CN114507650B

CN114507650B - Leucine dehydrogenase mutant and application thereof in synthesis of (S) -o-chlorophenylglycine

Info

Publication number: CN114507650B
Application number: CN202210103919.8A
Authority: CN
Inventors: 王亚军; 谢为邦; 程峰; 郑裕国
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2022-01-28
Filing date: 2022-01-28
Publication date: 2023-09-05
Anticipated expiration: 2042-01-28
Also published as: CN114507650A

Abstract

The invention discloses a leucine dehydrogenase mutant and application thereof in synthesizing (S) -o-chlorophenylglycine, wherein the leucine dehydrogenase mutant is obtained by site-directed saturation mutation of 362 th site of an amino acid sequence shown in SEQ ID NO. 2. The activity of the leucine dehydrogenase mutant EsLeuDH-F362L constructed by the invention is improved by 32 times compared with that of the wild leucine dehydrogenase, wherein the feeding amount of o-chlorobenzoic acid serving as a substrate of the mutant EsLeuDH-F362L can reach 500mM, the concentration of a product is gradually increased along with the time, the reaction can be completed within 4.0h, the substrate conversion rate is more than 99%, and the e.e value of the product is always kept above 99.5%. Therefore, the leucyl dehydrogenase mutant EsLeuDH-F362L has industrial application prospect.

Description

Leucine dehydrogenase mutant and application thereof in synthesis of (S) -o-chlorophenylglycine

Field of the art

The invention relates to a mutant of leucine dehydrogenase EsLeuDH from Microbacterium sibiricum, recombinant bacteria for developing the leucine dehydrogenase mutant and application of the enzyme in clopidogrel chiral intermediate (S) -o-chlorophenylglycine catalytic synthesis.

(II) background art

(S) -Clopidogrel (Clopidogrel), a thienopyridine drug developed by Sanofi, inc. of French in 1986, is known as methyl (S) - (+) -2- (2-chloro) -2 (4, 5,6, 7-tetrahydrothiophene [3,2-c ] naphthyridine-5) acetate. In 1998, the U.S. FDA approved clopidogrel for sale under the trade name "Bolivin" (Plavix). Clopidogrel is also currently one of the most marketed drugs worldwide, with annual sales of up to $64 billion.

(S) -O-chlorophenyl glycine, also known as (S) -2-amino- (2-chlorophenyl) acetic acid, is a biologically active unnatural chiral amino acid, one of the most important uses of which is for the synthesis of clopidogrel. The synthesis method of (S) -o-chlorophenylglycine mainly comprises chemical synthesis and enzymatic synthesis. The chemical resolution method is the main method for producing (S) -o-chlorophenylglycine industrially at present, but the method still has the defects of low theoretical yield, poor optical purity of the product and the like. Compared with a chemical resolution method, the enzymatic method has the advantages of high catalytic efficiency, mild condition, simple process, good selectivity, environmental friendliness and the like. Therefore, the synthesis of (S) -o-chlorophenylglycine by using the biological enzyme method has a relatively broad prospect.

Thanks to technological advances in protein engineering, biocatalysis has been widely used in industrial production. According to the invention, a mutation library is constructed through semi-rational design, and the optimal mutant EsLeuDH-F362L with the strongest catalytic performance is further screened and obtained, so that the molecular mechanism of the improvement of the catalytic performance of the mutant is further analyzed, the reaction process parameters are optimized, and the mutant is applied to the chemical-enzymatic catalytic synthesis of clopidogrel chiral intermediate (S) -o-chlorophenylglycine.

(III) summary of the invention

Aiming at the problems of low asymmetric amination activity of the existing leucine dehydrogenase on o-chlorobenzoic acid and low substrate feeding amount, the invention provides a stereoselective leucine dehydrogenase mutant and application of the leucine dehydrogenase mutant gene recombinant bacteria in synthesizing (S) -o-chlorobenzoic acid, the activity of a catalyst under the condition of pH9.0 is improved by 32 times compared with the activity of a wild leucine dehydrogenase EsLeuDH under the condition of pH7.0, the substrate feeding amount is improved to 500mM, the obtained mutant is also applied to construction of a route for synthesizing (S) -o-chlorobenzoic acid by a chemical-enzymatic method, and a novel synthesis method and a novel biocatalyst are provided for chiral intermediate (S) -o-chlorobenzoic acid of (S) -clopidogrel.

The technical scheme adopted by the invention is as follows:

the invention provides a leucine dehydrogenase EsLeuDH mutant, which is obtained by site-directed saturation mutation of 362 th site of an amino acid sequence shown in SEQ ID NO. 2. The amino acid sequence shown in SEQ ID NO.2 is derived from Microbacterium sibiricum (Exiguobacterium sibiricum), and the corresponding nucleotide sequence of the coding gene is shown in SEQ ID NO. 1. Preferably, the leucine dehydrogenase EsLeuDH mutant is obtained by mutating phenylalanine at position 362 of the amino acid sequence shown in SEQ ID NO.2 into leucine (F362L).

The invention also relates to a coding gene of the leucine dehydrogenase EsLeuDH mutant, a recombinant vector containing the coding gene and a genetic engineering bacterium containing the recombinant vector, wherein the recombinant vector uses pET28a (+) as a basic vector, and the genetic engineering bacterium uses E.coli BL21 (DE 3) as a host bacterium.

The invention also provides an application of the leucine dehydrogenase EsLeuDH mutant in the preparation of (S) -o-chlorobenzoic acid by asymmetric catalytic amination, and the application method comprises the following steps: mixing wet thalli obtained by induction culture of leucine dehydrogenase EsLeuDH mutant genetically engineered bacteria and wet thalli obtained by induction culture of glucose dehydrogenase genetically engineered bacteria, taking the mixed thalli as a catalyst, o-chlorobenzoic acid as a substrate, glucose as an auxiliary substrate, ammonium sulfate as an amino donor, and adding NAD ⁺ The conversion system is formed by taking buffer solution with pH of 7-10 (preferably Tris-HCl buffer solution with pH of 9.0 and 100 mM) as a reaction medium, and the reaction is carried out at 30-50 ℃ and 500-800rpm (preferably 40 ℃ and 600 rpm) to obtain (S) -o-chlorophenylglycine after the reaction is finished and the reaction solution is separated and purified.

