Carbamoylase mutant

Abstract

The invention discloses a carbamoylase mutant SEQ ID NO: 3, which can efficiently catalyze the hydrolysis of N-carbamoyl-D-p-hydroxyphenylglycine to D-p-hydroxyphenylglycine, has high thermal stability, and can be used for industrial dual-enzyme method to catalyze the generation of D-p-hydroxyphenylglycine.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme catalysis, and in particular relates to a carbamoylase mutant and application thereof in the synthesis of D-p-hydroxyphenylglycine. Background Art

[0002] D-p-hydroxyphenylglycine (also known as L-p-hydroxyphenylglycine, English name Dp-hydroxyphenylglycine, D-HPG) is an important side chain raw material compound for the synthesis of β-lactam semi-synthetic broad-spectrum antibiotics such as amoxicillin, cefadroxil, cefoperazone, hydroxyamoxazole cefuroxime, cefuroxime, ceftriaxone, etc. It is also an important intermediate for the synthesis of antibacterial and antiviral drugs and artificial sweeteners, and has wide commercial application value.

[0003] D-HPG synthesis methods can be roughly divided into two categories, one is chemical synthesis method, and the other is bioenzyme method. There are three chemical methods for synthesizing D-HPG. The first is to obtain mixed DL-HPG by cyclization, pressurized alkaline hydrolysis and demethylation of p-methoxybenzaldehyde and sodium cyanide in aqueous solution or alcohol solution; the second is to react glyoxylic acid with phenol to generate p-hydroxy α-hydroxyphenylacetic acid, and then react at 50-70°C in acidic or alkaline conditions, condense to synthesize DL-p-hydroxyphenylhydantoin, and then hydrolyze to generate DL-HPG; the third is to use glyoxylic acid aqueous solution, phenol and ammonium salt as raw materials to synthesize DL-HPG in one step. However, the products synthesized by all chemical methods are mixed DL-HPG, and they must be chirally resolved by chemical resolution agents such as bromocamphorsulfonic acid, o-toluenesulfonic acid, sulfosalicylic acid, phenylethanesulfonic acid, etc. to obtain D-HPG. The chemical synthesis method has the problems of high price of racemic chiral resolution agents, low overall reaction yield, and high energy consumption, which is not in line with the current concept of green manufacturing.

[0004] The bioenzymatic method is a splitting method that is currently being studied more at home and abroad. The bioenzymatic method has the advantages of single optical activity, low energy consumption, and low pollution of "three wastes". For example, DL-p-hydroxyphenyl hydroxylamine is used as a raw material, and D-HPG is catalyzed by the action of D-hydantoinase and carbamoylase. First, D-hydantoinase can selectively hydrolyze the D-type in racemic DL-p-hydroxyphenyl hydroxylamine into N-carbamoyl-D-p-hydroxyphenylglycine (NC-D-HPG), and the residual L-p-hydroxyphenyl hydroxylamine will rapidly and spontaneously racemize to DL-p-hydroxyphenyl hydroxylamine under neutral or alkaline conditions. The racemic DL-p-hydroxyphenyl hydroxylamine is eventually almost completely converted into D-HPG, which saves the tedious steps of synthesizing the racemic mixture first and then splitting it, and the desired product can be directly obtained. In recent years, the preparation of D-amino acids by the dual enzyme method has attracted much attention due to its potential application value due to its advantages such as economy, environmental friendliness and wide substrate range.

[0005] Patent CN201510457039.0 discloses that N-carbamyl-D-p-hydroxyphenylglycine hydrolase (DCase) and its mutants derived from marine Streptomyces (Streptomyces sp.7-145 (CGMCCNO.7914)) can be used to hydrolyze NC-D-HPG into D-HPG. Summary of the invention

[0006] When we conducted experimental tests on carbamoylase / DCase reported in the prior art, we found that these enzymes either had substrate or product inhibition or poor thermal stability when catalyzing the hydrolysis reaction of substrate NC-D-HPG, and were generally easily inactivated at reaction temperatures above 40°C, making it difficult to maintain enzyme activity for a long time. For example, the enzyme activity of DCase and its mutants disclosed in CN201510457039.0 tended to decrease significantly after the initial concentration of substrate NC-D-HPG reached 5%, and it was difficult to increase the reaction rate accordingly by adding more enzymes to the reaction system, indicating the existence of substrate or product D-HPG inhibition.

[0007] The reaction rate of carbamoylase-catalyzed hydrolysis of substrate NC-D-HPG is generally not very fast, and generally takes a long time. However, the reaction process is easily damaged by oxidation, resulting in the production of by-products, which seriously affects the yield and quality of the product. Therefore, the ideal reaction process is to have a reaction rate as high as possible, such as using a higher reaction temperature, while avoiding high temperature-induced enzyme denaturation and inactivation, and avoiding the presence of oxygen.

