084] The present invention also includes a nucleic acid molecule whose polynucleotide sequence is the coding sequence of the hydantoinase mutant of the present invention or its complementary sequence. In some embodiments, the polynucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12.

[0085] The present invention also includes nucleic acid constructs, which contain nucleic acid molecules of the present invention. In some embodiments, the nucleic acid construct is a gene expression cassette. In addition to the nucleic acid molecules, the gene expression cassette may also contain a transcription termination sequence and a promoter. The promoter may be any of various promoters known in the art, as long as it is suitable for expressing the hydantoinase of the present invention in a desired host. Those skilled in the art may select appropriate promoters according to the host cell used to construct a gene expression cassette of the present invention and a recombinant vector described below.

[0086] The present invention also includes a recombinant vector. The recombinant vector may contain a nucleic acid molecule or a nucleic acid construct as described in any embodiment of the present invention. The recombinant vector may be a recombinant cloning vector or a recombinant eukaryotic expression vector. The recombinant vector may contain other regulatory elements, including but not limited to enhancers, multiple cloning sites, transcription terminators, resistance genes, etc. The corresponding vector backbone with the required regulatory elements may be selected according to different purposes, and the nucleic acid molecule or nucleic acid construct of the present invention may be cloned into the backbone to construct the recombinant vector of the present invention.

[0087] The hydantoinase described herein can be prepared by preparing nucleic acid molecules, constructing nucleic acid constructs and recombinant vectors using methods well known in the art, and expressing using conventional methods.

[0088] In some embodiments, the present invention also provides a host cell, which contains a nucleic acid molecule, a nucleic acid construct and / or a recombinant vector as described in any embodiment of the present invention, or expresses a hydantoinase as described in any embodiment of the present invention. Any host known in the art suitable for expressing a target protein can be used in the present invention, and exemplary host cells include Escherichia coli cells, insect cells, yeast cells and mammalian cells.

[0089] The method of the present invention comprises the steps of hydrolyzing a chiral monomer compound represented by the following formula (iii) or preparing a chiral monomer compound represented by the following formula (ii) using hydantoin in a buffer system containing metal ions:

[0090]

[0091] Wherein, the R group in formula (ii) or formula (iii) is halogen, unsubstituted or substituted with one or more R 2 Substituted phenyl, amino, Among them, R1 C 1-6 The alkyl group is either unsubstituted or substituted with one or more R 1-1 Substituted phenyl; each R 1-1 are independently halogen, unsubstituted or substituted with one or more hydroxyl groups. 3-6 cycloalkyl, 3-6 membered heterocycloalkyl which is unsubstituted or substituted by one or more hydroxyl groups, or Each R 2 are independently halogen, unsubstituted or substituted with one or more hydroxyl groups. 3-6 The cycloalkyl radical is a 3-6 membered heterocycloalkyl radical which is unsubstituted or substituted by one or more hydroxyl groups; the heteroatom of the 3-6 membered heterocycloalkyl radical is N, O or S, and the number of the heteroatom is 1.

[0092] In a particularly preferred embodiment, the R group in formula (ii) or formula (iii) is:

[0093] -NH2 or Br.

[0094] The mass of hydantoinase in the reaction system is 0.5-100 times, such as 1-4 times, 0.5-2 times or 2.5-5 times, of the mass of the substrate.

[0095] The reaction system may also contain a cosolvent. Cosolvents commonly used in experimental research and development can be used in the present invention. Usually, the cosolvent is an organic solvent, for example, a cosolvent selected from the group consisting of dimethyl sulfoxide, toluene, an alcohol solvent, or a combination thereof. The alcohol solvent includes but is not limited to isopropanol. Preferably, the cosolvent is selected from dimethyl sulfoxide and isopropanol. The cosolvent used should be miscible with water to further increase the solubility of the substrate.

[0096] In the present invention, the reaction system is a buffered saline solution system. The pH of the reaction system is controlled by a buffer. Commonly used buffers include, but are not limited to, phosphate buffer, Tris-HCl buffer, etc. Preferably, the reaction system uses Tris-HCl buffer at a concentration of 10-200mM, preferably 50-100mM. Preferably, the pH of the reaction system is 6-10, preferably 7-9, and more preferably 7-8. In some embodiments, the pH of the reaction system is 7.0-7.5.