Further, in the conversion system, the final concentration of the substrate is 20-500mM (preferably 100 mM), the final concentration of glucose is 24-600mM (preferably 120 mM), the final concentration of ammonium sulfate is 30-750mM (preferably 150 mM), and NAD is calculated ⁺ The final concentration is 0.2-5mM (preferably 1 mM), and the catalyst is used in an amount of 1-10g DCW/L (DCW cell dry weight) based on the total dry weight of the mixed cells, preferably 3g DCW/L; the wet thalli obtained by the induction culture of the leucine dehydrogenase EsLeuDH mutant genetic engineering bacteria in the mixed thalli and the wet thalli obtained by the induction culture of the glucose dehydrogenase genetic engineering bacteria are mixed at a dry weight ratio of 1-5:1 (w/w), preferably 2:1. The glucose dehydrogenase gene is derived from bacillus megatherium (Bacillus megaterium) IWG3, the nucleotide sequence of the glucose dehydrogenase gene is shown as SEQ ID NO.3, and the amino acid sequence of the glucose dehydrogenase gene is shown as SEQ ID NO. 4.

Further, the wet cell is prepared as follows: inoculating a leucine dehydrogenase EsLeuDH mutant genetically engineered bacterium into an LB liquid culture medium containing 50 mug/mL kanamycin at a final concentration, culturing at 37 ℃ for 10 hours, inoculating the strain into a fresh LB liquid culture medium containing 50 mug/mL kanamycin at a final concentration of 1.5% by volume, culturing at 37 ℃ at 180rpm for 2 hours, adding Isopropyl thiogalactoside (IPTG) with a final concentration of 0.10mM into the culture solution, culturing at 25 ℃ for 12 hours, and centrifuging at 4 ℃ at 8000rpm for 10 minutes to obtain a leucine dehydrogenase mutant wet bacterial strain; the preparation method of wet bacteria obtained by induced culture of the glucose dehydrogenase genetically engineered bacteria is the same as that of leucine dehydrogenase mutant wet bacteria.

The invention also provides an application of the leucine dehydrogenase mutant in synthesizing (S) -o-chlorophenylglycine by a chemical-enzymatic method, wherein the application method comprises the following steps: (1) Using acetophenone as substrate, selenium dioxide as oxidant, anhydrous pyridine as solvent, and adding the mixture into N ₂ After refluxing at 100 ℃ for 15 hours under protection, the solvent was removed under reduced pressure, and the reaction solution was adjusted to ph=12 with 2M aqueous sodium hydroxide solution and stirred for 0.5 hour; extracting with water for 2 times, and adjusting pH to=2 with 40% hydrochloric acid aqueous solution; extracting with ethyl acetate for 4 times, mixing organic phases, drying with anhydrous sodium sulfate, and removing solvent under reduced pressure to obtain o-chlorobenzoyl formic acid; (2) Mixing wet thalli obtained by induction culture of engineering bacteria containing leucine enzyme mutant genes and wet thalli obtained by induction culture of engineering bacteria containing glucose dehydrogenase genes, taking the mixed thalli as a catalyst, taking o-chlorobenzoyl formic acid prepared in the step (1) as a substrate, taking glucose as an auxiliary substrate, and adding NAD ⁺ The method comprises the steps of forming a conversion system by taking ammonium sulfate as an amino donor and taking buffer solution (preferably Tris-HCl buffer solution with pH of 9.0 and 100 mM) with pH of 7-10 as a reaction medium, carrying out reaction at 30-50 ℃ and 500-800rpm (preferably 40 ℃ and 600 rpm), and separating and purifying reaction liquid after the reaction is finished to obtain the (S) -o-chlorophenylglycine.

The ratio of the acetophenone to the selenium dioxide feeding material in the step (1) is 1:2; the volume of the anhydrous pyridine was 1.25mL/mmol based on the amount of acetophenone species.

In the conversion system according to step (2), the final concentration of substrate is 20-500mM (preferred100 mM), glucose final concentration 24-600mM (preferably 120 mM), NAD ⁺ The final concentration is 0.2-5mM (preferably 1 mM), the final concentration of ammonium sulfate is 30-750mM (preferably 150 mM), the catalyst dosage is 1-10g DCW/L (preferably 3g DCW/L) based on the total dry weight of the mixed bacteria, and the dry weight ratio of wet bacteria obtained by induction culture of the engineering bacteria containing leucine dehydrogenase mutant genes in the mixed bacteria to wet bacteria obtained by induction culture of the engineering bacteria containing glucose dehydrogenase genes is 1-5:1 (preferably 2:1).

The leucine dehydrogenase mutant pure enzyme of the invention is prepared by the following method: (1) crude enzyme solution: the wet bacterial strain of the leucine dehydrogenase EsLeuDH mutant gene engineering bacteria subjected to induction culture is suspended in a Tris-HCl buffer solution with the pH value of 9.0 and the concentration of 100mM, and is subjected to ultrasonic crushing on an ice-water mixture for 10min under the condition of ultrasonic crushing: the power is 400W, crushing is carried out for 1s, suspension is carried out for 1s, and the crushed mixed solution is taken to obtain crude enzyme solution; (2) pure enzyme: centrifuging the crushed mixture at 8000rpm at 4deg.C for 10min, and collecting supernatant; after the supernatant was subjected to microfiltration through a 0.45 μm membrane, the filtrate was purified at 4℃using a Ni affinity column (1.6X10 cm, bio-Rad Co., USA) as a loading solution, and the procedure was as follows: (1) preequilibration with buffer A (pH 9.0 containing 0.3M NaCl, 20mM Tris-HCl buffer); (2) loading the sample liquid at the speed of 0.5mL/min, wherein the loading amount is 10mL; unbound impurities were washed away with buffer B (pH 9.0 containing 0.3M NaCl, 20mM imidazole, 20mM Tris-HCl buffer) at a flow rate of 1.0mL/min, eluting at 10 column volumes until conductivity was stable; (3) then, the target protein was eluted with buffer C (pH 8.0 containing 0.3M NaCl, 500mM imidazole, 20mM Tris-HCl buffer) at an elution rate of 1mL/min, an elution amount of 10 column volumes, and collection was started when the UV absorbance reached 0.1 and started to rise, and started to fall after the absorbance reached about 1.0, and stopped when the absorbance was again reduced to 0.1; (4) the collected eluate was dialyzed overnight against 20mM Tris-HCl buffer (pH 9.0) using a dialysis bag (MD 34, viskase, USA) to obtain a retentate as pure enzyme.

The full length of the base sequences of the leucine dehydrogenase EsLeuDH and the leucine dehydrogenase EsLeuDH mutant of the invention is 1125bp, the starting codon is ATG and the stopping codon is TGA from the first base to 1125 bases.

