Hydantoinase mutants resistant to neutral environments and use thereof

Abstract

The application discloses a hydantoinase mutant SEQ ID NO:3, which can efficiently catalyze hydrolysis of D-p-hydroxyphenylhydantoin into N-carbamoyl-D-p-hydroxyphenylglycine in a pH 6.5-7.5 neutral reaction system, and can be combined with carbamoyl hydrolase to catalyze synthesis of D-p-hydroxyphenylglycine from D,L-p-hydroxyphenylhydantoin, and has a good industrial application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biocatalysis technology, specifically, it relates to a hydantoin mutant and its application in the preparation of N-carbamoyl-D-p-hydroxyphenylglycine and D-p-hydroxyphenylglycine. Background Technology

[0002] D-p-hydroxyphenylglycine (D-HPG), also known as levorotatory p-hydroxyphenylglycine, has the molecular formula C8H9NO3 and the structural formula shown below:

[0003]

[0004] D-HPG is an important intermediate in the synthesis of β-lactam semi-synthetic broad-spectrum antibiotics, such as amoxicillin, cefadroxil, and amoxicillin-cephalosporins. These antibiotics possess excellent physiological functions, including a broad antibacterial spectrum, low adverse effects, low allergenicity, good oral efficacy, and long duration of action. Furthermore, D-HPG is also an intermediate in the production of some antibacterial and antiviral drugs, as well as artificial sweeteners.

[0005] The synthesis methods of D-HPG include chemical synthesis and bioenzymatic methods. Chemical methods can be divided into benzaldehyde method, β-cyclodextrin method, p-hydroxyphenylhydantoin method, active amide compound reaction method and glyoxylic acid method, etc. (Research on the synthesis of D,L-p-hydroxyphenylglycine. Applied Chemical Industry, 2003, 32(05):46-49). However, chemical synthesis methods have disadvantages such as low reaction yield, high energy consumption, and high price of racemic chiral resolving agents, which do not conform to the current concept of green manufacturing. The bioenzymatic method uses D,L-p-hydroxyphenylglycine as a raw material, and D-hydantoinase and carbamoyl hydrolase play a catalytic role in the resolution and hydrolysis to generate D-HPG. It has the advantages of fewer reaction steps, high catalytic efficiency and environmental friendliness, and has attracted much attention in recent years (Production of Dp-Hydroxyphenylglycine by N-Carbamoyl-D-aminoAcid Amidohydrolase-Overproducing Escherichia coli Strains. Biotechnol Prog, 1999, 15(4): 603-607. Wang Yameng, Ban Rui, Liu Lu, Shen Yu. Construction of recombinant Bacillus subtilis co-expressed with heterologous D-hydantoinase and N-carbamoyl hydrolase. Acta Microbiologica Sinica, 2017, 57(1): 54-65.).

[0006] D-hydantoin can selectively hydrolyze D-p-hydroxyphenylhydantoin to N-carbamoyl-D-p-hydroxyphenylglycine (NC-D-HPG). The remaining L-p-hydroxyphenylhydantoin will rapidly and spontaneously race to D,L-p-hydroxyphenylhydantoin under neutral or alkaline conditions. NC-D-HPG is then converted to D-HPG by carbamoyl hydrolase.

[0007]

[0008] Biological enzymatic methods can be divided into two-strain / two-enzyme methods and one-strain two-enzyme methods, which are reported in patent documents CN103993010A, CN110396484A, CN110699396A, CN111621452A, and CN112080532A. p-Hydroxyphenylhydantoin is unstable under alkaline conditions and undergoes spontaneous racemization; therefore, nitrogen gas is required to isolate it from air during the enzymatic reaction (D-p-hydroxyphenylglycine synthesis by two-enzyme method. Journal of China Pharmaceutical University, 2001, 32(2):155-158). Furthermore, carbamoyl hydrolase (DCase) has high activity under neutral conditions, but under alkaline conditions, it is affected by the byproduct NH4+. + Inhibition leads to decreased activity (Biotechnol Prog, 1999, 15(4): 603-607). Hydantoin (DHase) has better stability at higher pH values ​​(pH 8-9), but it is rapidly inactivated at pH values ​​below 8.0. The adaptability of these two enzymes to different pH values ​​is contradictory. In order to prepare D-p-hydroxyphenylglycine from the raw material p-hydroxyphenylhydantoin and improve the enzyme conversion efficiency of the substrate, CN112080532A provides a cascade enzyme reaction. Using D,L-p-hydroxyphenylhydantoin as the substrate, hydantoin is first added, and the reaction is carried out in an anaerobic environment at pH 9.5-11 to obtain an intermediate reaction solution. The intermediate reaction solution is then subjected to ultrafiltration to remove impurities, and then N-carbamoyl hydrolase is added to the ultrafiltration permeate. The reaction is carried out in an anaerobic environment at pH 7.0-8.5 until the end to obtain D-p-hydroxyphenylglycine. The cascade reaction also has obvious drawbacks. First, each step requires tedious post-processing. Second, the enzyme activities and feed amounts of the two enzymes need to be experimentally matched, inevitably leading to increased costs. Overall, the single-strain dual-enzyme method using co-expression of two enzymes is a more ideal choice. Summary of the Invention