[0008] The characteristics of this reaction require that the carbamoylase used has no substrate / product inhibition or very low inhibition, has high thermal stability, is insensitive to oxygen, has the highest possible enzyme activity, and has high stereoselectivity. In order to find an enzyme that meets these requirements, the inventors compared and screened the carbamoylase / Dcase or their isozymes reported in many documents, and finally found that the N-carbamyl-D-amino acid amide hydrolase (akDcase, SEQ ID NO: 1) from Agrobacterium sp. Strain KNK712 reported by Hirokazu NANBA et al. in the document (Hirokazu NANBA, Yasuhiro IKENAKA, et al., Isolation of Agrobacterium sp. Strain KNK712 That Produces N-Carbamyl-D-Amino Acid Amido hydrolase, Cloning of the Gene for this Enzyme, and Properties of the Enzyme. Bioscience, Biotechnology, and Biochemistry, Volume 62, Issue 5, 1 January 1998. DOI: 10.1271 / bbb.62.875.) NO:1) exhibits excellent substrate and product tolerance in catalyzing the hydrolysis of NC-D-HPG, is resistant to oxidative environments, and has high stereoselectivity. Therefore, the inventors focus on studying it as a carbamoylase.

[0009] In order to improve the thermal stability and enzyme activity of the initial carbamoylase akDCase, we carried out structural modification by mutation. After a large number of screenings, we obtained several mutants with excellent comprehensive performance, which not only have good thermal stability but also have almost no substrate / product inhibition, and have the potential to be applied to the enzymatic production of D-p-hydroxyphenylglycine. Specifically, the present invention includes the following technical solutions:

[0010] A carbamoylase mutant, which is a polypeptide selected from the following:

[0011] (a) a polypeptide having an amino acid sequence of SEQ ID NO: 3;

[0012] (b) A polypeptide having an amino acid sequence that is 85% or more, preferably 90% or more, preferably 95% or more, preferably 98% or more, and more preferably 99% or more homologous to SEQ ID NO: 3, and having an enzyme activity that is increased in a reaction environment of 45° C. or more compared to SEQ ID NO: 3.

[0013] MTRQMILAVGQQGPIARAETREQVVVRLLDMLTKAASRGANFIVFPELALTTFFPRWYFTDEAELDSFYETEMPGPVVRPLFEKAAELGIGFNLGYAELVVEGGVKRRFNTSILVDKSGKIVGKYRKIHLPGHKETEAYRDFQHLEKRVFEPGD LGFPVYDVDAAKMGMFICNDRRWPEAWRVMGLRGAEIICGGYNTPTHNPEVPQHDHLTSFHHLLSMQAGSYQNGAWSAAAGKAGMEENCMLLGHSCIVAPTGEIVALTTTLEDEVITAAVDLDRCRELREHIFNFKQHRQPQHYGLIAEL(SEQ ID NO:3).

[0014] Herein, the carbamoylase mutant having an amino acid sequence of SEQ ID NO: 3 is numbered 2-C4, which is a mutant in which the tyrosine (Y) at position 136 in the initial carbamoylase SEQ ID NO: 1 is replaced by threonine (T), the proline (P) at position 141 is replaced by aspartic acid (D), and the tyrosine (Y) at position 149 is replaced by valine (V).

[0015] The above enzyme activity refers to the enzyme activity when catalyzing the hydrolysis of N-carbamyl-D-p-hydroxyphenylglycine (NC-D-HPG) into D-p-hydroxyphenylglycine (D-HPG).

[0016] Another aspect of the present invention also provides a gene encoding the above-mentioned carbamoylase mutant.

[0017] For example, the gene encoding the carbamoylase mutant SEQ ID NO:3 can be a polynucleotide whose nucleotide sequence is as shown in SEQ ID NO:4, or a polynucleotide whose nucleotide sequence has 90% or more, preferably 92% or more, preferably 95% or more, preferably 97% or more, preferably 98% or more, and more preferably 99% or more homology with SEQ ID NO:4.

[0018] The present invention also provides a plasmid comprising the above coding gene. For example, the above plasmid can be a pET vector such as pET22b, pET24a, pET28a, or other commonly used vectors such as pSH plasmid.

[0019] Another aspect of the present invention provides a microorganism for expressing the above-mentioned carbamoylase mutant such as SEQ ID NO: 3, wherein the above-mentioned coding gene such as SEQ ID NO: 4 is integrated into its genome, or a microorganism transformed with the above-mentioned plasmid.

[0020] The transformation of the above plasmid can be carried out by conventional chemical transformation or electroporation into the competent cell. The above gene editing technology is selected from the following groups: homologous double exchange, TALEN system, CRISPR-Cas9 system, CRISPR-Cpf1 system, CRISPR-Cas12 system, CRISPR-BEST system, MuGENT (multiplex genome editing by natural transformation), etc.

