D-carbamoylase mutant, gene, expression vector and application thereof

Abstract

The invention discloses a D-carbamoylase mutant, a gene, an expression vector and an application thereof, and belongs to the field of genetic engineering technology. The invention improves the catalytic activity of D-carbamoylase by a protein engineering method, and the catalytic efficiency of the mutant is at least 2.6 times higher than that of the wild type, thereby reducing the amount of enzyme added. In the cascade reaction catalysis of L-indolemethylhydantoin, the yield of more than 90% can still be achieved when the amount of enzyme added is low, which is much higher than 79.3% of the wild type, and has good industrial application prospects.

Description

[0001] This application is a divisional application with application number: 202010382460.0, application date: May 8, 2020, and application name: D-carbamoylase mutants and their application in the synthesis of D-aromatic amino acids. Technical Field

[0002] The invention relates to a D-carbamoylase mutant, a gene, an expression vector and application thereof, and belongs to the technical field of genetic engineering. Background Art

[0003] As a non-natural amino acid, D-amino acid is often used to synthesize various pharmaceutical intermediates. For example, D-p-hydroxyphenylglycine is often used to synthesize the precursor of cephalosporin (Syldatk C., Advances in Biochemical Engineering Biotechnology, 1990, 29–75); D-tryptophan (D-Trp) can be used to synthesize octreotide and tadalafil Cialis, which are important drugs for the treatment of acromegaly and erectile dysfunction; D-Trp can also be used to synthesize peptide drugs for the treatment of dermatitis, including Tyrocidines C (D-Phe-Pro-Trp-D-Trp-Asn-Gln-Tyr-Val-Orn-Leu) and Thymodepressin (γ-D-Glu-D-Trp), etc. (Martínez-Rodríguez et al., Chem. Biodivers, 2010, 7, 1531–1548). At the same time, it can also be used as a non-nutritive sweetener and is very popular in the food industry.

[0004] DN-carbamoylase is the sixth class of the nitrilase superfamily. It can hydrolyze DN-carbamoylamino acids to obtain D-amino acids. It is often used to form a cascade reaction with hydantoin racemase and hydantoinase to prepare optically pure D-amino acids.

[0005] In recent years, the synthesis research of D-aromatic amino acids is mainly D-tryptophan and D-p-hydroxyphenylglycine. For example, in 1998, Yamamoto et al. used DN-amidase to selectively hydrolyze D-tryptophan amide and then obtained D-Trp by chemical hydrolysis (EP0853128A1). Similar to the amidase method, in 1957, Greenstein et al. used L-aminoacylase to prepare D-Trp, in which only the L-enantiomer in N-acetyl-DL-tryptophan amide was hydrolyzed, and the remaining enantiomer could be chemically deacetylated to form D-Trp (Greenstein, JP, Methods In Enzymology, 1957, 3: 554–570). In 1995, Yamamoto H et al. reported that D-tryptophanase can degrade L-Trp from DL-Trp racemate to produce D-Trp, but the byproducts of the reaction, pyruvate and indole, can inhibit the activity of tryptophanase and thus need to be removed (Kawasaki et al. Bioscience Biotechnology and Biochemistry, 1995, 59: 1938–1943). The methods reported so far all have the disadvantages of a theoretical yield of 50%, a complicated process, low yield and enantioselectivity. Although in 1949, Eadie G et al. reported that optically pure D-Trp can be prepared by a method in which D-amino acid transaminase reacts the substrate indolepyruvate and D-alanine, the method has a theoretical yield of 100%, but the activity is low, and the final yield is only 13% (Eadie Get al. Journal of Biological Chemistry, 1949, 181: 449–458). The hydantoinase process is a three-enzyme cascade reaction consisting of hydantoin racemase, hydantoinase and carbamoylase. It has been considered as an efficient and economical method for preparing optically pure D-amino acids because it can break the traditional 50% conversion rate limit and thus has a higher yield.In my previous research, I have screened a D-carbamoylase from Arthrobacter crystallinum, patent number (201711097767.0), which can catalyze the almost complete conversion of 300mM L-indolemethylhydantoin to the corresponding D-tryptophan. Although the enzyme can achieve the conversion of 300mM substrate in the cascade reaction, a large amount of enzyme (50kU / L AtHyuA, 25kU / L AtHyuH, 50kU / L AcHyuC) needs to be added. In addition, it was found in the previous study that the enzyme activity of D-carbamoylase is significantly lower than that of the first two steps. When the substrate concentration is further increased, a large amount of D-carbamoylase needs to be further added to complete the conversion, which makes this process lose its original economy. Therefore, in the previous study, this research group obtained a D-carbamoylase from the Indian Ocean nitrate-reducing bacterium Nitratireductor indicusC115 through gene mining, named NiHyuC, which can achieve the conversion of 1M The complete conversion of L-indolemethylhydantoin, but also has the problem of low D-carbamoyl hydrolase and high enzyme load (50kU / L AaHyuA, 25kU / L AtHyuH, 50kU / LNiHyuC). Summary of the invention

[0006] To solve the above problems, the present invention improves the catalytic activity of D-carbamoylase by protein engineering, thereby reducing the amount of enzyme added, which is crucial for the efficient preparation of D-amino acids using a hydantoinase process.