The invention relates to a preparation method of leucine dehydrogenase EsLeuDH mutant, which adopts error-prone PCR technology and site-directed saturation mutation technology, uses the technology to mutate EsLeuDH leucine dehydrogenase gene (SEQ ID NO. 1), transfers the obtained mutated plasmid into E.coli BL21 (DE 3) competent cells in a heat shock mode, inoculates, transfers, induces and recovers the obtained strain, and utilizes a resuspension bacterial liquid to catalyze o-chlorobenzoic acid to carry out asymmetric amination to prepare optical pure (S) -o-chlorobenzoic acid, and the specific method is as follows: in the first step, the control bacteria were activated to obtain control bacteria EsLeuDH, and plasmid pET28a (+) -esLeudh was extracted and stored at-20 ℃. The second step is based on the amino acid sequence of EsLeuDH wild enzyme as template, and Homology modeling is carried out on Protein Homology/analogy Recognition Engine V2.0 (Phyre 2) and molecular docking is carried out. Then selecting proper mutation site, wherein the selected site is mainly amino acid residue on the hinge structure of leucine dehydrogenase. Designing a mutant primer, taking pET28a (+) -EsLeuDH as a template plasmid, obtaining a mutant plasmid through error-prone PCR and site-directed saturation mutation, transforming, screening dominant mutant bacteria to obtain a leucine dehydrogenase mutant EsLeuDH-F362L, and carrying out sample sequencing and preservation on the dominant mutant.

The leucine dehydrogenase mutant and glucose dehydrogenase genetically engineered bacteria of the invention can be inoculated, transferred, induced and recovered, and the culture medium can be any culture medium which can enable the bacteria to grow and generate the invention, preferably LB culture medium: 10g/L tryptone, 5g/L yeast extract, 10g/L NaCl, and distilled water to adjust the pH to 7.0. The culture method and the culture conditions are not particularly limited, and may be appropriately selected according to the general knowledge in the art depending on the type of host, the culture method and the like.

Compared with the prior art, the invention has the main beneficial effects that: compared with the wild leucine dehydrogenase, the leucine dehydrogenase mutant EsLeuDH-F362L constructed by the invention has 2.2 times of the enzyme activity, wherein the feeding amount of o-chlorobenzoyl formic acid of a substrate of the mutant EsLeuDH-F362L can reach 500mM, the concentration of the product gradually increases along with the time, the reaction can be completed within 4.0h, the substrate conversion rate is more than 99%, and the e.e value of the product is always kept above 99.5%. The invention adopts a chemical-enzymatic method to prepare o-chlorophenylglycine precursor ketone by a chemical method from simple raw materials, and then utilizes a leucine dehydrogenase mutant to perform asymmetric amination to synthesize (S) -o-chlorophenylglycine. Compared with full chemical synthesis, the method has the advantages of mild reaction conditions, high catalytic rate, high specificity and high e.e value of the product. Therefore, the leucyl dehydrogenase mutant EsLeuDH-F362L has industrial application prospect.

(IV) description of the drawings

FIG. 1 is a schematic illustration of the reaction of leucine dehydrogenase and glucose dehydrogenase BmGDH coupled to catalyze asymmetric amination of o-chlorobenzoic acid to produce (S) -o-chlorobenzoic acid.

FIG. 2 is a SDS-PAGE electrophoresis of leucine dehydrogenase and its mutant pure enzyme. Lane 1: control EsLeuDH pure enzyme; lane 2: esLeuDH-F362L pure enzyme; m: standard protein molecules.

FIG. 3 is a time course diagram for the preparation of (S) -o-chlorobenzoic acid using leucine dehydrogenase EsLeuDH coupled with BmGDH in example 6; a is a substrate, and the feeding amount of o-chlorobenzoyl formic acid is 100mM; b is the substrate, and the feeding amount of o-chlorobenzoyl formic acid is 500mM.

FIG. 4 is a graph of the time course of preparation of (S) -o-chlorobenzoic acid from asymmetric amination of o-chlorobenzoic acid using leucine dehydrogenase EsLeuDH-F362L coupled BmGDH in example 7; a is a substrate, and the feeding amount of o-chlorobenzoyl formic acid is 100mM; b is the substrate, and the feeding amount of o-chlorobenzoyl formic acid is 500mM.

FIG. 5 is a mass spectrum of crude o-chlorobenzoic acid prepared in example 8.

FIG. 6 is a nuclear magnetic resonance hydrogen spectrum of crude o-chlorobenzoic acid prepared in example 8.

(fifth) detailed description of the invention

The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:

in the embodiment of the invention, E.coli BL21 (DE 3)/pET 28a (+) -esleudh is constructed according to the article (Journal of Molecular Catalysis B: enzymic 2014 (105) 11-17), wherein the amino acid sequence of leucine dehydrogenase esleudh derived from micro bacillus sibirica (Exiguobacterium sibiricum) is shown as SEQ ID NO.2, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 1.

Example 1: construction and screening of leucine dehydrogenase random mutation library

A random mutation library is constructed by using a vector pET28a (+) -esleudh (leucine dehydrogenase esleudh amino acid sequence is shown as SEQ ID NO.2, encoding gene nucleotide sequence is shown as SEQ ID NO. 1) in an original strain E.coli BL21 (DE 3)/pET 28a (+) -esleudh as a template, and using epPCR-F and epPCR-R in Table 1 as primers and using an error-prone PCR kit (purchased from Tianzenze company, namely, an error-prone PCR kit).

EsLeuDH nucleotide sequence:

atggtggaaacgaacgttgaggcgcgcttctcgattttcgagacgatggcgatggaagactacgaacaggtggttttttgccatgacaaagtgagcgggctgaaagcaattattgcgattcatgataccaccctgggtccggcactgggtggcctgagaatgtggaattatgcaagcgatgaagaagcattaattgatgcattacgcctggcaaaaggtatgacctacaaaaacgcagcagcaggcctgaatttaggtggtggtaaagcggttataatcggggacgccaaaacccagaaaagcgaagcactgtttagagcatttggtcgttacgttcagagcctgaacggaagatatattacagcagaggatgttaatacaacagttgcagatatggattatatccatatggaaacagatttcgttaccggtgtgagtccggcatttggcagttcaggaaatccgagcccagtcaccgcctatggggtttatcgcggcatgaaagcagcagcaaaggaggtttatggtaccgatagcctggggggtaaaacggttgcaattcagggtgttggtaatgttgcctttaatttatgccgtcatctgcatgaggaaggtgcaaaactgatagtaacagatattaaccaggatgcactgcgtcgtgcagaagaagcatttggtgccctggttgttggaccggatgagatctatagcgttgatgccgatatatttgcgccgtgtgctctgggagcaaccctgaatgacgaaaccattcctcaactgaaagtgaaaattatagcaggtgcagcaaataaccagctgaaagaagatcgtcatggtgatatgctgcaggaacgtggaattctgtatacccctgactttgtgattaacgcaggtggtgtgattaatgttgcggatgaattagatggctataaccgtgaacgggcaatgaaaaaagttgaactggtttatgatgcagttgcaaaagtgattgaaattgcgaaaagagatcatctgccgacctaccgtgcagcagaaaaaatggcagaagaaagaattgcaacaatgggtagcgcccggagccagttcttaagacgtgataaaaatattctgggcagtcggggataa。