[0021] Preferably, the microorganism is a microorganism with a fast growth rate and suitable for expressing exogenous recombinant proteins, for example, selected from Bacillus subtilis, Lactobacillus brevis, Escherichia coli, Candida magnolii, Pichia pastoris, and Saccharomyces cerevisiae. Preferably, the microorganism is Escherichia coli, more preferably Escherichia coli BL21 (DE3).

[0022] The carbamoylase mutant or the microorganism can be used to produce D-p-hydroxyphenylglycine. For example, N-carbamoyl-D-p-hydroxyphenylglycine (NC-D-HPG) is used as a reaction substrate, and the carbamoylase mutant or the microorganism is used to catalyze the hydrolysis reaction to obtain D-p-hydroxyphenylglycine (D-HPG).

[0023] Optionally, the above catalytic reaction is carried out in a buffer solution reaction system containing β-mercaptoethanol.

[0024] In one embodiment, the reaction system is filled with an inert gas such as nitrogen or argon to protect the reaction from the presence of oxygen.

[0025] Preferably, the above buffer solution is a phosphate buffer solution.

[0026] The hydrolysis reaction temperature may be 35-60° C., preferably 38-55° C., preferably 39-52° C., preferably 40-50° C., preferably about 45° C. The reaction pH may be 6.5-8.5, preferably pH 6.8-8.0, more preferably pH 7.0-7.5.

[0027] Optionally, the above carbamoylase mutant or the microorganism expressing the same and D-hydantoinase form a dual enzyme system, which jointly catalyzes the reaction of the substrate DL-p-hydroxyphenylhydantoin to produce D-p-hydroxyphenylglycine (D-HPG).

[0028] When the carbamoylase mutant obtained by mutation screening in the present invention catalyzes the hydrolysis of substrate NC-D-HPG to generate D-HPG, the substrate concentration can reach at least 10wt%, there is almost no substrate / product inhibition, and the mutant has high thermal stability, and can catalyze for a long time in a reaction system above 45°C without inactivation, to obtain a product D-HPG with high optical purity, thereby improving the generation efficiency of D-HPG and showing good industrial development and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a map of the plasmid pET28a-akDCase constructed for expressing the initial type carbamoyl hydrolase akDCase. DETAILED DESCRIPTION

[0030] The carbamoylase and its mutants described in the present invention are enzymes with stereoselectivity, that is, they hydrolyze the D-type substrate N-carbamoyl-D-p-hydroxyphenylglycine into D-p-hydroxyphenylglycine which is still in the D-type, so the carbamoylase (mutant) can also be called D-carbamoylase (mutant).

[0031] The initial D-carbamoylase akDCase from Agrobacterium sp. Strain KNK712 with the amino acid sequence of SEQ ID NO: 1 is the starting point for the improvement of the present invention. The inventors screened out some mutants with significantly improved enzyme activity by mutating the initial D-carbamoylase akDCase.

[0032] MTRQMILAVGQQGPIARAETREQVVVRLLDMLTKAASRGANFIVFPELALTTFFPRWYFTDEAELDSFYETEMPGPVVRPLFEKAAELGIGFNLGYAELVVEGGVKRRFNTSILVDKSGKIVGKYRKIHLPGHKEYEAYRPFQHLEKRYFEPGD LGFPVYDVDAAKMGMFICNDRRWPEAWRVMGLRGAEIICGGYNTPTHNPEVPQHDHLTSFHHLLSMQAGSYQNGAWSAAAGKAGMEENCMLLGHSCIVAPTGEIVALTTTLEDEVITAAVDLDRCRELREHIFNFKQHRQPQHYGLIAEL(SEQ ID NO:1).

[0033] In this article, the terms "initial (type)", "initial enzyme", and "initial type enzyme" have the same meaning, all referring to the initial type of carbamoylase akDCase. Sometimes for the sake of convenience, the initial enzyme and its mutants such as SEQ ID NO: 3 can be collectively referred to as "carbamoylase" or "D-carbamoylase" in this article.

[0034] As used herein, the term "(enzyme activity) increase" or "increase" as used above means an increase of at least 100% compared to a reference level, for example, an increase of at least about 1 times, at least about 2 times, or at least about 3 times, or at least about 5 times, or at least about 10 times, or at least about 20 times compared to a reference level.