[0007] The first object of the present invention is to provide a D-carbamoylase mutant, wherein the D-carbamoylase mutant is a D-carbamoylase with the amino acid sequence as shown in SEQ ID NO.1, wherein the 200th alanine is mutated to serine (A200S), or the 200th alanine is mutated to asparagine (A200N), or the 200th alanine is mutated to glutamic acid (A200E), or the 200th alanine is mutated to histidine (A200H). Specifically, the amino acid sequence shown in SEQ ID NO.1 is as follows:

[0008] MTRRIRIGGAQMGAISRSDSKKEIVDRLIALLRQASEKGCELVVFPELALSTFFPRWYAERDGMDGYFEDGMPNAATLPLFEEARRLGIGFSLGYAELVQEDGRVRRFNTTVLVERNGEIVGKYRKIHLPGHAEYEPERSHQHLEKRYFEVGNT GFQVWDAFGGRVGMAICNDRRWVETYRVMGLQDVELILIGYNTPVADSLSGESETLRMFHNHLTMQAGAYQNSTWVVGVAKAGVEDGHRLMGGSVIVAPTGEIVAQAMTEGDELIVADCDLDRCRYYKSHIFNFAAHRRPEFYQRITSQTGVE.

[0009] The second object of the present invention is to provide a gene encoding the D-carbamoylase mutant.

[0010] The third object of the present invention is to provide an expression vector carrying the gene encoding the D-carbamoylase mutant.

[0011] The fourth object of the present invention is to provide a cell expressing the D-carbamoylase mutant.

[0012] Furthermore, the cell is a bacterium, a fungus, a plant cell or an animal cell.

[0013] Furthermore, when the host cell is a bacterium, the method for constructing the recombinant bacterium is as follows: the nucleic acid molecule encoding the D-carbamoylase mutant is cloned into a recombinant vector, and the obtained recombinant vector is transformed into the host bacterium to obtain the recombinant bacterium.

[0014] Furthermore, the host of the recombinant bacteria is Escherichia coli, and the plasmid is pET28a(+).

[0015] Furthermore, the host of the recombinant bacteria is E. coli BL21 (DE3).

[0016] The fifth object of the present invention is to provide a method for producing D-carbamoylase by the recombinant bacteria, which comprises the following steps: inoculating the recombinant bacteria into LB medium containing 40-60 μg / mL kanamycin sulfate, culturing at 30-40°C and 100-200 rpm on a shaking table, and determining the absorbance OD of the culture solution. 600 When the concentration reaches 0.5-1.0, add 0.05-1.0 mM IPTG for induction at a temperature of 16-30°C. After induction for 5-12 hours, recombinant D-carbamoylase can be obtained.

[0017] The sixth object of the present invention is to provide the use of the D-carbamoylase mutant in the preparation of optically pure D-amino acids.

[0018] Furthermore, the application is to use the D-carbamoylase mutant as a catalyst to catalyze the substrate to produce D-amino acids, and the substrate is DL-N-carbamoyltryptophan, DL-N-carbamoylphenylalanine and DL-N-carbamoylphenylglycine, DL-N-carbamoylmethionine, DL-N-carbamoyl-tryptophan, DL-N-carbamoyl-o-chlorophenylglycine, DL-N-carbamoylleucine or DL-N-carbamoylisoleucine.

[0019] Furthermore, the application specifically includes the following steps: constructing a reaction system, the L-indolemethyl concentration is 10mM to 1M, the D-carbamoylase mutant dosage is 1 to 10kU / L, and the concentration of phosphate buffer is 0.05 to 0.15M; reacting at 30 to 35°C and pH 6 to 8 for 1 to 24 hours.

[0020] Beneficial effects of the present invention:

[0021] The D-carbamoylase mutant of the present invention has high activity for various N-carbamoyl amino acids, and can catalyze various aliphatic or aromatic substituted amino acid substrates, especially DN-carbamoyl amino acid substrates with large steric hindrance. The catalytic efficiency of the D-carbamoylase mutant of the present invention is at least 2.6 times higher than that of the wild type. In the cascade reaction catalysis of L-indolemethylhydantoin, the yield of more than 90% can still be achieved under the condition of low enzyme addition amount, which is much higher than 79.3% of the wild type. It can be seen that the D-carbamoylase mutant obtained by the present invention is particularly suitable for preparing optically pure D-amino acids by hydantoin enzyme process cascade reaction, and has good industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the nucleic acid electrophoresis diagram of the whole plasmid PCR of D-carbamoylase mutant;