EsLeuDH amino acid sequence:

MVETNVEARFSIFETMAMEDYEQVVFCHDKVSGLKAIIAIHDTTLGPALGGLRMWNYASDEEALIDALRLAKGMTYKNAAAGLNLGGGKAVIIGDAKTQKSEALFRAFGRYVQSLNGRYITAEDVNTTVADMDYIHMETDFVTGVSPAFGSSGNPSPVTAYGVYRGMKAAAKEVYGTDSLGGKTVAIQGVGNVAFNLCRHLHEEGAKLIVTDINQDALRRAEEAFGALVVGPDEIYSVDADIFAPCALGATLNDETIPQLKVKIIAGAANNQLKEDRHGDMLQERGILYTPDFVINAGGVINVADELDGYNRERAMKKVELVYDAVAKVIEIAKRDHLPTYRAAEKMAEERIATMGSARSQFLRRDKNILGSRG。

error-prone PCR reaction System (30. Mu.L): 1. Mu.L of forward primer (100. Mu.M), 1. Mu.L of reverse primer (100. Mu.M), 3. Mu.L of 10X error-prone PCR Mix, 3. Mu.L of 10X error-prone PCR-specific Dntp, 1. Mu.L of plasmid template, 1.5. Mu.L of error-prone PCR-specific MnCl ₂ 0.5. Mu.L error-prone PCR-specific Taq DNA polymerase and 19. Mu.L ultrapure water. The PCR procedure was as follows: pre-denaturation at 95℃for 5min, followed by 30 cycles (denaturation at 95℃for 15s, annealing at 55℃for 15s, extension at 72℃for 7 s), final extension at 72℃for 10min, incubation at 16 ℃. And (3) detecting the PCR product by agarose gel electrophoresis, and recovering the PCR product by using a PCR gel recovery kit to obtain the gene DNA fragment required by the experiment. The gene DNA fragment and vector pET28a (+) empty plasmid are subjected to double digestion and digestion by NcoI and XhoI, then are connected, are transformed into E.coli BL21 (DE 3) competent cells, are coated on LB plate containing 50 mug/mug kanamycin (Kana) and are cultured overnight at 37 ℃ for 12 hours, and the product is obtained to be more than 8 multiplied by 10 ³ Random mutation library of individual clones.

9600 single clones from the random mutation library were selected and inoculated into 100 96-well plates. 1mL of LB medium containing 50. Mu.g/mL kanamycin was added to each well, and the mixture was cultured at 37℃for 10 hours at 180rpm to obtain a seed solution. 50. Mu.L of each seed solution was transferred to a new 96-well plate, 1mL of LB medium containing 50. Mu.g/mL kanamycin was added to each well, and after shaking culture at 37℃and 180rpm for 4 hours, IPTG was added to each well at a final concentration of 0.15mM, and the wells were transferred to 28℃for 16 hours. Each 96-well plate was centrifuged at 4000rpm at 4℃for 10min by a 96-well plate centrifuge, the supernatant was discarded, and 200. Mu.L of Tris-HCl solution (0.1 mM, pH=9.0) was added to each well to suspend the cells. Lysozyme (. Gtoreq.20000U/mg) was prepared with Tris-HCl solution (0.1 mM, pH=9.0) to a final concentration of 0.3mg/mL lysozyme solution, and then 100. Mu.L of lysozyme solution was added to each well of the 96-well plate and reacted at 37℃for 1 hour. After the thalli are fully cracked, the thalli are centrifuged for 10min at the temperature of 4 ℃ by a 96-well plate centrifuge at 4000rpm, and the supernatant is taken to obtain cell lysate. A clean 96-well plate was prepared, 150. Mu.L of a reaction solution (0.8 mM NADH,0.1mM Tris-HCl buffer, pH9.0, 8mM o-chlorobenzoic acid and 16mM ammonium sulfate) was added to each well, and 50. Mu.L of the above-obtained cell lysate was added thereto to react at 40℃and 180rpm for 1 minute, and the absorbance of the system at 340nm at the end of the reaction was measured by an enzyme-labeled instrument.

The strain E.coli BL21 (DE 3)/pET 28a (+) -esleudh is used as a control group, and the strain with activity higher than that of the control group is screened (namely, the change of absorbance value before and after 1min of reaction is larger than that of the control strain). The dominant strain obtained was sent to Hangzhou qing biotechnology limited for sequencing and stored in a-80℃refrigerator. Through the construction and screening of the random mutant, the leucine dehydrogenase mutant with the activity improved by 12 times of that of 1 strain is obtained; the other 9000 mutants showed an increase in activity of 2-fold or more, and were therefore discarded. Sequencing shows that the base T at the 1084 position of the mutant is changed into C, the corresponding phenylalanine at the 362 th position is changed into leucine, and the mutant is named EsLeuDH-F362L.

TABLE 1 design of leucine dehydrogenase random mutagenesis primer

Example 2: inducible expression of wild-type leucine dehydrogenase, mutant and glucose dehydrogenase

Glucose dehydrogenase genetically engineered bacteria: according to a glucose dehydrogenase gene (Genbank accession number: 1 RBB_A) from bacillus megatherium (Bacillus megaterium) IWG3, artificially synthesizing a glucose dehydrogenase bmgdh gene (the nucleotide sequence is shown as SEQ ID NO.3, the amino acid sequence of the encoded protein is shown as SEQ ID NO. 4), inserting the gene between two cleavage sites of Nco I and Xho I of pET28b (+) to construct a recombinant expression vector, and transferring the expression vector into E.coli BL21 (DE 3) to prepare E.coli BL21 (DE 3)/pET 28b (+) -bmgdh.