[0097] The present invention finds that hydantoinase needs metal ions for activation. Metal ions need to be added to the reaction system, and commonly used metal ions include zinc ions, manganese ions or cobalt ions, etc. The mass of the metal ions is 0.01-0.5% of the mass of the substrate, preferably 0.05-0.2%.

[0098] The reaction temperature of the catalytic reaction of the present invention may be 10° C. to 50° C., preferably 20° C. to 45° C., more preferably 20° C. to 35° C. In some embodiments, the reaction temperature is room temperature, i.e., 25±3° C. The reaction time may be determined depending on the amount of reactants, and may generally be 0.1 to 120 hours, such as 0.5 to 48 hours or 12 to 25 hours.

[0099] The present invention finds that when the hydantoinase mutant of the present invention is used, especially the mutant obtained by mutation at one or more key positions of M63A, M63S or M63V; P64I; T70I; F92Y or F92M; L94Y; H123M; S318I; F319L; and R345Q of SEQ ID NO: 1 as described herein, the conversion rate and chirality are relatively high when the target chiral monomer compound is prepared. Therefore, in some embodiments of the present invention, the method of hydrolyzing the chiral monomer compound shown in formula (iii) or preparing the chiral monomer compound shown in formula (ii) of the present invention comprises: using the hydantoinase mutant described in any embodiment of the present invention to catalyze the substrate racemate shown in formula (i), selectively hydrolyze one of the configurations (R configuration) to obtain a compound chiral monomer of another configuration (S configuration). Preferably, the reaction system of the reaction also contains metal ions. In a particularly preferred embodiment, the hydantoinase mutant is a hydantoinase having an amino acid sequence as shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9.

[0100] The present invention also includes the aforementioned hydantoinase mutants, their coding sequences (nucleic acid molecules), nucleic acid constructs, recombinant vectors and host cells.

[0101] In some embodiments, the enzyme preparation is a buffer containing the hydantoinase mutant. Preferably, the buffer is a Tris-HCl buffer with a pH of 6 to 10, preferably 6.5 to 7.5. In some embodiments, the concentration of the buffer is 50 to 200 mM.

[0102] The present invention also provides the use of the hydantoinase mutant, its coding sequence (nucleic acid molecule), nucleic acid construct, recombinant vector and host cell described in any of the above embodiments in hydrolyzing the chiral monomer compound shown in formula (iii) or preparing the chiral monomer compound shown in formula (ii). In some embodiments, the reagent is the enzyme preparation described in any of the embodiments herein.

[0103] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0104] Preparation Example

[0105] The sequence of SEQ ID NO:1 (coding sequence as shown in SEQ ID NO:4) was used as the parent, and the directed evolution transformation was carried out by roller PCR, iterative saturation mutation, and combination mutation strategies. Then the mutant was transformed into Escherichia coli BL21 (DE3) competent cells, and evenly coated on LB agar plates with 50ug / ml kanamycin, and placed in a 37℃ incubator for static culture for 18h. The mutants on the transformed plate were picked into a 96-well plate with a sterile toothpick and cultured overnight in a shaker at 37℃ and 250rpm. 50 microliters of bacterial solution were drawn from the holes of the primary plate and inoculated into the corresponding holes of the secondary plate. After culturing at 37℃ and 250rpm for 2-3h, IPTG with a final concentration of 0.2mM was added, and the mutants were cultured at 30℃ for 20h to obtain the corresponding mutants for high-throughput screening. Combined with HPLC and GC detection rescreening, mutants with significantly improved activity and stability were identified for gene sequencing. The sequencing results are shown in SEQ ID NO:2 and SEQ ID NO:3, and the coding sequences are shown in SEQ ID NO:5 and SEQ ID NO:6.

[0106] SEQ ID NO: 1, wild-type hydantoinase

[0107] MTKLIKNGTIVTATDIYEADLLIQDGKIAVIGRNLDESGAEVIDATGCYVFPGGIDPHTHLDMPFGGTVTKDDFESGTIAAAFGGTTTIIDFCLTNKGEPLKKAIETWHNKATGKAVIDYGFHLMISEITDDVLEELPKVIEEEGITSFKVFMAYKDVFQADDGTLYRTLVAAKELGALVMVHAENGDVIDYLTKKALEDGHTDPIYHALTRPPELEGEATGRACQLTELAGSQLYVVHVSCAQAVEKIAEARNKGLNVWGETCPQYLVLDQSYLEKPNFEGAKYVWSPPLREKWHQEVLWNALKNGQLQTLGSDQCSFDFKGQKELGRGDFTKIPNGGPIIEDRVSILFSEGVKKGRITLNQFVDIVSTRIAKLFGLFPKKGTIAVGADADLVIFDPTVERVISAETHHMAVDYNPFEGMKVTGEPVSVLCRGEFVVRDKQFVGKPGYGQYVKRAKYGALMADQDVVKMS