[0023] Figure 2 The chiral liquid chromatogram of DN-carbamyltryptophan catalyzed by the D-carbamoylase mutant;

[0024] Figure 3 This is the chiral liquid chromatogram of DN-carbamoyl-p-hydroxyphenylalanine catalyzed by the D-carbamoylase mutant. DETAILED DESCRIPTION

[0025] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0026] Determine the enzyme activity of DN-carbamoylase on the substrate DL-N-carbamoyltryptophan. The assay system is: appropriate amount of enzyme solution, 10mmol·L -1 DL-N-carbamyltryptophan. Let the reaction stand at 30°C for 10 minutes. After the reaction is completed, take a sample for liquid phase detection. Liquid phase detection conditions: the chromatographic column is Diamonsil Plus C18 (25cm×4.6mm, 5μm), the mobile phase is acetonitrile: potassium dihydrogen phosphate (25:75), and the flow rate is 0.2~1mL·min -1 , the detection wavelength is 210nm.

[0027] Definition of enzyme activity unit (U):

[0028] At 30°C, the amount of enzyme required for DN-carbamoylase to catalyze the substrate DL-N-carbamoyltryptophan to produce 1 μmol of D-tryptophan is defined as one enzyme activity unit (U).

[0029] Example 1: Construction of D-carbamoylase mutant gene and recombinant expression transformant

[0030] The whole plasmid PCR method was used to perform site-directed saturation mutagenesis on the amino acid residues of Ala200. The primers designed are shown in Table 1 (all described in the 5'-3' direction), and the underline represents the mutation site.

[0031] Table 1 Site-directed saturation mutagenesis primer design

[0032]

[0033] The PCR reaction system was as follows: the PCR reaction system (50 μL) included KOD enzyme (2.5 U / mL) 1.0 μL, template (5-50 ng) 1.0 μL, dNTP 4.0 μL, 10× reaction buffer 5.0 μL, upstream and downstream primers 1.0 μL each, and ddH2O was added to 50 μL.

[0034] The PCR amplification procedure was as follows: (1) denaturation at 94°C for 3 min, (2) denaturation at 94°C for 30 sec, (3) annealing at 54°C for 30 sec, and (4) extension at 72°C for 150 sec. Steps (2) to (4) were repeated for 10-15 cycles, and finally extension was performed at 72°C for 10 min. The PCR amplification product was stored at 4°C.

[0035] After PCR, add DpnI restriction endonuclease to the reaction mixture and incubate at 37°C for 1 h. Use CaCl2 thermal conversion method to transfer 10 μL of digested PCR reaction solution into 50 μL E. coli BL21 (DE3) competent cells and evenly spread on LB agar plates containing 50 μg / ml kanamycin sulfate and inverted culture at 37°C for 12 h.

[0036] Example 2: Expression and purification of D-carbamoylase and its mutants

[0037] The recombinant E. coli containing the mutant plasmid was inoculated into LB medium containing kanamycin sulfate (50 μg / mL) at a transfer volume of 2%, and cultured in a shaking incubator at 37°C and 200 rpm. The absorbance of the culture solution was OD 600 When the p-value reached 0.8, 0.2 mM IPTG was added for induction at 25°C. After 12 h of induction, the cells were centrifuged at 8000 rpm for 5 min to obtain the cells efficiently expressing the recombinant D-carbamoylase mutant. The collected cells were suspended in Tris-HCl buffer (100 mM, pH 8.0) and ultrasonically disrupted.

[0038] The column used for purification is a nickel affinity column HisTrap HP 5mL, and affinity chromatography is performed using the histidine tag on the recombinant protein. First, the nickel column is balanced with A liquid, the crude enzyme solution is loaded, and the penetration peak is eluted using A liquid (25mM Tris, 500mM NaCl, 20mM imidazole, pH 7.4). After equilibration, the recombinant protein bound to the nickel column is eluted with B liquid (25mM Tris, 500mM NaCl, 500mM imidazole, pH 7.4) for gradient elution to obtain a recombinant D-carbamoylase mutant. The purified protein is subjected to activity assay and SDS-PAGE analysis. After nickel column purification, a single band is displayed at about 38kDa, and there are fewer foreign proteins, indicating that the column purification effect is good. The purified D-carbamoylase protein was then replaced with Tris-HCl (100 mM, pH 8.0) buffer using a His Trap Desalting column (GE Healthcare).

[0039] Example 3: Analysis of kinetic parameters and cascade reaction conversion effects of D-carbamoylase mutants

[0040] The kinetic parameters of NiHyuC and its mutants for the substrate DL-N-carbamyl-tryptophan were determined. The kinetic parameter determination system is listed as follows: Tris-HCl buffer (100mmol·L -1 , pH 8.0), DL-N-carbamyl-tryptophan (0~20mmol·L -1). The reaction rate is characterized by calculating the specific enzyme activity, thereby calculating the kinetic parameters.