[0009] To optimize the process route for the synthesis of D-p-hydroxyphenylglycine from p-hydroxyphenylhydantoin using a single-strain dual-enzyme method and to balance the conflicting pH adaptability of hydantoin and carbamoyl hydrolase, the inventors explored altering the pH preference of hydantoin. Through extensive screening and mutagenesis techniques, they finally obtained several hydantoin mutants tolerant to neutral reaction environments. Furthermore, based on the high-activity carbamoyl hydrolase (CN202310044861.9) already obtained by the inventors, the hydantoin mutants and carbamoyl hydrolase (SEQ ID NO:5) were co-expressed in *E. coli*, achieving the cellular catalytic synthesis of D-p-hydroxyphenylglycine under neutral pH conditions. Specifically, this invention includes the following technical solutions.

[0010] The first aspect of the present invention provides a hydantoin mutant, which is a mutant formed by mutations at five or more, preferably six or more, sites in the amino acid sequence of wild-type hydantoin SEQ ID NO:1: Q25L, D45T, D45A, T69A, H124N, L310P, G354R, L394I, P399S. This hydantoin mutant has the function of catalyzing the hydrolysis of D-p-hydroxyphenylhydantoin (i.e., D-5-(4-hydroxyphenyl)hydantoin) to N-carbamoyl-D-p-hydroxyphenylglycine (NC-D-HPG) in an environment below pH 7.5.

[0011] Preferably, the above-mentioned hydantoin mutant is a mutant formed by mutations at five or more, preferably six or more, sites in the amino acid sequence of wild-type hydantoin SEQ ID NO:1: Q25L, D45T, T69A, H124N, L310P, G354R, L394I, P399S.

[0012] MMTKLIKNGTIVTATDIYEADLLIQDGKIAVIGRNLDESGAEVIDATGCYVFPGGIDPHTHLDMPFGGTVTKDDFESGTIAAAFGGTTTIIDFCLTNKGEPLKKAIETWHNKATGKAVI DYGFHLMISEITDDVLEELPKVIEEEGITSFKVFMAYKDVFQADDGTLYRTLVAAKELGALVMVHAENGDVIDYLTKKALEDGHTDPIYHALTRPPELEGEEATGRACQLTELAGSQLYV VHVSCAQAVEKIAEARNKGLNVWGETCPQYLVLDQSYLEKPNFEGAKYVWSPPLREKWHQEVLWNALKNGQLQTLGSDQCSFDFKGQKELGRGDFTKIPNGGPIIEDRVSILFSEGVKK GRITLNQFVDIVSTRIAKLFGLFPKKGTIAVGADADLVIFDPTVERVISAETHHMAVDYNPFEGMKVTGEPVSVLCRGEFVVRDKQFVGKPGYGQYVKRAKYGALMADQDVVKMS(SEQ ID NO:1).

[0013] More preferably, the amino acid sequence of the above-mentioned hydantoin mutant is SEQ ID NO:3, which is a mutant formed by mutations at the following six sites in the amino acid sequence of wild-type hydantoin SEQ ID NO:1: Q25L, D45T, H124N, G354R, L394I, P399S.

[0014] A second aspect of the invention provides a gene encoding the aforementioned heinz mutant.

[0015] For example, the nucleotide sequence of the gene encoding the amino acid sequence of the hydantoin mutant SEQ ID NO:3 can be SEQ ID NO:4.

[0016] MMTKLIKNGTIVTATDIYEADLILLDGKIAVIGRNLDESGAEVITATGCYVFPGGIDPHTHLDMPFGGTVTKDDFESGTIAAAFGGTTTIIDFCLTNKGEPLKKAIETWHNKATGKAVI DYGFNLMISEITDDVLEELPKVIEEEGITSFKVFMAYKDVFQADDGTLYRTLVAAKELGALVMVHAENGDVIDYLTKKALEDGHTDPIYHALTRPPELEGEEATGRACQLTELAGSQLYV VHVSCAQAVEKIAEARNKGLNVWGETCPQYLVLDQSYLEKPNFEGAKYVWSPPLREKWHQEVLWNALKNGQLQTLGSDQCSFDFKGQKELGRGDFTKIPNGGPIIEDRVSILFSERVKK GRITLNQFVDIVSTRIAKLFGLFPKKGTIAVGADADIVIFDSTVERVISAETHHMAVDYNPFEGMKVTGEPVSVLCRGEFVVRDKQFVGKPGYGQYVKRAKYGALMADQDVVKMS(SEQ ID NO:3).

[0017] Furthermore, the present invention also provides a plasmid containing the above-mentioned gene for expressing the above-mentioned hydantoin mutant in microorganisms.

[0018] The aforementioned plasmid vectors can be selected from pET vectors, pSH plasmids, pRSFDuet plasmids, or other vectors. For example, the pET vectors are pET22b, pET24a, and pET28a.

[0019] A third aspect of the invention provides the use of the above-described hydantoin mutant in the preparation of N-carbamoyl-D-p-hydroxyphenylglycine (NC-D-HPG) and / or D-p-hydroxyphenylglycine (D-HPG).