The bmgdh nucleotide sequence:

ATGTACAAGGACCTTGAGGGAAAGGTCGTCGTCATTACTGGATCTTCTACTGGACTGGGAAA

GTCTATGGCTATTCGATTCGCTACTGAGAAGGCTAAGGTCGTCGTGAACTACCGATCTAAGGA

GGACGAGGCTAACTCTGTCCTTGAGGAGATTAAGAAGGTCGGAGGAGAGGCTATTGCTGTC

AAGGGTGACGTCACTGTCGAGTCTGACGTCATTAACCTGGTCCAGTCTGCTATTAAGGAGTT

CGGAAAGCTGGACGTCATGATTAACAACGCTGGACTTGAGAACCCTGTGTCCTCTCACGAG

ATGTCTCTGTCTGACTGGAACAAGGTCATTGACACTAACCTGACTGGTGCTTTCCTGGGATC

TCGAGAGGCTATTAAGTACTTCGTCGAGAACGACATTAAGGGAACTGTCATTAACATGTCCT

CTGTCCACGAGAAGATTCCTTGGCCTCTGTTCGTCCACTACGCTGCTTCTAAGGGTGGAATG

AAGCTGATGACTAAGACTCTGGCTCTTGAGTACGCTCCTAAGGGTATTCGAGTCAACAACAT

TGGACCTGGTGCTATTAACACTCCTATTAACGCTGAGAAGTTCGCTGACCCTGAGCAGCGAG

CTGACGTCGAGTCTATGATTCCTATGGGTTACATTGGAGAGCCTGAGGAGATTGCTGCTGTCG

CTGCTTGGCTGGCTTCTTCTGAGGCTTCTTACGTCACTGGAATTACTCTGTTCGCTGACGGTGGAATGACTCTTTACCCTTCGTTCCAGGCTGGACGAGGATAG.

the bmgdh amino acid sequence:

MYKDLEGKVVVITGSSTGLGKSMAIRFATEKAKVVVNYRSKEDEANSVLEEIKKVGGEAIAVK

GDVTVESDVINLVQSAIKEFGKLDVMINNAGLENPVSSHEMSLSDWNKVIDTNLTGAFLGSREA

IKYFVENDIKGTVINMSSVHEKIPWPLFVHYAASKGGMKLMTKTLALEYAPKGIRVNNIGPGAIN

TPINAEKFADPEQRADVESMIPMGYIGEPEEIAAVAAWLASSEASYVTGITLFADGGMTLYPSFQAGRG.

the starting strain E.coli BL21 (DE 3)/pET 28a (+) -esleudh of example 1 and the leucine dehydrogenase mutant strain selected in example 1 were inoculated into LB liquid medium containing kanamycin at a final concentration of 50. Mu.g/mL, respectively, cultured at 37℃for 10 hours, the culture broth was inoculated into fresh LB liquid medium containing kanamycin at a final concentration of 50. Mu.g/mL at an inoculum size of 1.5% (v/v), cultured at 37℃for 2 hours at 180rpm, IPTG at a final concentration of 0.1mM was added to the culture broth, cultured at 28℃for 12 hours, centrifuged at 4℃for 10 minutes at 8000rpm, and washed twice with saline at 0.9% (w/v), to obtain the corresponding wet cell. The obtained cells produce corresponding proteins, and can be used for preparing protein pure enzyme liquid and preparing (S) -o-chlorobenzoic acid by catalyzing asymmetric reduction of o-chlorobenzoic acid by crude enzyme liquid.

Example 3: enzyme activity assay for wild-type leucine dehydrogenase

1. Enzyme activity assay

The starting strain E.coli BL21 (DE 3)/pET 28a (+) -esleudh wet cell and E.coli BL21 (DE 3)/pET 28b (+) -bmgdh wet cell of the induced expression of example 2 were mixed at a dry weight ratio of 2:1 (w/w) to give a starting mixed cell.

The enzyme activity unit (U) is defined as: the amount of enzyme required per minute to produce 1. Mu. Mole of (S) -o-chlorophenylglycine at 40℃and pH9.0 was defined as one enzyme activity unit, U. Specific enzyme activity is defined as the number of units of activity, U/mg, per milligram of enzyme protein.

The enzyme activity determination method comprises the following steps: the mixed thallus is taken as a catalyst, o-chlorobenzoyl formic acid is taken as a substrate, glucose is taken as an auxiliary substrate, and a small amount of exogenous NAD is added ⁺ A coenzyme circulation system was established using a 100mM Tris-HCl buffer solution at pH9.0 as a reaction medium. The reaction system is 1mL, the catalyst dosage is 3g/L based on the total dry weight of the mixed bacteria, the final concentration of the substrate is 20mM, the final concentration of glucose is 24mM, the final concentration of ammonium sulfate is 30mM, and NAD is calculated ⁺ The final concentration was 0.2mM, pH9.0, 100mM Tris-HCl buffer was used as a reaction medium to construct a transformation system, and the reaction was stopped with 6.0M HCl at 40℃and 600rpm for 10min. The sample is diluted by 20 times by centrifugation (12,000 rpm, 1 min) and ultrapure water, and after passing through a microfiltration membrane of 0.22 mu m, the filtrate is taken to detect the peak area of o-chlorobenzoic acid and (S) -o-chlorobenzoic acid and glycine by HPLC, and the content of the o-chlorobenzoic acid and the (S) -o-chlorobenzoic acid is obtained according to a corresponding standard curve.

O-chlorobenzoic acid standard curve equation: y=28.054x+0.8675 (R ² ＝0.999)。

(S) -o-chlorophenylglycine standard curve equation: y=44.89x+4.1074 (R ² ＝0.999)

Enzyme activity calculation formula:

wherein, [ S ], concentration of product (S) -o-chlorophenylglycine, μmol/mL; v, total reaction volume, mL; t, reaction time, min; [E] concentration of enzyme protein, mg/mL.

HPLC detection conditions: chromatographic columnCrownpack CR (+) (0.4X115 cm,5 μm), 10mM perchloric acid aqueous solution (pH=2.0) as mobile phase, 0.6mL/min flow rate, 220nm ultraviolet detection wavelength, 10. Mu.L sample injection amount, and 25℃column temperature. The retention time of the elution peak of the o-chlorobenzoic acid and the (S) -o-chlorobenzoic glycine is 15min and 10min respectively.

2. Enzyme activity determination of starting strain

According to the enzyme activity measuring method in the step 1, the enzyme activity of the original strain E.coli BL21 (DE 3)/pET 28a (+) -esleudh wet bacterial strain is measured under the conditions of 40 ℃ and pH7.0 PB buffer, and the enzyme activity is 8U/mg.