[0108] SEQ ID NO:2, Hydantoinase Mutant

[0109] MTKLIKNGTIVTATDIYEADLLIQDGKIAVIGRNLDESGAEVIDATGCYVFPGGIDPHTHLDAPFGGTVTKDDFESGTIAAAFGGTTTIIDFCLTNKGEPLKKAIETWHNKATGKAVIDYGFHLMISEITDDVLEELPKVIEEEGITSFKVFMAYKDVFQADDGTLYRTLVAAKELGALVMVHAENGDVIDYLTKKALEDGHTDPIYHALTRPPELEGEATGRACQLTELAGSQLYVVHVSCAQAVEKIAEARNKGLNVWGETCPQYLVLDQSYLEKPNFEGAKYVWSPPLREKWHQEVLWNALKNGQLQTLGSDQCSFDFKGQKELGRGDFTKIPNGGPIIEDRVSILFSEGVKKGRITLNQFVDIVSTRIAKLFGLFPKKGTIAVGADADLVIFDPTVERVISAETHHMAVDYNPFEGMKVTGEPVSVLCRGEFVVRDKQFVGKPGYGQYVKRAKYGALMADQDVVKMS

[0110] SEQ ID NO:3, Hydantoinase Mutant

[0111] MTKLIKNGTIVTATDIYEADLLIQDGKIAVIGRNLDESGAEVIDATGCYVFPGGIDPHTHLDAIFGGTVTKDDFESGTIAAAFGGTTTIIDFCLTNKGEPLKKAIETWHNKATGKAVIDYGFHLMISEITDDVLEELPKVIEEEGITSFKVFMAYKDVFQADDGTLYRTLVAAKELGALVMVHAENGDVIDYLTKKALEDGHTDPIYHALTRPPELEGEATGRACQLTELAGSQLYVVHVSCAQAVEKIAEARNKGLNVWGETCPQYLVLDQSYLEKPNFEGAKYVWSPPLREKWHQEVLWNALKNGQLQTLGSDQCSFDFKGQKELGRGDFTKIPNGGPIIEDRVSILFSEGVKKGRITLNQFVDIVSTRIAKLFGLFPKKGTIAVGADADLVIFDPTVERVISAETHHMAVDYNPFEGMKVTGEPVSVLCRGEFVVRDKQFVGKPGYGQYVKRAKYGALMADQDVVKMS

[0112] SEQ ID NO:4, Coding DNA sequence of wild-type hydantoinase

[0113]

[0114] SEQ ID NO:5, DNA sequence encoding a mutant of hydantoinase

[0115]

[0116] SEQ ID NO:6, DNA sequence encoding a mutant of hydantoinase

[0117]

[0118] SEQ ID NO:7, Hydantoinase Mutant

[0119] MTKLIKNGTIVTATDIYEADLLIQDGKIAVIGRNLDESGAEVIDATGCYVFPGGIDPHTHLDMPFGGTVTKDDFESGTIAAAFGGTTTIIDFCYTNKGEPLKKAIETWHNKATGKAVIDYGFMLMISEITDDVLEELPKVIEEEGITSFKVFMAYKDVFQADDGTLYRTLVAAKELGALVMVHAENGDVIDYLTKKALEDGHTDPIYHALTRPPELEGEATGRACQLTELAGSQLYVVHVSCAQAVEKIAEARNKGLNVWGETCPQYLVLDQSYLEKPNFEGAKYVWSPPLREKWHQEVLWNALKNGQLQTLGSDQCSFDFKGQKELGRGDFTKIPNGGPIIEDRVSILFSEGVKKGRITLNQFVDIVSTRIAKLFGLFPKKGTIAVGADADLVIFDPTVERVISAETHHMAVDYNPFEGMKVTGEPVSVLCRGEFVVRDKQFVGKPGYGQYVKRAKYGALMADQDVVKMS