[0020] In the above reactions for preparing NC-D-HPG and / or D-HPG, the pH of the reaction system can be neutral, ranging from pH 6.5 to 7.5.

[0021] In a preferred embodiment, when the above-mentioned hydantoin mutant is used to prepare D-p-hydroxyphenylglycine, it is used in combination with carbamoyl hydrolase (the carbamoyl hydrolase mutant reported in patent document CN202310044861.9) with the amino acid sequence SEQ ID NO:5, wherein the carbamoyl hydrolase is used to hydrolyze N-carbamoyl-D-p-hydroxyphenylglycine (NC-D-HPG) to D-p-hydroxyphenylglycine (D-HPG).

[0022] MTRQMILAVGQQGPIARAETREQVVVRLLDMLTKAASRGANFIVFPELALTTFFPRWYFTDEAELDSFYETEMPGPVVRPLFEKAAELGIGFNLGYAELVVEGGVKRRFNTSILVDKSGKIVGKYRKIHLPGHKETEAYRDFQHLEKRVFEPGD LGFPVYDVDAAKMGMFICNDRRWPEAWRVMGLRGAEIICGGYNTPTHNPEVPQHDHLTSFHHLLSMQAGSYQNGAWSAAAGKAGMEENCMLLGHSCIVAPTGEIVALTTTLEDEVITAAVDLDRCRELREHIFNFKQHRQPQHYGLIAEL(SEQ ID NO:5).

[0023] Preferably, the above-mentioned hydantoin mutant and the above-mentioned carbamoyl hydrolase are co-expressed in microorganisms. The fermented cells or crude enzyme can be directly used as a catalyst to hydrolyze the substrate D-p-hydroxyphenylhydantoin to obtain D-p-hydroxyphenylglycine, thereby avoiding the trouble of matching the enzyme activity and feed amount of the two isolated enzymes or expression strains in advance. It is equivalent to the "one-pot method" to prepare the final product D-p-hydroxyphenylglycine from the raw material D-p-hydroxyphenylhydantoin.

[0024] For example, the microorganisms that co-express hydantoin and carbamoyl hydrolase can be transformants of the hydantoin mutant and the carbamoyl hydrolase co-expression plasmid.

[0025] The aforementioned microbial hosts can be selected from Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae, or Escherichia coli, with Escherichia coli BL21(DE3) being the preferred choice.

[0026] In the above-mentioned application method, when microorganisms co-expressing the hydantoin mutant and the carbamoyl hydrolase are used as catalysts, they can be added to the reaction system in the form of microbial cells or their cell fragments to carry out whole-cell catalysis.

[0027] That is, in the reaction system, D-p-hydroxyphenylhydantoin (D-5-(4-hydroxyphenyl)hydantoin) is used as the substrate raw material, and the hydantoin mutant and the carbamoyl hydrolase or their expressing microorganisms are used to catalyze the hydrolysis reaction to obtain D-p-hydroxyphenylglycine (D-HPG).

[0028] During the catalytic hydrolysis of D-p-hydroxybenzenehydantoin, the pH of the reaction system is 6.5-7.5. The reaction temperature is 35-45℃, preferably 40℃.

[0029] The concentration of the substrate D-p-hydroxyphenylhydantoin can be 2-60 g / L, preferably 40 g / L.

[0030] Another aspect of the present invention provides a plasmid that co-expresses the above-mentioned hydantoin mutant and the above-mentioned carbamoyl hydrolase.

[0031] Preferably, the plasmid contains a hydantoin mutant encoding gene with SEQ ID NO:4 and a carbamoyl hydrolase encoding gene with SEQ ID NO:6.

[0032] The present invention provides a hydantoin mutant, such as SEQ ID NO:3, obtained through genetic engineering mutation, which overcomes the defects of ordinary hydantoin, such as intolerance to neutral environments, easy inactivation in neutral environments, or significant decrease in enzyme activity. This hydantoin mutant SEQ ID NO:3 can be combined with carbamoyl hydrolase SEQ ID NO:5, for example, by co-expression in microorganisms, for efficient catalysis of D-p-hydroxyphenylhydantoin hydrolysis to D-p-hydroxyphenylglycine in a neutral reaction system. Attached Figure Description

[0033] Figure 1 This is a schematic diagram of the structure of plasmid pET-HY-0, which is used to express wild-type hydantoin.

[0034] Figure 2 This is an HPLC chromatogram of the hydrolysis reaction of D-p-hydroxyphenylhydantoin catalyzed by the hydantoin mutant and carbamoyl hydrolase co-expression strain constructed in this invention. Detailed Implementation

[0035] Because D-p-hydroxyphenylhydantoin is unstable under alkaline conditions with high pH and undergoes spontaneous racemization, in order to change the fact that hydantoin enzymes (DHases) are generally adapted to alkaline environments with high pH (pH 8-9), but are rapidly inactivated at neutral pH below 8.0 and are therefore unsuitable for catalyzing the hydrolysis of D-p-hydroxyphenylhydantoin in neutral reaction systems to produce high-optical-purity chiral products N-carbamoyl-D-p-hydroxyphenylglycine (NC-D-HPG) and D-p-hydroxyphenylglycine (D-HPG), the inventors screened various hydantoin enzymes from microbial sources reported in the literature and found that the hydantoin enzyme derived from *Geobacillus skaustophilus* (GenBank accession number BAD75708.1, amino acid sequence SEQ ID NO:1) has a wider pH range than other reported hydantoin enzymes.