[0041] Since there are few literature reports on the modification of carbamoylase activity and there are no mutation sites for reference, this patent uses EasyModeller to perform homology modeling using D-carbamoylase from Agrobacterium radiobacter (PDB number: 1fo6) as a template, and then verifies and evaluates the model.

[0042] After obtaining the protein structure, molecular docking was used to dock the substrate DN-carbamyltryptophan with the model protein structure, and then the substrate was selected. The saturation mutation library was constructed and screened for sites within the range. The mutation sites involved included Ala200. The superior mutants obtained in the initial screening were screened in shake flasks, and the mutants with improved activity in the rescreening were purified and the kinetic parameters were determined. The results are shown in Table 2. Four mutants, A200E, A200N, A200S, and A200H, were screened. The kinetic parameters of each mutant were: A200E, its k cat / K m 96.4min -1 mM -1 , is WT(k cat / K m 25.7min -1 mM -1 ) is 3.8 times that of A200N; its k cat / K m 88.0min -1 mM -1 , which is 3.4 times that of WT; A200S, its k cat / K m 109min -1 mM -1 , which is 4.2 times of WT; A200H, its k cat / K m 67.7min -1 mM -1 , which is 2.6 times of WT. cat / K m The single point mutants that were improved compared to WT were verified by cascade reaction to catalyze the conversion of 200mM L-indolemethylhydantoin. The enzyme addition amounts of the three enzymes were 15kU / L AaHyuA, 20kU / L AtHyuH, and 5kU / LNiHyuC, and the product yields were 97.8%, 92.7%, 94.2%, and 85.6%, respectively, of which the yield of WT was 79.3%.

[0043] Table 2D- Kinetic parameters of carbamoylase single-point mutants and validation of catalytic 200mM substrate cascade reaction

[0044]

[0045] Example 4: Time course of D-carbamoylase mutants used in cascade reaction to produce D-tryptophan

[0046] 5 kU / L DN-carbamoylase mutant, 15 kU / L hydantoin racemase (AaHyuA), and 20 kU / L D-hydantoinase (AtHyuH) were mixed in Tris-HCl buffer (pH 6-8, 100 mmol / L -1 ), add 20% PEG400, 500mmol·L -1 L-indolemethylhydantoin, the total volume of the reaction solution is 10 mL. The reaction is placed at 30°C, and sampling is performed to detect the conversion process. The conditions are as follows: Diamonsil Plus C18 column (25 cm × 4.6 mm, 5 μm), the detection wavelength is 210 nm, the mobile phase is acetonitrile: potassium dihydrogen phosphate (10-30: 90-70), and the flow rate is 0.5-1 mL / min.

[0047] Example 5: Time course of application of D-carbamoylase mutants in cascade reaction to prepare D-p-hydroxyphenylalanine

[0048] 10 kU / L DN-carbamoylase mutant, 15 kU / L hydantoin racemase (AaHyuA), and 20 kU / L D-hydantoinase (AtHyuH) were mixed in Tris-HCl buffer (pH 6-8, 100 mmol / L -1 ), add 5-10% PEG400, 500mmol·L -1 L-p-hydroxyphenylhydantoin, the total volume of the reaction solution is 10 mL. The reaction is placed at 30°C, and sampling is performed to detect the conversion process. The conditions are as follows: Diamonsil Plus C18 column (25 cm × 4.6 mm, 5 μm), the detection wavelength is 210 nm, the mobile phase is acetonitrile: potassium dihydrogen phosphate (10-30: 90-70), and the flow rate is 0.5-1 mL / min.

[0049] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A D-carbamoylase mutant, characterized in that: The D-carbamoylase mutant is obtained by mutating the 200th position alanine of the D-carbamoylase shown in the amino acid sequence of SEQ ID NO.1 to serine, glutamic acid, asparagine or histidine.

2. A gene encoding the D-carbamoylase mutant of claim 1.

3. An expression vector carrying the gene according to claim 2.

4. A cell expressing the D-carbamoylase mutant of claim 1, characterized in that: The cells are bacteria or fungi.

5. The cell according to claim 4, characterized in that: The bacteria is Escherichia coli.

6. The cell according to claim 5, characterized in that: The Escherichia coli includes E. coli BL21(DE3).

7. The cell according to claim 5, characterized in that: The bacteria use pET28a(+) as an expression vector.

8. Use of the D-carbamoylase mutant according to claim 1 in the preparation of optically pure D-amino acids.

9. The use according to claim 8, characterized in that: The application is to use the D-carbamoylase mutant as a catalyst to catalyze the substrate to generate D-amino acid.

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