[0035] The "mutation" includes but is not limited to replacement, deletion, insertion, chemical modification of amino acid residues, preferably a forward mutation, i.e. a mutation that improves enzyme activity. The substitution can be a non-conservative substitution, a conservative substitution, or a combination of a non-conservative substitution and a conservative substitution. "Conservative" amino acid substitution or mutation refers to the interchangeability of residues with similar side chains, and therefore generally includes replacing the amino acids in the polypeptide with the same or similar amino acid definition categories. However, as used herein, if a conservative mutation can be replaced by aliphatic to aliphatic, non-polar to non-polar, polar to polar, acidic to acidic, basic to basic, aromatic to aromatic, or restricted residue to restricted residue substitution, then a conservative mutation does not include a hydrophilic to hydrophilic, hydrophobic to hydrophobic, hydroxyl-containing to hydroxyl-containing or small residue to small residue substitution. It is well known in the art that common cases of conservative substitutions include: mutual substitution between aromatic amino acids F, W, and Y; mutual substitution between hydrophobic amino acids L, I, and V; mutual substitution between polar amino acids Q and N; mutual substitution between basic amino acids K, R, and H; mutual substitution between acidic amino acids D and E; and mutual substitution between hydroxyl amino acids S and T. In addition, A, V, L, or I can be conservatively mutated to another aliphatic residue or another non-polar residue. Exemplary conservative substitutions can be made, for example, according to the following table, in which amino acids belonging to the same partition in the second column can be substituted for each other, and in preferred cases, amino acids in the same row in the third column can be substituted for each other:

[0036]

[0037] "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 substitutions may use amino acids between, rather than within, the defined groups listed above. In one embodiment, a non-conservative mutation affects (a) the structure of the peptide backbone in the area of ​​the substitution (e.g., proline for glycine), (b) charge or hydrophobicity, or (c) side chain bulk.

[0038] "Deletion" refers to a modification of a polypeptide by removing one or more amino acids from a reference polypeptide. Deletion can include removal of 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids constituting the reference enzyme, while retaining enzymatic activity and / or retaining the improved properties of the engineered aldolase. Deletion can be directed to the interior and / or ends of a polypeptide. In various embodiments, deletion can comprise a continuous segment or can be discontinuous.

[0039] "Insertion" refers to the modification of a polypeptide by adding one or more amino acids from a reference polypeptide. In some embodiments, the improved engineered aldolase enzymes include the insertion of one or more amino acids into naturally occurring aldolase enzymes and the insertion of one or more amino acids into other improved aldolase polypeptides. The insertion can be internal to the polypeptide, or at the carboxyl or amino termini. Insertion as used herein includes 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.

[0040] In order to perform site-directed mutagenesis, a three-dimensional model (modeling) of carbamoylase can be constructed based on bioinformatics technology to infer its enzyme catalytic active center, substrate / product entry and exit channels, rigid / flexible structural regions, etc., select possible structural influencing sites and try saturation mutation.

[0041] For the initial carbamoylase akDCase, we selected 6 key amino acid sites F59, Y136, P141, Y149, H201, and H285 in the surface corner region of the protein structure of SEQ ID NO: 1 for single-point saturation mutation and constructed a single-point mutant library. From the single-point mutant library, mutants with improved enzyme activity and improved thermal stability (for example, the enzyme activity was basically not reduced in 0.1M phosphate buffer pH7.5 at 45°C for 12 hours) were screened, and it was found that the activities of the five mutations F59L, Y136T, P141D, Y149V, and H201T were higher. Based on this, multi-site combined mutations were carried out and a combined mutant library was constructed to screen out mutants with further improved enzyme activity and improved thermal stability (for example, the enzyme activity was basically not reduced within 12 hours in 0.1M phosphate buffer pH 7.5 at 45°C). Among them, it was found that the mutant enzyme SEQ ID NO: 3 had many advantages and also maintained the substrate / product inhibition characteristics and high stereoselectivity of the initial enzyme akDCase, and was suitable for the higher temperature hydrolysis reaction system of the present invention.

[0042] The study found that L-cysteine ​​exists in the active center of the D-carbamoyl hydrolase, and the addition of a reducing agent such as β-mercaptoethanol helps to improve the thermal stability of the structure of the active center of the enzyme and plays a role in protecting the enzyme.

[0043] On the other hand, those skilled in the art can expect that the mutant enzyme SEQ ID NO: 3 can also form a dual enzyme system with the existing D-hydantoinase, and jointly catalyze the reaction of the substrate DL-p-hydroxyphenylhydantoin to produce D-p-hydroxyphenylglycine (D-HPG), thereby realizing the dual enzyme method for preparing D-HPG using DL-p-hydroxyphenylhydantoin as the raw material.

[0044] The amino acid number of the carbamoylase mutant SEQ ID NO: 3 of the present invention is 304, and the structure is clear, so it is easy for those skilled in the art to obtain its encoding gene, expression cassettes and plasmids containing these genes, and transformants containing the plasmids. These genes, expression cassettes, plasmids, and transformants can be obtained by genetic engineering construction methods well known to those skilled in the art.