[0036] Experiments revealed that when wild-type hydantoin SEQ ID NO:1 catalyzes the hydrolysis of D-p-hydroxyphenylhydantoin in a reaction system at around pH 7.0, although the enzyme exhibits high stereoselectivity and produces N-carbamoyl-D-p-hydroxyphenylglycine with high optical purity, the catalytic reaction rate is very slow and the enzyme activity decreases rapidly, indicating that it is not well-suited to an environment at around pH 7.0.

[0037] Therefore, it is necessary to modify its structure through mutation in order to obtain mutant hydantoinase that is tolerant to neutral environments.

[0038] In this paper, the terms "wild-type," "wild-type (hydantoin) enzyme," and "wild-type (hydantoin) enzyme" have the same meaning, referring to the hydantoin enzyme derived from *Bacillus thermophilus* with the amino acid sequence SEQ ID NO:1, designated HY-0 in this paper. Correspondingly, the terms "mutant," "mutant (hydantoin) enzyme," and "hydantoin enzyme mutant" have the same meaning, referring to enzymes that retain the same catalytic reaction characteristics after amino acid sequence modification of wild-type hydantoin enzymes, especially enzymes with increased enzyme activity and / or enhanced enzyme activity stability, such as the mutant with the amino acid sequence SEQ ID NO:3. For ease of expression, wild-type hydantoin enzymes and their mutants are collectively referred to as "hydantoin enzymes" in this paper.

[0039] In this document, the terms “(enzyme activity) increase” and / or “(stability) increase” as used above mean an increase of at least 100% compared to the reference level, such as an increase of at least 1, at least 2, or at least 3, or at least 5, or at least 10, or at least 20 times compared to the reference level.

[0040] In the description of the technical solutions of this invention, the term "and / or" used in terms such as "A and / or B" or "A and / or B" is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to cover each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); C (alone).

[0041] The term "mutation" as used herein includes, but is not limited to, the substitution, deletion, insertion, or chemical modification of amino acid residues, preferably positive mutations, i.e., mutations that increase enzyme activity. The substitution can be a non-conservative substitution, a conserved substitution, or a combination of both. "Conservative" amino acid substitution or mutation refers to the interchangeability of residues with similar side chains, and therefore generally includes the substitution of amino acids in polypeptides with amino acids from the same or similar amino acid definition class. However, as used herein, if a conserved mutation can alternatively be an aliphatic to aliphatic, nonpolar to nonpolar, polar to polar, acidic to acidic, basic to basic, aromatic to aromatic, or restriction residue to restriction residue substitution, then a conserved mutation does not include hydrophilic to hydrophilic, hydrophobic to hydrophobic, hydroxyl-containing to hydroxyl-containing, or small residue to small residue substitution. As is known in the art, common examples of conservative substitutions include: substitutions between aromatic amino acids F, W, and Y; substitutions between hydrophobic amino acids L, I, and V; substitutions between polar amino acids Q and N; substitutions between basic amino acids K, R, and H; substitutions between acidic amino acids D and E; and substitutions between hydroxyl amino acids S and T. Furthermore, A, V, L, or I can be conservatively mutated to another aliphatic residue or another nonpolar residue. Exemplary conservative substitutions can be performed according to the table below, wherein amino acids belonging to the same partition in the second column can be substituted for each other, and preferably, amino acids in the same row in the third column can be substituted for each other:

[0042]

[0043] "Non-conservative substitution" refers to the substitution or mutation of an amino acid in a polypeptide with an amino acid having significantly different side chain properties. Non-conservative substitution can be performed between, rather than within, the amino acids defined above. In one embodiment, a non-conservative mutation affects (a) the structure of the peptide backbone in the substituted region (e.g., proline replacing glycine), (b) charge or hydrophobicity, or (c) side chain volume.

[0044] "Deletion" refers to a modification of a peptide by removing one or more amino acids from a reference peptide. Deletion may include the removal of one or more amino acids, two or more amino acids, five or more amino acids, ten or more amino acids, fifteen or more amino acids, or twenty or more amino acids, up to 10% of the total number of amino acids constituting the reference enzyme, while preserving enzyme activity and / or the modified properties of the engineered hydantoin. Deletion may target the interior and / or ends of the peptide. In several embodiments, the deletion may comprise a continuous segment or may be discontinuous.

[0045] "Insertion" refers to a modification of a polypeptide by adding one or more amino acids to a reference polypeptide. In some embodiments, modified engineered hydantoins include inserting one or more amino acids into a naturally occurring hydantoin as well as inserting one or more amino acids into other modified hydantoin polypeptides. The insertion can be internal to the polypeptide, or at the carboxyl terminus or amino terminus. Insertions as used herein include fusion proteins as known in the art. The insertion can be a continuous amino acid segment or separated by one or more amino acids in a naturally occurring polypeptide.