[0120] SEQ ID NO:8, Hydantoinase Mutant

[0121] MTKLIKNGTIVTATDIYEADLLIQDGKIAVIGRNLDESGAEVIDATGCYVFPGGIDPHTHLDSIFGGTVTKDDFESGTIAAAFGGTTTIIDFCLTNKGEPLKKAIETWHNKATGKAVIDYGFHLMISEITDDVLEELPKVIEEEGITSFKVFMAYKDVFQADDGTLYRTLVAAKELGALVMVHAENGDVIDYLTKKALEDGHTDPIYHALTRPPELEGEATGRACQLTELAGSQLYVVHVSCAQAVEKIAEARNKGLNVWGETCPQYLVLDQSYLEKPNFEGAKYVWSPPLREKWHQEVLWNALKNGQLQTLGSDQCIFDFKGQKELGRGDFTKIPNGGPIIEDQVSILFSEGVKKGRITLNQFVDIVSTRIAKLFGLFPKKGTIAVGADADLVIFDPTVERVISAETHHMAVDYNPFEGMKVTGEPVSVLCRGEFVVRDKQFVGKPGYGQYVKRAKYGALMADQDVVKMS

[0122] SEQ ID NO:9, Hyaluronidase Mutant

[0123] It should be noted that the content you provided seems to be a sequence or related information. If there are specific requirements for further processing or understanding of this translation, more context and instructions would be helpful. Also, the term "海因酶" in Chinese is translated as "Hyaluronidase" here, but it might need to be verified according to the actual situation as it may not be a common and accurate translation in all cases. If it is a specific enzyme name with a more accurate translation, it should be adjusted accordingly.MTKLIKNGTIVTATDIYEADLLIQDGKIAVIGRNLDESGAEVIDATGCYVFPGGIDPHTHLDVPFGGTVIKDDFESGTIAAAFGGTTTIIDFCLTNKGEPLKKAIETWHNKATGKAVIDYGFHLMISEITDDVLEELPKVIEEEGITSFKVFMAYKDVFQADDGTLYRTLVAAKELGALVMVHAENGDVIDYLTKKALEDGHTDPIYHALTRPPELEGEATGRACQLTELAGSQLYVVHVSCAQAVEKIAEARNKGLNVWGETCPQYLVLDQSYLEKPNFEGAKYVWSPPLREKWHQEVLWNALKNGQLQTLGSDQCSFDFKGQKELGRGDFTKIPNGGPIIEDRVSILFSEGVKKGRITLNQFVDIVSTRIAKLFGLFPKKGTIAVGADADLVIFDPTVERVISAETHHMAVDYNPFEGMKVTGEPVSVLCRGEFVVRDKQFVGKPGYGQYVKRAKYGALMADQDVVKMS

[0124] SEQ ID NO:10, Coding DNA sequence of the hydantoinase mutant

[0125]

[0126] SEQ ID NO: 11, DNA sequence encoding a mutant of hydantoinase

[0127]

[0128] SEQ ID NO: 12, DNA sequence encoding a mutant of hydantoinase

[0129]

[0130] The present invention will be described below in the form of specific examples. It should be understood that these examples are merely illustrative and are not intended to limit the present invention. The methods, materials and reagents used in the examples are, unless otherwise stated, conventional methods in the art, and materials and reagents available from commercial sources.

[0131] Example 1

[0132] Prepare 50mM Tris-HCl buffer solution: weigh 6.057g Tris into a 1L glass bottle, stir at room temperature until the solid is dissolved, and dilute to 1L with ddH2O. Adjust the pH to 7.0 with HCL / NaOH.

[0133] 0.01 mg ZnCl2 and 20 mg hydantoinase powder (amino acid sequence as shown in SEQ ID NO: 1) were weighed in sequence and added into a 4 mL glass tube containing 2 mL 50 mM Tris-HCl buffer solution, and vortexed to mix.

[0134] Weigh 20 mg of substrate (3-aminopiperidine-2,6-dione) and add it to 100 uL of dimethyl sulfoxide (DMSO) and mix well. Add the mixed liquid to the aforementioned 4 mL glass tube and vortex to mix well.

[0135] Cover the glass tube tightly, put it in a shaker at 30℃ and 1000rpm, and take it out after 20h. Take 100uL of the reaction solution in a 1.5mL centrifuge tube, add 1mL of acetonitrile, vortex and shake, mix, and then centrifuge at 12000rpm for 2min. Take 300uL of the supernatant for SFC detection. The peak at t=1.513min is the target compound (S)-3-aminopiperidine-2,6-dione, with an ee value of 14.04%, S / DMSO=32.62%. The results are as follows Figure 1 shown.