[0045] In order to express carbamoylase mutant optimally in the most frequently used Escherichia coli in genetic engineering, the expression genes of these enzymes can be codon optimized. Codon optimization is a technology that can be used for maximizing protein expression in organisms by increasing the translation efficiency of gene of interest. Different organisms usually show the special preference for one of some codons of the same amino acid of encoding due to mutation tendency and natural selection. For example, in fast-growing microorganisms such as Escherichia coli, the optimization codon reflects the composition of its genome tRNA library separately. Therefore, in fast-growing microorganisms, low-frequency codons of amino acid can be used for the same amino acid but high-frequency codon displacement. Therefore, the expression of optimized DNA sequence dna is improved in fast-growing microorganisms.

[0046] For example, to express the carbamoylase mutant in E. coli, the gene encoding the codon-optimized original carbamoylase SEQ ID NO: 1 can be SEQ ID NO: 2; and the gene encoding the carbamoylase mutant SEQ ID NO: 3 can be SEQ ID NO: 4.

[0047] The above-mentioned transformant host can be any microorganism suitable for expressing carbamoylase, including bacteria and fungi. Preferred microorganisms are Bacillus subtilis, Corynebacterium glutamicum, Pichia pastoris, Saccharomyces cerevisiae, or Escherichia coli, preferably Escherichia coli, more preferably Escherichia coli BL21 (DE3).

[0048] When used as a biocatalyst for producing D-p-hydroxyphenylglycine, the carbamoylase mutant of the present invention can be in the form of an enzyme or a bacterial cell. The enzyme forms include free enzymes, immobilized enzymes, including purified enzymes, crude enzymes, fermentation broths, enzymes immobilized on carriers, etc.; the bacterial cells include living bacterial cells, dead bacterial cells, immobilized bacterial cells, etc.

[0049] When microorganisms such as Bacillus subtilis, Corynebacterium glutamicum, Pichia pastoris, Saccharomyces cerevisiae or Escherichia coli are no longer fermented and proliferated, but used for enzyme catalysis, they are themselves a natural immobilized enzyme, and do not need to be crushed or even extracted and purified, and can be used as an enzyme preparation for catalytic reactions. Since both the reaction substrate and the reaction product are small molecules, they can easily pass through the biological barrier of the bacteria - the cell membrane, so there is no need to crush the bacteria, which is economically beneficial.

[0050] The present invention is further described in detail below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0051] Example

[0052] This article involves the addition amount, content and concentration of various substances, and the percentages mentioned here refer to the mass percentages unless otherwise specified.

[0053] Materials and methods

[0054] The whole gene synthesis, primer synthesis and sequencing in the examples were all completed by Suzhou Genewise Biotechnology Co., Ltd.

[0055] The molecular biology experiments in the embodiments include plasmid construction, enzyme digestion, ligation, competent cell preparation, transformation, culture medium preparation, etc., and are mainly carried out with reference to Molecular Cloning Experiment Guide (3rd edition), edited by J. Sambrook and DW Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002). If necessary, the specific experimental conditions can be determined by simple experiments.

[0056] PCR amplification experiments were performed according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions. If necessary, adjustments could be made through simple experiments.

[0057] LB medium: 10g / L tryptone, 5g / L yeast extract, 10g / L sodium chloride, pH 7.2. (LB solid medium plus 20g / L agar powder.)

[0058] TB medium: 24g / L yeast extract, 12g / L tryptone, 16.43g / L K2HPO4.3H2O, 2.31g / L KH2PO4, 5g / L glycerol, pH 7.0-7.5. (TB solid medium plus 20g / L agar powder.)

[0059] HPLC detection conditions of substrate N-carbamyl-D-p-hydroxyphenylglycine and product D-p-hydroxyphenylglycine: Agilent 1260, waters Symmetry-C18 (250×4.6 mm, 5 um), mobile phase: 0.05 M sodium acetate-acetic acid (PH 4.2): methanol (90:10), 1.0 ml / min, 254 nm, 20 ul, column temperature 30°C.

[0060] It should be noted that, for the sake of convenience of description, in the embodiments, the strain number, plasmid number, enzyme number, and enzyme encoding gene number may share one number, which is easily understood by those skilled in the art, that is, the same number can refer to different biological forms in different environments.

[0061] Example 1: Construction of initial carbamoylase-expressing recombinant Escherichia coli

[0062] According to the amino acid sequence of D-carbamoylase from Agrobacterium sp. KNK 712 published in the literature (Hirokazu NANBA, Yasuhiro IKENAKA, et al.. Biotechnology, and Biochemistry, Volume 62, Issue 5, 1 January 1998. DOI: 10.1271 / bbb.62.875.), i.e., SEQ ID NO: 1, Escherichia coli-preferred codon optimization was performed, and the coding gene sequence SEQ ID NO: 2 was fully synthesized, and restriction endonuclease sites Nde I and Xho I were designed at both ends of the gene, and subcloned into the corresponding sites of the vector pET28a (Novagen) to obtain the recombinant plasmid pET28a-akDCase, whose structure is shown in 1.