[0046] Through multiple rounds of error-prone PCR random mutagenesis, several mutants adapted to a neutral environment of approximately pH 7.0 were screened. Effective mutation sites included: Y18H, Q25L, D45T, D45A, T69A, H124N, L135R, L310P, G354R, F380Y, L394I, and P399S. Among these, the mutants with mutations at the following six sites exhibited the best enzyme activity and stability: Q25L, D45T, T69A, H124N, L310P, G354R, L394I, and P399S. The amino acid sequence of this mutant is SEQ ID NO:3.

[0047] The amino acid sequence of the hydantoin mutant of the present invention is well known, so those skilled in the art can easily obtain its encoding gene, expression cassette and plasmid containing these genes, and transformants containing the plasmid.

[0048] To optimally express hydantoin or its mutants in E. coli, the most commonly used gene for genetic engineering, codon optimization can be performed on the expression genes of these enzymes.

[0049] Codon optimization is a technique used to maximize protein expression in an organism by increasing the translation efficiency of genes of interest. Different organisms often exhibit a particular preference for one of a set of codons encoding the same amino acid due to mutational predisposition and natural selection. For example, in fast-growing microorganisms such as *E. coli*, optimized codons reflect the composition of their respective genomic tRNA repertoires. Thus, in fast-growing microorganisms, low-frequency codons for amino acids can be used for high-frequency codon substitutions of the same amino acid. Consequently, the expression of optimized DNA sequences is improved in fast-growing microorganisms.

[0050] For example, in order to express carbamoyl hydrolase in Escherichia coli, the gene encoding carbamoyl hydrolase SEQ ID NO:5, which has been codon-optimized, can be SEQ ID NO:6.

[0051] These genes, expression cassettes, plasmids, and transformants can be obtained through genetic engineering construction methods well known to those skilled in the art.

[0052] When used in the synthesis of D-p-hydroxyphenylglycine (D-HPG), hydantoin needs to be combined with carbamoyl hydrolase to catalyze a cascade hydrolysis reaction of D,L-p-hydroxyphenylhydantoin. The inventors have already reported a high-performance carbamoyl hydrolase in patent document CN202310044861.9, whose amino acid sequence is SEQ ID NO.5.

[0053] In a preferred embodiment, hydantoinase and carbamoyl hydrolase can be co-expressed in the same microbial strain, eliminating the need to add the two isolated enzymes in sequence according to the reaction process during the above-mentioned hydrolysis reaction, thus saving the trouble of adding the two enzymes or expression cells in proportion.

[0054] The aforementioned microorganisms can be transformants of hydantoin expression plasmids and carbamoyl hydrolase expression plasmids. The hydantoin mutant encoding gene and the carbamoyl hydrolase encoding gene can be cloned on different plasmids, or they can be cloned together on the same plasmid vector to form a co-expression plasmid.

[0055] The transformant host can be any microorganism suitable for expressing hydantoin and / or carbamoyl hydrolase, including bacteria and fungi. Preferred microorganisms are Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae, or Escherichia coli, with Escherichia coli being preferred, and Escherichia coli BL21(DE3) being more preferred.

[0056] When used as a biocatalyst, the hydantoin and / or carbamoyl hydrolase of the present invention can be in the form of an enzyme or in the form of a bacterial cell. The enzyme form includes free enzymes and immobilized enzymes, including purified enzymes, crude enzymes, fermentation broth, enzymes immobilized on a carrier, etc.; the bacterial cell form includes live cells, dead cells, immobilized cells, etc.

[0057] As another alternative implementation, microbial cells expressing the aforementioned hydantoin and / or carbamoyl hydrolase can be used as biocatalysts for the enzyme-catalyzed reaction. The microorganisms can be in the form of cells or their cell fragments. Cellular forms include both live and dead cells. When microorganisms such as Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae, or Escherichia coli are no longer fermenting and multiplying but are used for enzyme-catalyzed reactions, they are themselves a naturally occurring immobilized enzyme. Furthermore, they do not require disruption or even extraction and purification and can be used as an enzyme preparation for catalyzing the reaction. Since the reaction substrates and products are small molecule compounds, they can easily cross the biological barrier of the cell membrane, thus eliminating the need for cell disruption, which is economically advantageous.

[0058] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0059] Example

[0060] This article involves the addition amount, content and concentration of various substances. Unless otherwise specified, the percentage content mentioned refers to the mass percentage.

[0061] Materials and methods

[0062] The whole gene synthesis, primer synthesis and sequencing in the examples were completed by Suzhou Genewiz Biotechnology Co., Ltd.

[0063] The molecular biology experiments in the examples included plasmid construction, enzyme digestion, ligation, preparation of competent cells, transformation, and culture medium preparation, etc., mainly referring to "Molecular Cloning: A Laboratory Manual," 3rd edition (J. Sambrook and DW Russell, eds., translated by Huang Peitang et al., Science Press, Beijing, 2002). Specific experimental conditions could be determined through simple experiments if necessary.

[0064] PCR amplification experiments should be performed according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions. Adjustments can be made through simple experiments if necessary.