[0136] Example 2

[0137] Prepare 50mM Tris-HCl buffer solution: weigh 6.057g Tris into a 1L glass bottle, stir at room temperature until the solid is dissolved, and dilute to 1L with ddH2O. Adjust the pH to 7.0 with HCL / NaOH.

[0138] 0.01 mg ZnCl2 and 20 mg hydantoinase powder (amino acid sequence as shown in SEQ ID NO: 2) were weighed in sequence and added into a 4 mL glass tube containing 2 mL 50 mM Tris-HCl buffer solution, and vortexed to mix.

[0139] Weigh 20 mg of substrate (3-aminopiperidine-2,6-dione) and add it to 100 uL of dimethyl sulfoxide (DMSO) and mix well. Add the mixed liquid to the aforementioned 4 mL glass tube and vortex to mix well.

[0140] Cap the glass tube tightly, put it in a shaker at 30℃ and 1000rpm, and take it out after 20h. Take 100uL of the reaction solution in a 1.5mL centrifuge tube, add 1mL of acetonitrile, vortex and shake, mix, and then centrifuge at 12000rpm for 2min. Take 300uL of the supernatant for SFC detection. The peak at t=1.508min is the target compound (S)-3-aminopiperidine-2,6-dione, with an ee value of 32.28%, S / DMSO=33.17%. The results are as follows Figure 2 shown.

[0141] Example 3

[0142] Take about 20 mg of bacteria (after induction expression, containing hydantoinase shown in the amino acid sequence SEQ ID NO: 3), treat the cells with 270 uL 50 mM Tris-HCl buffer solution (containing 1 mg / mL nuclease and 2 mg / mL lysozyme), and put them in a 30°C 1000 rpm shaker for 2 hours. Take out and centrifuge at 4000 rpm for 15 minutes, take 200 uL of the supernatant and add it to a new 4 mL glass tube.

[0143] Weigh 2 mg of substrate (3-aminopiperidine-2,6-dione) and add it to 10 uL of dimethyl sulfoxide (DMSO) and mix well. Add the mixed liquid to the aforementioned 4 mL glass tube and vortex to mix well.

[0144] Weigh 0.001 mg ZnCl2 and add it into the aforementioned 4 mL glass tube, vortex and mix.

[0145] Cap the glass tube tightly, put it in a shaker at 30℃ and 1000rpm, and take it out after 20h. Take 200uL of the reaction solution in a 1.5mL centrifuge tube, add 1mL of acetonitrile, vortex and shake, mix, and then centrifuge at 12000rpm for 2min. Take 300uL of the supernatant for SFC detection. The peak at t=1.544min is the target compound (S)-3-aminopiperidine-2,6-dione, with an ee value of 25.20%, S / DMSO=35.44%. The results are as follows Figure 3 shown.

[0146] Example 4

[0147] Prepare 50mM Tris-HCl buffer solution: weigh 6.057g Tris into a 1L glass bottle, stir at room temperature until the solid is dissolved, and dilute to 1L with ddH2O. Adjust the pH to 7.0 with HCL / NaOH.

[0148] 0.01 mg ZnCl2 and 80 mg hydantoinase powder (amino acid sequence as shown in SEQ ID NO: 2) were weighed in sequence and added into a 4 mL glass tube containing 2 mL 50 mM Tris-HCl buffer solution, and vortexed to mix.

[0149] Weigh 20 mg of substrate (3-aminopiperidine-2,6-dione) and add it to 100 uL of dimethyl sulfoxide (DMSO) and mix well. Add the mixed liquid to the aforementioned 4 mL glass tube and vortex to mix well.

[0150] The glass tube was capped tightly and placed in a 30°C 1000 rpm shaker. It was taken out after 20 hours. 100uL of the reaction solution was placed in a 1.5mL centrifuge tube, 1mL of acetonitrile was added, vortexed, mixed, and then centrifuged at 12000rpm for 2 minutes. 300uL of the supernatant was taken for SFC detection. The peak at t=1.508min was the target compound (S)-3-aminopiperidine-2,6-dione, with an ee value of 94.14%, S / DMSO=34.60%, and the results were as follows: Figure 4 shown.

[0151] Example 5

[0152] Prepare 50mM Tris-HCl buffer solution: weigh 6.057g Tris into a 1L glass bottle, stir at room temperature until the solid is dissolved, and dilute to 1L with ddH2O. Adjust the pH to 7.0 with HCL / NaOH.