[0063] The recombinant plasmid pET28a-akDCase was transformed into the expression host Escherichia coli BL21 (DE3) competent cells by electroporation to obtain the recombinant Escherichia coli akDCase expressing the initial D-carbamoyl hydrolase.

[0064] Example 2: Establishment and screening of single-site saturation mutation library

[0065] According to the protein structure model of carbamoylase SEQ ID NO: 1, six amino acid sites F59, Y136, P141, Y149, H201, and H285 in the surface corner region of the protein structure were selected for single-point saturation mutation.

[0066] 2.1 Establishment of saturation mutation point library

[0067] Using the initial enzyme coding gene SEQ ID NO: 2 as a template, the degenerate codon primer pairs F59-F / F59-R, Y136-F / Y136-R, P141-F / P141-R, Y149-F / Y149-R, H201-F / H201-R, and H285-F / H285-R were used to amplify the single-site saturation mutant library of 6 sites, F59, Y136, P141, Y149, H201, and H285. The primer sequences are shown in Table 1.

[0068] Table 1. Primer list of saturation mutation library

[0069] Primer Name Sequence (5’→3’) F59-F CTTCTTCCCGCGTTGGTACNNKACCGACGAAGCTGAAC F59-R GTTCAGCTTCGTCGGTMNNGTACCAACGCGGGAAGAAG Y136-F CTGCCGGGTCACAAAGAANNKGAAGCTTACCGTCCGTTC Y136-R GAACGGACGGTAAGCTTCMNNTTCTTTGTGACCCGGCAG P141-F GAATACGAAGCTTACCGTNNKTTCCAGCACCTGGAAAAAC P141-R GTTTTTCCAGGTGCTGGAAMNNACGGTAAGCTTCGTATTC Y149-F CAGCACCTGGAAAAACGTNNKTTCGAACCGGGTGACCTG Y149-R CAGGTCACCCGGTTCGAAMNNACGTTTTTCCAGGTGCTG H201-F GTTACAACACCCCGACCNNKAACCCGGAGGTTCCGCAG H201-R CTGCGGAACCTCCGGGTTMNNGGTCGGGGTGTTGTAAC H285-F GCCGTGAACTGCGTGAANNKATCTTCAACTTCAAACAG H285-R CTGTTTGAAGTTGAAGATMNNTTCACGCAGTTCACGGC

[0070] Where M=A / C, K=G / T, N=A / G / C / T.

[0071] The 50 μL PCR reaction system includes: 10 ng plasmid template, 10 pmol primer pair, 1xKOD plus buffer, 0.2 mM dNTP, 1.5 mM MgSO4, and 5 units of KOD-plus DNA polymerase.

[0072] PCR reaction conditions were: 95°C for 3 min; 98°C for 10 s, 57°C for 30 s, 68°C for 2 min / kbp; 30 cycles; 68°C for 10 min.

[0073] 1 μL DpnI was added to the amplified PCR product for digestion for 1 hour, and then electroporated into E. coli BL21 (DE3) competent cells to obtain more than 10 3 A random mutation library of 10 clones.

[0074] 2.2 High-throughput screening of mutant libraries

[0075] 160 transformants were randomly selected from each single-point saturation mutation library and inoculated into 500 μL of LB liquid medium containing 50 μg / mL kanamycin in a 96-well deep-well culture plate. The culture was then cultured overnight. Then 80 μl of the overnight culture was transferred to 800 μl of LB liquid medium containing 50 μg / mL kanamycin. After culturing at 37°C for 3 hours, IPTG was added at a final concentration of 0.5 mM, cooled to 25°C, and cultured overnight. The overnight culture liquid was centrifuged at 4000 rpm for 15 minutes, the supernatant was discarded, and the culture was placed in a -70°C freezer for 1 hour and thawed at room temperature for 30 minutes. Add 200 μL of 0.1 M potassium phosphate buffer (pH 8.0) containing 1 mg / ml lysozyme, 10 mg / ml DNase and 0.1% (v / v) β-mercaptoethanol, resuspend the bacteria, incubate at 37°C for 1 h, centrifuge at 5000 rpm at 4°C for 20 min, take 20 μl of the supernatant for enzyme activity determination of the unheat-treated sample, and take another 20 μl and incubate at 65°C for 30 min for enzyme activity determination.