[0065] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.2. (LB solid medium with an additional 20 g / L agar powder.)

[0066] TB medium: 24 g / L yeast extract, 12 g / L tryptone, 16.43 g / L K₂HPO₄·3H₂O, 2.31 g / L KH₂PO₄, 5 g / L glycerol, pH 7.0-7.5. (TB solid medium with an additional 20 g / L agar powder.)

[0067] Fermentation medium for a 5L fermenter: 24g / L yeast extract, 12g / L tryptone, 16.43g / L K2HPO4·3H2O, 2.31g / L KH2PO4, 5g / L glycerol, 0.5g / L defoamer, pH 7.0-7.5, with a fermentation broth volume of 2L per fermenter.

[0068] Feeding medium for a 5L fermenter: 60% glycerol.

[0069] Kanamycin (Kan, 50 μg / mL) should be used as directed based on the antibiotic gene carried by the plasmid.

[0070] HPLC detection conditions for substrate D,L-p-hydroxyphenylhydantoin, intermediate N-carbamoyl-D-p-hydroxyphenylglycine, and product D-p-hydroxyphenylglycine:

[0071] Shanghai Wufeng LC-100 liquid chromatograph, Waters Symmetry-C18 (250*4.6mm, 5μm), mobile phase: 10mM PBS solution (pH3.5): acetonitrile (90:10), 1.0mL / min, 230nm, 20ul, column temperature 30℃.

[0072] It should be noted that, for the sake of convenience, in the embodiments, the strain number, plasmid number, enzyme number, and enzyme-encoding gene number may share the same number. This is easily understood by those skilled in the art, that is, the same number can refer to different biological forms in different environments.

[0073] Example 1: Construction of an engineered Escherichia coli strain expressing wild-type hydantoinase

[0074] In this study, the hydantoin from *Geobacillus kaustophilus* was designated HY-0. Based on its amino acid sequence SEQ ID NO:1 (GenBank accession number BAD75708.1) and its coding gene sequence SEQ ID NO:2, the whole gene was synthesized by Suzhou Genewise Biotechnology Co., Ltd. Restriction endonuclease sites NdeI and BamHI were designed at both ends of the gene. The gene was subcloned into the corresponding sites of the vector pET24a (Novagen) to obtain the recombinant plasmid pET-HY-0, the structure of which is shown in Figure 1.

[0075] The recombinant plasmid pET-HY-0 was transformed into host Escherichia coli BL21(DE3) competent cells by electroporation to obtain recombinant Escherichia coli BL-HY-0 expressing wild-type hydantoinase.

[0076] Example 2: Establishment of a random mutant library of hydantoin and high-throughput screening in rounds 1-3

[0077] 1. Error-prone PCR method for constructing a random mutation library

[0078] Using plasmid pET-HY-0 as a template, a random mutant library was constructed using error-prone PCR technology.

[0079] Design the following primer pair HY-5 / HY-3:

[0080] Forward primer HY-5: 5'-ATGATGACAAAATTGATAAAAAATGG-3',

[0081] Reverse primer HY-3: 5'-TTAGGACATTTTCACCACATC-3'.

[0082] Using plasmid pET-HY-0 as a template, PCR amplification was performed to obtain a hydantoin mutant DNA sequence of approximately 1.4 kb.

[0083] The 50 μL error-prone PCR reaction system includes: 10 ng plasmid (pET-HY-0) template, 50 pmol primer pair HY-5 and HY-3, 1×Taq buffer, 0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP, 1 mM dTTP, 7 mM MgCl2, (0 mM, 0.05 mM, 0.1 mM, 0.15 mM, 0.2 mM) MnCl2, and 2.5 units of Taq enzyme (Takara).

[0084] The PCR reaction conditions were: 95℃ for 5 min; 94℃ for 30 s, 55℃ for 30 s, 72℃ for 2 min / kbp, 30 cycles; 72℃ for 10 min.

[0085] PCR products were electrophoresed and gel-recovered (Axygen DNA Gel Recovery Kit AP-GX-50). Using plasmid pET-HY-0 as a template and a recovered product (random mutant fragment) of approximately 1.4 kb as a large primer, MegaPrimer PCR was performed using KOD-plus DNA polymerase: 94℃ for 5 min; 98℃ for 10 s, 60℃ for 30 s, 68℃ for 2 min / kb, 25 cycles; 68℃ for 10 min. The plasmid template was digested with DpnI restriction endonuclease (Thermo Fisher Scientific), and electrotransformed into *E. coli* BL21(DE3) to obtain more than 10...4 A random mutation library of clones.