[0153] 0.01 mg ZnCl2 and 20 mg hydantoinase powder (amino acid sequence as shown in SEQ ID NO: 2) were weighed in sequence and added into a 4 mL glass tube containing 2 mL 50 mM Tris-HCl buffer solution, and vortexed to mix.

[0154] Weigh 10 mg of substrate (3-aminopiperidine-2,6-dione) and add it to 50 uL of dimethyl sulfoxide (DMSO) and mix well. Add the mixed liquid to the aforementioned 4 mL glass tube and vortex to mix well.

[0155] Cap the glass tube tightly, put it in a shaker at 30℃ and 1000rpm, and take it out after 20h. Take 100uL of the reaction solution in a 1.5mL centrifuge tube, add 1mL of acetonitrile, vortex and shake, mix, and then centrifuge at 12000rpm for 2min. Take 300uL of the supernatant for SFC detection. The peak at t=1.514min is the target compound (S)-3-aminopiperidine-2,6-dione, with an ee value of 49.16%, S / DMSO=34.38%. The results are as follows Figure 5 shown.

[0156] Example 6

[0157] Prepare 50mM Tris-HCl buffer solution: weigh 6.057g Tris into a 1L glass bottle, stir at room temperature until the solid is dissolved, and dilute to 1L with ddH2O. Adjust the pH to 7.0 with HCL / NaOH.

[0158] 0.01 mg ZnCl2 and 30 mg hydantoinase powder (amino acid sequence as shown in SEQ ID NO: 1) were weighed in sequence and added into a 4 mL glass tube containing 2 mL 50 mM Tris-HCl buffer solution, and vortexed to mix.

[0159] Weigh 10 mg of substrate (3-bromopiperidine-2,6-dione) and add it to 50 uL of dimethyl sulfoxide (DMSO) and mix well. Add the mixed liquid to the aforementioned 4 mL glass tube and vortex to mix well.

[0160] Cover the glass tube tightly, put it in a shaker at 30℃ and 1000rpm, and take it out after 20h. Take 100uL of the reaction solution in a 1.5mL centrifuge tube, add 1mL of acetonitrile, vortex and shake, mix, and then centrifuge at 12000rpm for 2min. Take 300uL of the supernatant for SFC detection. The peak at t=1.575min is the target compound (S)-3-bromopiperidine-2,6-dione, with an ee value of 84.78%, S / DMSO=23.45%. The results are as follows Figure 6 shown.

[0161] Example 7

[0162] Prepare 50mM Tris-HCl buffer solution: weigh 6.057g Tris into a 1L glass bottle, stir at room temperature until the solid is dissolved, and dilute to 1L with ddH2O. Adjust the pH to 7.0 with HCL / NaOH.

[0163] 0.01 mg ZnCl2 and 30 mg hydantoinase powder (amino acid sequence as shown in SEQ ID NO: 7) were weighed in sequence and added into a 4 mL glass tube containing 2 mL 50 mM Tris-HCl buffer solution, and vortexed to mix.

[0164] Weigh 10 mg of substrate (3-bromopiperidine-2,6-dione) and add it to 50 uL of dimethyl sulfoxide (DMSO) and mix well. Add the mixed liquid to the aforementioned 4 mL glass tube and vortex to mix well.

[0165] Cap the glass tube tightly, put it in a shaker at 30℃ and 1000rpm, and take it out after 20h. Take 100uL of the reaction solution in a 1.5mL centrifuge tube, add 1mL of acetonitrile, vortex and shake, mix, and then centrifuge at 12000rpm for 2min. Take 300uL of the supernatant for SFC detection. The peak at t=1.587min is the target compound (S)-3-bromopiperidine-2,6-dione, with an ee value of 96.10%, S / DMSO=18.52%. The results are as follows Figure 7 shown.

[0166] Example 8

[0167] Prepare 50mM Tris-HCl buffer solution: weigh 6.057g Tris into a 1L glass bottle, stir at room temperature until the solid is dissolved, and dilute to 1L with ddH2O. Adjust the pH to 7.0 with HCL / NaOH.

[0168] 0.01 mg ZnCl2 and 80 mg hydantoinase powder (amino acid sequence as shown in SEQ ID NO: 8) were weighed in sequence and added into a 4 mL glass tube containing 2 mL 50 mM Tris-HCl buffer solution, and vortexed to mix.