[0076] The screening reactions of the samples obtained by the above two treatment methods were carried out respectively, and the reaction system was as follows: 180 μL of substrate reaction solution (20 mM N-carbamyl-D-hydroxyphenylglycine (NC-D-HPG), 1 mM ethylenediaminetetraacetic acid (EDTA), 0.1% (v / v) β-mercaptoethanol, 0.01% phenol red, pH 5.8-6.0) was added to 20 μL of the above different treatment solutions, and the reaction was carried out at 45°C for 1-3 hours, and the absorbance change at 558 nm was detected.

[0077] Definition of enzyme activity: The amount of enzyme required to catalyze the hydrolysis of substrate to produce 1 micromole (μmol) of ammonia per minute at 45°C is defined as 1 unit (U).

[0078] The mutant clones of the six single-point saturation mutant libraries were screened, and strains with significantly enhanced enzyme activities in both the unheat-treated and heat-treated samples were selected, plasmids were extracted, and Suzhou Jinweizhi Company was commissioned to perform nucleic acid sequencing, and the carbamoylase-related fragments in the genome were compared with SEQ ID NO: 1 to determine the changes in the amino acid sequence of carbamoylase. The screening found that the akDCase-F59L, akDCase-Y136T, akDCase-P141D, akDCase-Y149V, and akDCase-H201T mutant strains had significantly enhanced enzyme activities relative to the starting akDCase unheat-treated and heat-treated samples, and the specific results are shown in Table 2.

[0079] Table 2. Relative activity results of mutant bacteria

[0080]

[0081]

[0082] Example 3: Combinatorial mutation library establishment and screening

[0083] 3.1 Establishment of combinatorial mutation library

[0084] The plasmid pET28a-akDCase containing the initial enzyme encoding gene SEQ ID NO: 2 was used as a template, and two primer pairs F59-F0 / Y136-P141-R0 and Y149-F0 / H201-R0 were used for amplification to amplify two fragments A and B, and then the A and B fragments were used as templates, and F59-F0 / H201-R0 was used as a primer pair for amplification to obtain a large fragment AB, and finally AB was used as a primer and pET28a-akDCase was used as a template for mega-primer PCR amplification to obtain a combined mutation library of 5 sites. Among them, the four primers F59-F0, Y136-P141-R0, Y149-F0, and H201-R0 were obtained by mixing two different primers in equal proportions, and the primer mixing preparation table is shown in Table 3 below.

[0085] Table 3. Primer list of combinatorial mutation library

[0086]

[0087] Among them, S=G / C, K=G / T.

[0088] The 50 μl PCR reaction system includes: 10 ng plasmid template, 40 pmol primer pair, 1xKOD plus buffer, 0.2 mM dNTP, 1.5 mM MgSO4, and 5 units of KOD-plus DNA polymerase.

[0089] The PCR reaction conditions were: 95°C for 1 min, 98°C for 10 s, 57°C for 30 s, 68°C for 1 min / kbp, 30 cycles, and 68°C for 10 min. Two fragments A and B were recovered from the gel. The size of fragment A was 288 bp, and the size of fragment B was 198 bp.

[0090] The second round of PCR was performed using fragments A and B as templates and primers F59-F0 and H201-R0 to obtain fragment AB, 466 bp in size, which was recovered by gel excision;

[0091] PCR reaction conditions were: 95°C for 3 min; 98°C for 10 s, 60°C for 30 s, 68°C for 1 min / kbp; 25 cycles; 68°C for 10 min.

[0092] MegaPrimer PCR was performed using fragment AB as the large primer, pET28a-akDCase as the template, and KOD-plus DNA polymerase: 94°C for 5 min, 98°C for 30 s, 60°C for 30 s, 68°C for 2 min / kbp, 25 cycles; 68°C for 10 min.

[0093] 1 μL DpnI was added to the amplified PCR product for digestion for 1 hour, and then electroporated into E. coli BL21 (DE3) competent cells to obtain more than 10 3 A random mutation library of 10 clones.

[0094] 3.2 High-throughput screening of mutant libraries

[0095] The method is the same as step 2.2 in Example 2. By screening 160 mutant clones, a strain 2-C4 was screened out. Compared with the starting strain akDCase, its enzyme activity under non-heat treatment and heat treatment conditions was significantly enhanced, that is, the enzyme activity and thermal stability were significantly improved. Through gene sequencing and comparison, it was determined that the 136th amino acid in the amino acid sequence of the carbamoylase of the strain was mutated from tyrosine to threonine, the 141st amino acid was mutated from proline to aspartic acid, and the 149th amino acid was mutated from tyrosine to valine.