Example 4: construction and screening of leucine dehydrogenase site-directed saturation mutation library

1. Site-directed saturation mutation of leucine dehydrogenase

The preparation of the leucine dehydrogenase mutant library is realized through 1 round of site-directed saturation mutation, the primer design is shown in table 2, the carrier pET28a (+) -EsLeuDH in E.coli BL21 (DE 3)/pET 28a (+) -EsLeuDH is used as a template, phe362-F and Phe362-R in table 2 are used as primers, site-directed saturation mutation PCR reaction is carried out, and the 362 th phenylalanine of the amino acid sequence of the leucine dehydrogenase EsLeuDH shown in SEQ ID No.2 is mutated into the rest 19 amino acids.

TABLE 2 Leu dehydrogenase site-directed saturation mutagenesis primer design

Note that: n in Table 2 represents A, T, G, C; s represents G, C.

PCR reaction System (50. Mu.L): 1. Mu.L of forward primer (100. Mu.M), 1. Mu.L of reverse primer (100. Mu.M), 25. Mu.L of 2 XPhanta buffer, 1. Mu.L of dNTP mix (10 mM each), 1. Mu.L of plasmid template, 1. Mu.L of LDNA polymerase and 21. Mu.L of ultrapure water. The PCR procedure set up according to the Phanta Super-Fidelity DNA polymerase manual was as follows: pre-denaturation at 95℃for 5min, followed by 30 cycles (denaturation at 95℃for 15s, annealing at 55℃for 15s, extension at 72℃for 7 s), final extension at 72℃for 10min, incubation at 16 ℃.

The recombinant plasmid obtained by the PCR reaction was transferred into competent cells of E.coli BL21 (DE 3) and the clones were cultured at 37℃for 12h. Positive clones were then picked up and transferred to 10mL of LB liquid medium containing 50. Mu.g/mL kanamycin, cultured at 37℃for 10 hours at 180rpm, and the culture broth centrifuged at 8000rpm for 10 minutes to collect the wet mutant cells.

2. Screening of dominant mutants

The mutant wet bacterial cells obtained in example 1 and E.coli BL21 (DE 3)/pET 28b (+) -bmgdh wet bacterial cells prepared by the method of example 2 were mixed at a dry weight ratio of 2:1 (w/w) to be used as mutant mixed bacterial cells for dominant mutant screening, and screening conditions were as follows: the mutant mixed cells were resuspended in Tris-HCl (100 mM) pH9.0 at a dry weight of 3g/L, followed by addition of 20mM o-chlorobenzoyl acid, 24mM glucose, 30mM ammonium sulfate, 0.2mM NAD ⁺ 1mL of the conversion system was constituted, and the reaction was stopped with 6.0M HCl at 40℃and 600rpm for 10 minutes. Sampling the content of o-chlorobenzoic acid and (S) -o-chlorobenzoic acid and glycine were measured by the method of example 3, dominant mutants were selected using the relative activity and e.e. of (S) -o-chlorobenzoic acid as indicators, and the experimental results are shown in Table 3. The results showed that the mutant EsLeuDH-F362L had a 10.4-fold increase in enzyme activity compared to the wild-type EsLeuDH at pH 9.0. The dominant strain obtained was sent to Hangzhou qing biotechnology limited for sequencing and stored in a-80℃refrigerator.

Under the same conditions, the starting mixed bacterial strain E.coli BL21 (DE 3)/pET 28a (+) -esleudh is used for replacing wet bacterial strain of the mutant bacterial strain to prepare the starting mixed bacterial strain.

e.e. the formula:

wherein C is _S-CPG And C _R-CPG Represents the molar concentration of the products (S) -o-chlorophenylglycine and (R) -o-chlorophenylglycine, mol/L.

TABLE 3 catalytic Properties and stereoselectivity of EsLeuDH and mutants thereof

Example 5: leucine dehydrogenase female parent and purification and relative enzyme activity of mutant F362L thereof

1. Pure enzyme

The optimal mutant obtained in example 4 (EsLeuDH-F362L in Table 3) was expressed by induction according to the method described in example 2 to obtain wet cells of leucine dehydrogenase mutant. 2g of wet cells were resuspended in 20mL of Tris-HCl buffer, pH9.0, 100mM, and sonicated on an ice-water mixture for 6min at a total amount of 100g/L wet cells under sonication conditions: the power was 400W, the mixture was crushed for 1s and suspended for 1s, 15ml of a crude enzyme solution of the mutant strain was obtained, and the mixture was centrifuged at 8000rpm and 4℃for 10 minutes, and the supernatant was collected, and after microfiltration through a 0.45 μm membrane, 10ml of a filtrate was collected as a sample solution, and the mutant protein was purified at 4℃using a Ni affinity column (1.6X10 cm, bio-Rad Co., USA) as follows: (1) the pre-equilibration was performed with buffer A (pH 9.0 containing 0.3M NaCl, 20mM Tris-HCl buffer). (2) Loading the sample liquid at the speed of 0.5mL/min, wherein the loading amount is 10mL; unbound impurities were washed away with buffer B (pH 9.0 containing 0.3M NaCl, 20mM imidazole, 20mM Tris-HCl buffer) at a flow rate of 1.0mL/min, eluting at 10 column volumes, until conductivity was stable. (3) Then, the target protein was eluted with buffer C (pH 9.0 containing 0.3M NaCl, 500mM imidazole, 20mM Tris-HCl buffer) at an elution rate of 1mL/min and an elution amount of 10 column volumes, and the collection was started when the UV absorbance was 0.1 and started to rise, and stopped when the absorbance was reduced to 0.1. (4) Dialyzing the eluent collected in the step (3) with a Tris-HCl buffer solution with pH of 9.0 and 20mM overnight by using a dialysis bag (MD 34, viskase, USA), taking 5mL of the retentate as pure enzyme, and measuring the protein concentration to be 2.7mg/mL by using a biquinolinecarboxylic acid protein assay kit (Nanj Kaiki Biotechnology development Co., nanji).

Under the same conditions, pure enzyme of an original strain E.coli BL21 (DE 3)/pET 28a (+) -esleudh is prepared, and the protein concentration is 2.5mg/mL.

The protein size was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the electrophoresis results are shown in FIG. 2, and the target enzyme expression level of the mutant strain was not significantly changed from that of the E.coli BL21 (DE 3)/pET 28a (+) -esleudh strain, so that the improvement of the enzyme activity of the mutant was not caused by the increase of the enzyme expression level, and was related to the increase of the specific activity of the enzyme itself.

2. Relative enzyme activity

Standard conditions for enzyme activity detection: 10mM o-chlorobenzoic acid, 15mM (NH) ₄ ) ₂ SO ₄ The reaction was carried out at 1mM NADH, 50mg/L of enzyme solution (based on protein content) in a Tris-HCl buffer solution (20 mM Tris-HCl) at pH9.0 and at 600rpm for 10min at 40℃to examine the content of o-chlorobenzoic acid, (S) -o-chlorobenzoylglycine by the method of example 3, and the relative enzyme activities and ee values of the products were calculated, and the results are shown in Table 4.