[0169] Weigh 20 mg of substrate (3-aminopiperidine-2,6-dione) and add it to 100 uL of dimethyl sulfoxide (DMSO) and mix well. Add the mixed liquid to the aforementioned 4 mL glass tube and vortex to mix well.

[0170] Cap the glass tube tightly, put it in a shaker at 30℃ and 1000rpm, and take it out after 20h. Take 100uL of the reaction solution in a 1.5mL centrifuge tube, add 1mL of acetonitrile, vortex and shake, mix, and then centrifuge at 12000rpm for 2min. Take 300uL of the supernatant for SFC detection. The peak at t=1.466min is the target compound (S)-3-aminopiperidine-2,6-dione, with an ee value of 94.59%, S / DMSO=19.99%, and the results are as follows Figure 8 shown.

[0171] Example 9

[0172] Take about 20 mg of bacteria (after induction expression, containing hydantoinase shown in the amino acid sequence SEQ ID NO: 9), treat the cells with 270 uL 50 mM Tris-HCl buffer solution (containing 1 mg / mL nuclease and 2 mg / mL lysozyme), and put them in a shaker at 30°C and 1000 rpm for 2 hours. Take out and centrifuge at 4000 rpm for 15 minutes, take 200 uL of the supernatant and add it to a new 4 mL glass tube.

[0173] Weigh 0.2 mg of substrate (tert-butyl 2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindole-4-yl)oxy)acetate) and add it to 1 uL of dimethyl sulfoxide (DMSO) and mix well. Add the mixed liquid to the aforementioned 4 mL glass tube and vortex to mix well.

[0174] Weigh 0.001 mg ZnCl2 and add it into the aforementioned 4 mL glass tube, vortex and mix.

[0175] Cover the glass tube tightly, put it in a shaker at 30℃ and 1000rpm, and take it out after 20h. Take 200uL of the reaction solution in a 1.5mL centrifuge tube, add 1mL of acetonitrile, vortex and shake, mix well, and then centrifuge at 12000rpm for 2min. Take 300uL of the supernatant for SFC detection. The peak at t=1.250min is the target compound (S)-tert-butyl 2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoindole-4-yl)oxy)acetate, with an ee value of 60.91%, S / DMSO=25.05%. The results are as follows Fig. 9 shown.

[0176] In summary, the above embodiments are only preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A hydantoinase, characterized in that The amino acid sequence of the hydantoinase is different from the amino acid sequence shown in SEQ ID NO: 1 at one or more positions selected from the group consisting of: position 63, position 64, position 70, position 92, position 94, position 123, position 318, position 319 and position 345.

2. The hydantoinase according to claim 1, wherein The differences are selected from one or more of the following: M63A, M63S or M63V; P64I; T70I; F92Y or F92M; L94Y; H123M; S318I; F319L; and R345Q; Preferably, the amino acid sequence of the hydantoinase is shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:

9.

3. A nucleic acid molecule, the polynucleotide sequence of which is selected from: (1) a polynucleotide sequence encoding the hydantoinase according to claim 1 or 2; or (2) a complementary sequence of the polynucleotide sequence described in (1); Preferably, the polynucleotide sequence described in (1) is shown as SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:

12.

4. A nucleic acid construct comprising the nucleic acid molecule according to claim 3; preferably, the nucleic acid construct is a gene expression cassette.

5. A recombinant vector, comprising the nucleic acid molecule according to claim 3 or the nucleic acid construct according to claim 4; preferably, the recombinant vector is a recombinant cloning vector or a recombinant expression vector.

6. A host cell, comprising the nucleic acid molecule of claim 3, the nucleic acid construct of claim 4 or the recombinant vector of claim 5, and / or expressing the hydantoinase of claim 1 or 2; preferably, the host cell is selected from the group consisting of Escherichia coli cells, insect cells, yeast cells and mammalian cells.