[0096] Example 4: Comparison of bacterial strain catalytic production of D-p-hydroxyphenylglycine

[0097] 4.1 Bacterial fermentation

[0098] Single colonies were picked from the LB culture plates of akDCase and 2-C4, respectively, and inoculated into 5 mL of LB liquid medium containing 50 μg / mL kanamycin sulfate, and cultured at 37°C, 250 rpm overnight. 2 mL of overnight culture was inoculated into 200 mL of TB medium, and cultured at 37°C, 250 rpm for 2-3 h until OD 600 When the concentration of the cell was 0.6-0.8, 0.1 mM IPTG was added and cultured overnight at 28°C and 200 rpm. Then, the cells were collected by centrifugation at 4°C and 10,000 rpm for 10 min, and then resuspended in 0.1 M phosphate buffer (pH 7.5) to a bacterial concentration of 10%, and then ultrasonically disrupted (ultrasonic disruption conditions: 55% energy, 2 s of operation, 5 s of rest, and 20 min of operation).

[0099] 4.2 Enzyme activity determination

[0100] Prepare 0.1 M phosphate buffer pH 7.5 containing 3.5% substrate N-carbamyl-D-p-hydroxyphenylglycine and 0.1% v / v β-mercaptoethanol, add the bacterial ultrasonic disruption mixture at a final concentration of 1.5 v / v%, and react in a shaker at 40°C and 170 rpm for 30 min. Then take out 0.5 ml of the reaction mixture, add 0.5 ml of 10% hydrochloric acid to terminate the reaction, and filter and detect the substrate and product contents by HPLC.

[0101] 4.3 Thermal stability determination

[0102] The ultrasonic bacterial cell disruption mixture with a 10 wt% bacterial concentration was kept at 65°C for 30 min, and then the treated disrupted bacterial suspension was taken for activity determination, and the determination process was the same as step 4.2.

[0103] Compared with the initial strain akDCase (initial enzyme SEQ ID NO: 1), the enzyme activity of the D-carbamoylase mutant 2-C4 (SEQ ID NO: 3) of the mutant strain screened by the present invention is increased by 3.56 times, and the enzyme activity after heat treatment at 65° C. is increased by 8.12 times.

[0104] Example 5: Application of mutants in producing D-p-hydroxyphenylglycine

[0105] A 0.1M phosphate buffer solution containing 10% substrate N-carbamoyl-D-p-hydroxyphenylglycine and 0.1% v / v β-mercaptoethanol was prepared at pH 7.5, and a bacterial ultrasonic crushing mixture with a final concentration of 3.5 v / v% was added, and nitrogen protection was introduced throughout the process. The pH was controlled at 7.0-7.5 with 3M phosphoric acid and 2M sodium hydroxide solution, and the reaction was carried out at 45°C for 20-24 hours. The reaction sample was detected by HPLC, and the results showed that after 20 hours of reaction, the generation rate of D-p-hydroxyphenylglycine in the reaction system exceeded 97%.

[0106] The experimental results show that compared with the initial D-carbamoylase akDCase, the D-carbamoylase mutant SEQ ID NO:3 constructed in the present invention has significantly improved enzyme activity and thermal stability in catalyzing the reaction of N-carbamoyl-D-p-hydroxyphenylglycine to generate D-p-hydroxyphenylglycine, can catalyze for a long time in a reaction system above 45° C. without inactivation, has no substrate / product inhibition in the reaction, and the substrate concentration can reach at least 10wt% or more, and has potential for industrial development and application.

Claims

1. A carbamoylase mutant, whose amino acid sequence is SEQ ID NO:

3.

2. A gene encoding the carbamoylase mutant according to claim 1.

3. The gene according to claim 2, characterized in that The nucleotide sequence of the gene is SEQ ID NO:

4.

4. A plasmid comprising the gene as claimed in claim 3.

5. A microorganism expressing the carbamoylase mutant according to claim 1, characterized in that: The gene according to claim 3 is integrated into the genome of the microorganism, or the microorganism is a microorganism transformed with the plasmid according to claim 4.

6. The microorganism according to claim 5, characterized in that The microorganism is Escherichia coli.

7. Use of the carbamoylase mutant according to claim 1 or the microorganism according to claim 6 in producing D-p-hydroxyphenylglycine.

8. The use according to claim 7, characterized in that In a reaction system containing β-mercaptoethanol, N-carbamoyl-D-p-hydroxyphenylglycine is used as a reaction substrate, and a hydrolysis reaction is catalyzed by the carbamoylase mutant as claimed in claim 1 or the microorganism as claimed in claim 6 to obtain D-p-hydroxyphenylglycine.

9. The use according to claim 8, characterized in that The carbamoylase mutant according to claim 1 or the microorganism according to claim 6 and D-hydantoinase form a dual enzyme system, which jointly catalyzes the substrate DL-p-hydroxyphenylhydantoin to produce D-p-hydroxyphenylglycine.

Images