TABLE 4 relative enzyme activities of leucine dehydrogenase and mutant pure enzymes and e.e. values of the products thereof

Example 6: asymmetric aminated o-chlorobenzoyl formate of wild leucine dehydrogenase EsLeuDH

1. 100mM o-chlorobenzoic acid

Wild-type leucine dehydrogenase EsLeuDH and glucose dehydrogenase BmGDH wet cells were prepared according to the method of example 2, and the wild-type leucine dehydrogenase EsLeuDH wet cells and glucose dehydrogenase BmGDH wet cells were mixed at a dry weight ratio of 2:1 (w/w) to give mixed cells.

In a 10mL conversion system, firstly, the mixed thalli is resuspended by PB buffer solution with pH of 7.0 and 100mM, 3g DCW/L dry weight is added into the conversion system, when the feeding amount of the substrate o-chlorobenzoyl formic acid is 100mM, the glucose concentration is 120mM, the ammonium sulfate concentration is 150mM, and the NAD is added ⁺ At a concentration of 1mM, PB buffer solution with pH of 7.0 and 100mM is used as a reaction medium to form a conversion system, the reaction is carried out at 40 ℃ and 600rpm, the reaction progress is shown as A in figure 3, the product (S) -o-chlorophenylglycine can be basically converted into 40h (the conversion rate is 95%), the substrate conversion rate is more than 99%, and the e.e value of the product is always kept above 99.5%.

2. 500mM o-chlorobenzoic acid

The mixed thalli starting in the step 1 is adopted, in a 10mL conversion system, the starting mixed thalli is firstly resuspended by PB buffer solution with the pH of 7.0 and 100mM, the dry weight of the mixed thalli in the conversion system is added to be 6g DCW/L, the feeding amount of the substrate o-chlorobenzoyl formic acid is 500mM, the glucose concentration is 600mM, the ammonium sulfate concentration is 750mM, the NAD+ concentration is 5mM, the PB buffer solution with the pH of 7.0 and 100mM is used as a reaction medium to construct a conversion system 10mL, the reaction is carried out at 40 ℃ and 600rpm, the reaction process is shown as the B in the figure 3, the conversion rate is only 61% after 40h, and the product e.e value is more than 99.5%.

Example 7: asymmetric reduction of o-chlorobenzoyl formate by amino acid dehydrogenase mutant EsLeuDH-F362L

1. 100mM o-chlorobenzoic acid

Leucine dehydrogenase mutant EsLeuDH-F362L wet cells and glucose dehydrogenase BmGDH wet cells were prepared according to the method of example 2, and the wet cells EsLeuDH-F362L and glucose dehydrogenase BmGDH wet cells were mixed at a dry weight ratio of 2:1 (w/w) to give mutant mixed cells.

In a 10mL conversion system, firstly, the mutant mixed thalli is resuspended by Tris-HCl buffer solution with pH of 9.0 and 100mM, 3g DCW/L dry weight of mixed thalli is added into the conversion system, the feeding amount of substrate o-chlorobenzoyl formic acid is 100mM, the glucose concentration is 120mM, the ammonium sulfate concentration is 150mM, and the NAD is added into the conversion system ⁺ At a concentration of 1mM, a transformation system of 10mL was constructed with 100mM Tris-HCl buffer solution at pH9.0 as a reaction medium, and the reaction was carried out at 40℃and 600rpm, the progress of the reaction being shown in FIG. 4A,the substrate is completely converted into the product (S) -o-chlorophenylglycine in 4h, and the product e.e. value is>99.5％。

2. 500mM o-chlorobenzoic acid

The mutation mixed thalli of the step 1 is adopted, firstly, the mixed thalli is resuspended by using Tris-HCl buffer solution with the pH value of 9.0 and 100mM in a 10mL conversion system, the dry weight of the mixed thalli in the conversion system is added to be 6g DCW/L, the feeding amount of the substrate o-chlorobenzoyl formic acid is 500mM, the glucose concentration is 600mM, the ammonium sulfate concentration is 750mM, the NAD+ concentration is 5mM, the pH value of 9.0 and 100mM Tris-HCl buffer solution is used as a reaction medium to construct a conversion system 10mL, the reaction is carried out at 40 ℃ and 600rpm, the reaction progress is shown as the formula B in fig. 4, the substrate can be completely converted into the product (S) -o-chlorobenzoyl glycine in 4h, and the product e.e value is more than 99.5%.

Example 8: synthesis of (S) -O-chlorophenylglycine by chemical-enzyme combination

1. O-chlorobenzoyl acid produced by oxidation of o-chloroacetophenone

Into a 50mL round bottom flask was added 1.54g (10 mmol) of acetophenone dissolved in 12.5mL anhydrous pyridine, 2.22g (20 mmol) of selenium dioxide was added under N ₂ Reflux for 15h at 100℃under protection, the solution changed from colorless to yellow. After the reaction was completed, the solvent was removed under reduced pressure by TLC monitoring (developing solvent: methanol: dichloromethane=1:10, v/v), followed by addition of 2M aqueous sodium hydroxide solution to adjust to ph=12 and stirring for 0.5h. Extraction with water was performed 2 times, followed by addition of 40% aqueous hydrochloric acid to adjust to ph=2.0. The organic phases were then combined after 4 times of extraction with ethyl acetate, dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure to give 1.68g of crude o-chlorobenzoyl carboxylic acid in 91% molar yield. The mass spectrum is shown in figure 5, and the nuclear magnetic resonance hydrogen spectrum is shown in figure 6, which shows that the synthesized substance is the target product o-chlorobenzoyl formic acid.

2. Synthesis of (S) -O-chlorophenylglycine by chemical-enzyme combination

In a 10mL conversion system, firstly, the mutant mixed thalli prepared in the example 7 is resuspended by Tris-HCl buffer solution (namely reaction medium) with the pH of 9.0 and 100mM, 3g DCW/L of dry weight is added into the mutant mixed thalli in the conversion system, and then the crude o-chlorobenzoyl formic acid (0.185 g) which is the substrate prepared in the step 1 and has the feeding amount of 100mM is added into the conversion systemGlucose at a concentration of 120mM, ammonium sulfate at a final concentration of 150mM, NAD at a final concentration of 1mM ⁺ Reaction at 40℃and 600rpm, the substrate was completely converted to the product (S) -o-chlorophenylglycine, product e.e.value, at 4h, using HPLC as described in example 3>99.5%. The reaction solution after 4 hours of reaction was placed in an ice bath and adjusted to ph=2.0 with 6mol/L hydrochloric acid. Extraction 1 time with ethyl acetate and drying of the aqueous phase under reduced pressure gave a white solid powder. The white solid powder was dissolved in hot isopropanol and then spin-evaporated to dryness to give 0.15g of the product (S) -o-chlorophenylglycine in a mass yield of 81.8%.