7. An enzyme preparation comprising the hydantoinase according to claim 1 or 2.

8. A method for hydrolyzing a chiral monomer compound represented by the following formula (iii) or preparing a chiral monomer compound represented by the following formula (ii): The method comprises using a hydantoinase from Geobacillus kaustophilus or a hydantoinase having at least 95% sequence identity with its amino acid sequence or a hydantoinase according to claim 1 or 2 or an enzyme preparation according to claim 7 to catalyze the hydrolysis of a chiral monomer compound represented by formula (iii) in a substrate; When the method is to prepare a chiral monomer compound represented by formula (ii), the substrate is a mixture of chiral monomer compounds represented by formula (ii) and formula (iii), for example, the substrate is a racemate of a compound represented by formula (i) below; after catalysis, a chiral monomer compound represented by formula (ii) is obtained; in, The R group in formula (i), formula (ii) or formula (iii) is halogen, unsubstituted or substituted by one or more R 2 Substituted phenyl, amino, Among them, R 1 C 1-6 The alkyl group is either unsubstituted or substituted with one or more R 1-1 Substituted phenyl; each R 1-1 are independently halogen, unsubstituted or substituted with one or more hydroxyl groups. 3-6 cycloalkyl, 3-6 membered heterocycloalkyl which is unsubstituted or substituted by one or more hydroxyl groups, or Each R 2 are independently halogen, unsubstituted or substituted with one or more hydroxyl groups. 3-6 A cycloalkyl group, a 3-6-membered heterocycloalkyl group which is unsubstituted or substituted by one or more hydroxyl groups; the heteroatom of the 3-6-membered heterocycloalkyl group is N, O or S, and the number of the heteroatom is 1; Preferably, the method further comprises adding a cosolvent and / or a coenzyme; and / or, the R group in formula (i), formula (ii) or formula (iii) is: -NH2 or Br; More preferably, the R group in formula (i), formula (ii) or formula (iii) is -NH2; More preferably, the compound of formula (i) is 9. The method according to claim 8, characterized in that The amino acid sequence of the hydantoinase from Geobacillus alkaliphilus is shown in SEQ ID NO: 1, or is the hydantoinase with NCBI accession number of BAD75708.

10. The method according to claim 8, characterized in that The method has one or more characteristics selected from the following: The co-solvent is selected from: dimethyl sulfoxide, alcohol solvent, toluene, or a combination thereof; The reaction system contains metal ions: zinc ions, manganese ions or cobalt ions, preferably zinc ions; The reaction system uses Tris-HCl buffer with a concentration of 10-200 mM, preferably 50-100 mM; The pH of the reaction system is 6-10, preferably 6.5-7.5; The reaction temperature is 10°C to 50°C, preferably 20°C to 45°C, more preferably 25°C to 32°C; The reaction time is 0.1 to 96 hours, preferably 12 to 25 hours; The mass of hydantoinase in the reaction system is 0.5-100 times, preferably 1-4 times, the mass of the substrate; and, The mass of the metal ions in the reaction system is 0.01-0.5% of the mass of the substrate, preferably 0.05-0.2%.

11. The method according to any one of claims 8 to 10, characterized in that: In the method, the cosolvent is dimethyl sulfoxide, the metal ion used is zinc ion, the hydantoinase is from alkalophilic Bacillus, preferably the hydantoinase or its enzyme preparation whose amino acid sequence is shown in SEQ ID NO: 1, or the hydantoinase is the hydantoinase or its enzyme preparation as claimed in claim 1 or 2.

12. Use of a hydantoinase or an enzyme preparation thereof from alkalophilic Geobacillus and / or a hydantoinase or an enzyme preparation thereof as claimed in claim 1 or 2 in hydrolyzing a chiral monomer compound represented by the following formula (iii) or preparing a chiral monomer compound represented by the following formula (ii); in, The R group in formula (ii) or formula (iii) is halogen, unsubstituted or substituted by one or more R 2 Substituted phenyl, amino, Among them, R 1 C 1-6 The alkyl group is either unsubstituted or substituted with one or more R 1-1 Substituted phenyl; each R 1-1 are independently halogen, unsubstituted or substituted with one or more hydroxyl groups. 3-6 cycloalkyl, 3-6 membered heterocycloalkyl which is unsubstituted or substituted by one or more hydroxyl groups, or Each R 2 are independently halogen, unsubstituted or substituted with one or more hydroxyl groups. 3-6 A cycloalkyl group, a 3-6-membered heterocycloalkyl group which is unsubstituted or substituted by one or more hydroxyl groups; the heteroatom of the 3-6-membered heterocycloalkyl group is N, O or S, and the number of the heteroatom is 1; Preferably, the amino acid sequence of the hydantoinase from Geobacillus alkaliphilus is as shown in SEQ ID NO: 1, or is the hydantoinase with NCBI accession number BAD75708; and / or, the R group in formula (ii) or formula (iii) is: -NH2 or Br; More preferably, the R group in formula (ii) or (iii) is -NH2.

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