[0086] 2. The first round of high-throughput screening of the mutant library

[0087] Single colonies were picked and transferred to 96-well plates (each well containing 110 μL of liquid LB-Kan medium). After incubation at 37°C and 400 rpm for 5 h, 60 μL of the bacterial culture from each well was transferred to 96-well deep-well plates (each well containing 240 μL of liquid TB-Kan-0.2 mM IPTG). The plates were incubated at 25°C and 400 rpm for 12–16 h. The cells were collected by centrifugation at 4°C and 4000 rpm for 10 min, and the supernatant was discarded. The cells were then washed with pre-cooled physiological saline and collected by centrifugation at 4°C and 4000 rpm for 10 min, and the supernatant was discarded. The cells were resuspended in 200 μL of enzyme reaction solution (20 mM D,L-p-hydroxyphenylhydantoin, 100 mM phosphate buffer, pH 8.0) in each well and incubated at 45°C and 250 rpm for 2–5 h. After the reaction was completed, 100 μL of the reaction solution was taken and an equal volume of colorimetric reagent (10% p-dimethylaminobenzaldehyde (PDAB) dissolved in 6 mol / L hydrochloric acid) was added. Positive strains showed a yellow color, and the deeper the yellow color, the higher the enzyme activity. The detection wavelength was 510 nm.

[0088] Strains with significantly enhanced enzyme activity were selected, plasmids were extracted, and nucleic acid sequencing was performed by Suzhou Genewiz Company. The hydantoin-related fragments in the genome were compared with SEQ ID NO:1 to determine the amino acid mutation sites. The strain with the highest activity improvement was used as the starting strain for the next round of random mutant library construction. The construction of the random mutant library and the high-throughput reaction screening with D,L-p-hydroxyphenylhydantoin as a substrate were repeated.

[0089] 3. Second round of high-throughput screening of the mutant library

[0090] The high-throughput screening method used in the first round of the mutant library was followed, but the pH of the enzyme reaction system was adjusted to 7.5, and 100 μL of 100 mM phosphate buffer (pH 7.5) was added before the reaction. The reaction was carried out after incubation at 45°C for 3 hours. Other enzyme reaction conditions were the same as in the first round of screening, i.e., 200 μL of enzyme reaction solution (20 mM D,L-p-hydroxyphenylhydantoin, 100 mM phosphate buffer, pH 7.5) was used to resuspend the bacterial cells in each well, and the cells were incubated at 45°C and 250 rpm for 2–5 hours.

[0091] 4. The third round of high-throughput screening of the mutant library

[0092] The high-throughput screening method used in the first round of the mutant library was followed, but the pH of the enzyme reaction was adjusted to 7.0, and 100 μL of 100 mM phosphate buffer (pH 7.0) was added before the reaction. The reaction was carried out after incubation at 45°C for 5 h. Other enzyme reaction conditions were the same as in the first round of screening, i.e., 200 μL of enzyme reaction solution (20 mM D,L-p-hydroxyphenylhydantoin, 100 mM phosphate buffer, pH 7.5) was used to resuspend the bacterial cells in each well, and the cells were incubated at 45°C and 250 rpm for 2–5 h.

[0093] Through three rounds of screening, a strain BL-HY-16 with the highest enzyme activity at pH 7.0 was selected. Gene sequencing and amino acid site alignment confirmed the amino acid sequence of the HY-16 heynase mutant as SEQ ID NO:3 and the corresponding nucleic acid sequence as SEQ ID NO:4. See Table 1.

[0094] Table 1. Results of high-throughput screening of random mutant libraries in rounds 1-3

[0095]

[0096]

[0097] In Table 1, "+" represents a viability percentage relative to the original strain that is greater than 0% and less than or equal to 50%; "++" represents a viability percentage relative to the original strain that is greater than 50% and less than or equal to 100%; "++++" represents a viability percentage relative to the original strain that is greater than 100% and less than or equal to 200%; and "++++" represents a viability percentage relative to the original strain that is greater than 200%.

[0098] Three rounds of mutation results showed that the following site mutations in the wild-type enzyme were effective positive mutations for pH neutrality adaptation: Y18H, Q25L, D45T, D45A, T69A, H124N, L135R, L310P, G354R, F380Y, L394I, and P399S. The mutant enzyme HY-16, which includes the mutations Q25L, D45T, H124N, G354R, L394I, and P399S, exhibited the highest enzyme activity at pH 7.0.

[0099] The following examples focus on the hydantoin mutant HY-16.

[0100] Example 3: Construction of an Escherichia coli strain co-expressing carbamoyl hydrolase and hydantoinase

[0101] 3.1 Following the method described in Example 1, the plasmid pET-HY-16, which can express the hydantoin mutant HY-16 in Escherichia coli, was constructed.

[0102] 3.2 To construct an engineered bacterium capable of directly catalyzing the synthesis of D-p-hydroxyphenylglycine from D,L-p-hydroxyphenylhydantoin, it is necessary to co-express carbamoyl hydrolase and hydantoinase in the strain. Based on patent document CN202310044861.9, the high-performance carbamoyl hydrolase numbered 2-C4 was selected. Its amino acid sequence in this paper is SEQ ID NO:5, and its encoding gene nucleotide sequence is SEQ ID NO:6.

[0103] Based on the sequence SEQ ID NO:6, a fragment 5'-ACTAGTCCGCGGATCC-3' was added to the 3' end to introduce the SpeI restriction site. Suzhou Genewiz Biotechnology Co., Ltd. was commissioned to synthesize and clone it into the NdeI and BamHI sites of plasmid pET24a to obtain plasmid pET-SJ.

[0104] Plasmids pET-HY-0 and pET-HY-16 were digested with XbaI and BamHI, respectively, to obtain approximately 1.5 kb DNA sequences containing the hydantoin gene. These two 1.5 kb DNA sequences were then ligated to the SpeI and BamHI sites of plasmid pET-SJ, respectively, to obtain co-expression plasmids pET-SJ-HY-0 and pET-SJ-HY-16, which simultaneously express carbamoyl hydrolase and hydantoin. Plasmids pET-SJ-HY-0 and pET-SJ-HY-16 were transformed into E. coli BL21(DE3) competent cells, respectively, to obtain engineered strains BL-SJ-HY-0 and BL-SJ-HY-16, which co-express carbamoyl hydrolase and hydantoin, respectively. Strain BL-SJ-HY-0 expresses carbamoyl hydrolase and wild-type hydantoin HY-0; strain BL-SJ-HY-16 expresses carbamoyl hydrolase and mutant hydantoin HY-16.

[0105] Example 4: Fermentation culture of engineered Escherichia coli

[0106] Fermentation cultures of strains BL-SJ-HY-0 and BL-SJ-HY-16 were conducted in a 5L fermenter. Single colonies were picked from LB agar plates and transferred to 5 mL of liquid LB medium containing Kans, and cultured overnight at 37°C and 220 rpm. The following day, a 5% (v / v) inoculum was transferred to shake flasks containing 100 mL of liquid TB medium and cultured at 37°C and 220 rpm until the OD600nm reached 6. This seed culture was then transferred to the 5L fermenter. After inoculation, the culture was maintained at 400–800 rpm / min and 37°C, with dissolved oxygen controlled at 20–30%. When the bacterial OD600nm reached 20, IPTG was added to induce enzyme expression. The final IPTG concentration was 0.2 mM, and the culture was continued at 28–30°C for 16–24 hours. Throughout the fermentation process, ammonia was used to maintain the pH at 6.8–7.2, and the aeration rate was controlled at 2.5–3.5. After fermentation, the cells were collected by centrifugation at 10,000 rpm for 10 min at 4 °C. The cells were then resuspended in PBS buffer at a concentration of 0.1 g / mL and then subjected to high-pressure disruption to obtain whole-cell reaction solution, which was stored at 2 °C for later use.

[0107] Example 5: Production of D-p-hydroxyphenylglycine by a dual-enzyme catalysis method

[0108] The 1L reaction system included: 0.1M phosphate buffer (pH 7.5), 100mL whole-cell reaction solution, 40g / L substrate D,L-p-hydroxyphenylglycine, 1mM manganese chloride, and 0.1% (v / v) β-mercaptoethanol. Nitrogen gas was used for protection throughout the reaction. The pH was maintained at approximately 7.0 using 3M phosphoric acid and 1M sodium hydroxide solution. The reaction was carried out at 40℃ for 48h. After the reaction, a sample was taken, and 10% hydrochloric acid was added to terminate the reaction. The sample was filtered and analyzed by HPLC. The results showed that after 48h of reaction, the formation rate of D-p-hydroxyphenylglycine in the BL-SJ-HY-16 whole-cell reaction solution exceeded 92% (e.g., ...). Figure 2 As shown in the figure, the D-p-hydroxyphenylglycine production rate in the BL-SJ-HY-0 whole-cell reaction system was only 23%, indicating that the hydantoin mutant HY-16 exhibits significantly higher enzyme activity for D,L-p-hydroxyphenylhydantoin at around pH 7.0 than the wild-type enzyme HY-0. Furthermore, HY-16 and carbamoyl hydrolase can form a better combination for the synthesis of D-p-hydroxyphenylglycine, showing promising prospects for industrial application development.

[0109] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A hydantoin mutant, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

3.

2. The gene encoding the heinzyme mutant as described in claim 1.

3. The gene as described in claim 2, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO:

4.

4. The use of the hydantoin mutant as described in claim 1 in the preparation of N-carbamoyl-D-p-hydroxyphenylglycine.

5. The application of the hydantoin mutant as described in claim 1 in the preparation of D-p-hydroxyphenylglycine, characterized in that, The hydantoin mutant is used in combination with carbamoyl hydrolase, whose amino acid sequence is shown in SEQ ID NO:

5.

6. The application as described in claim 5, characterized in that, The hydantoin mutant and the carbamoyl hydrolase are co-expressed in microorganisms.

7. The application as described in claim 5, characterized in that, In the reaction system, D-p-hydroxyphenylhydantoin is used as a substrate, and the hydantoin mutant and the carbamoyl hydrolase or their expression microorganisms are used to catalyze the hydrolysis reaction to obtain D-p-hydroxyphenylglycine.

8. The application as described in claim 6, characterized in that, The microorganism is a transformant of the co-expression plasmid of the hydantoin mutant and the carbamoyl hydrolase.

9. The application as described in claim 8, characterized in that, The microorganisms mentioned are selected from Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae, or Escherichia coli.

10. The application as described in claim 8, characterized in that, The microorganism mentioned is Escherichia coli BL21(DE3).

11. A plasmid co-expressing the hydantoin mutant as described in claim 1 and the carbamoyl hydrolase as described in claim 5.

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