Sequence listing

<110> Zhejiang university of industry

<120> leucine dehydrogenase mutant and its use in synthesis of (S) -o-chlorophenylglycine

<160> 4

<170> SIPOSequenceListing 1.0

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catgatacca ccctgggtcc ggcactgggt ggcctgagaa tgtggaatta tgcaagcgat 180

gaagaagcat taattgatgc attacgcctg gcaaaaggta tgacctacaa aaacgcagca 240

gcaggcctga atttaggtgg tggtaaagcg gttataatcg gggacgccaa aacccagaaa 300

agcgaagcac tgtttagagc atttggtcgt tacgttcaga gcctgaacgg aagatatatt 360

acagcagagg atgttaatac aacagttgca gatatggatt atatccatat ggaaacagat 420

ttcgttaccg gtgtgagtcc ggcatttggc agttcaggaa atccgagccc agtcaccgcc 480

tatggggttt atcgcggcat gaaagcagca gcaaaggagg tttatggtac cgatagcctg 540

gggggtaaaa cggttgcaat tcagggtgtt ggtaatgttg cctttaattt atgccgtcat 600

ctgcatgagg aaggtgcaaa actgatagta acagatatta accaggatgc actgcgtcgt 660

gcagaagaag catttggtgc cctggttgtt ggaccggatg agatctatag cgttgatgcc 720

gatatatttg cgccgtgtgc tctgggagca accctgaatg acgaaaccat tcctcaactg 780

aaagtgaaaa ttatagcagg tgcagcaaat aaccagctga aagaagatcg tcatggtgat 840

atgctgcagg aacgtggaat tctgtatacc cctgactttg tgattaacgc aggtggtgtg 900

attaatgttg cggatgaatt agatggctat aaccgtgaac gggcaatgaa aaaagttgaa 960

ctggtttatg atgcagttgc aaaagtgatt gaaattgcga aaagagatca tctgccgacc 1020

taccgtgcag cagaaaaaat ggcagaagaa agaattgcaa caatgggtag cgcccggagc 1080

cagttcttaa gacgtgataa aaatattctg ggcagtcggg gataa 1125

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<213> Siberian micro-bacillus (Exiguobacterium sibiricum)

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Met Val Glu Thr Asn Val Glu Ala Arg Phe Ser Ile Phe Glu Thr Met

1 5 10 15

Ala Met Glu Asp Tyr Glu Gln Val Val Phe Cys His Asp Lys Val Ser

20 25 30

Gly Leu Lys Ala Ile Ile Ala Ile His Asp Thr Thr Leu Gly Pro Ala

35 40 45

Leu Gly Gly Leu Arg Met Trp Asn Tyr Ala Ser Asp Glu Glu Ala Leu

50 55 60

Ile Asp Ala Leu Arg Leu Ala Lys Gly Met Thr Tyr Lys Asn Ala Ala

65 70 75 80

Ala Gly Leu Asn Leu Gly Gly Gly Lys Ala Val Ile Ile Gly Asp Ala

85 90 95

Lys Thr Gln Lys Ser Glu Ala Leu Phe Arg Ala Phe Gly Arg Tyr Val

100 105 110

Gln Ser Leu Asn Gly Arg Tyr Ile Thr Ala Glu Asp Val Asn Thr Thr

115 120 125

Val Ala Asp Met Asp Tyr Ile His Met Glu Thr Asp Phe Val Thr Gly

130 135 140

Val Ser Pro Ala Phe Gly Ser Ser Gly Asn Pro Ser Pro Val Thr Ala

145 150 155 160

Tyr Gly Val Tyr Arg Gly Met Lys Ala Ala Ala Lys Glu Val Tyr Gly

165 170 175

Thr Asp Ser Leu Gly Gly Lys Thr Val Ala Ile Gln Gly Val Gly Asn

180 185 190

Val Ala Phe Asn Leu Cys Arg His Leu His Glu Glu Gly Ala Lys Leu

195 200 205

Ile Val Thr Asp Ile Asn Gln Asp Ala Leu Arg Arg Ala Glu Glu Ala

210 215 220

Phe Gly Ala Leu Val Val Gly Pro Asp Glu Ile Tyr Ser Val Asp Ala

225 230 235 240

Asp Ile Phe Ala Pro Cys Ala Leu Gly Ala Thr Leu Asn Asp Glu Thr

245 250 255

Ile Pro Gln Leu Lys Val Lys Ile Ile Ala Gly Ala Ala Asn Asn Gln

260 265 270

Leu Lys Glu Asp Arg His Gly Asp Met Leu Gln Glu Arg Gly Ile Leu

275 280 285

Tyr Thr Pro Asp Phe Val Ile Asn Ala Gly Gly Val Ile Asn Val Ala

290 295 300

Asp Glu Leu Asp Gly Tyr Asn Arg Glu Arg Ala Met Lys Lys Val Glu

305 310 315 320

Leu Val Tyr Asp Ala Val Ala Lys Val Ile Glu Ile Ala Lys Arg Asp

325 330 335

His Leu Pro Thr Tyr Arg Ala Ala Glu Lys Met Ala Glu Glu Arg Ile

340 345 350

Ala Thr Met Gly Ser Ala Arg Ser Gln Phe Leu Arg Arg Asp Lys Asn

355 360 365

Ile Leu Gly Ser Arg Gly

370

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atgtacaagg accttgaggg aaaggtcgtc gtcattactg gatcttctac tggactggga 60

aagtctatgg ctattcgatt cgctactgag aaggctaagg tcgtcgtgaa ctaccgatct 120

aaggaggacg aggctaactc tgtccttgag gagattaaga aggtcggagg agaggctatt 180

gctgtcaagg gtgacgtcac tgtcgagtct gacgtcatta acctggtcca gtctgctatt 240

aaggagttcg gaaagctgga cgtcatgatt aacaacgctg gacttgagaa ccctgtgtcc 300

tctcacgaga tgtctctgtc tgactggaac aaggtcattg acactaacct gactggtgct 360

ttcctgggat ctcgagaggc tattaagtac ttcgtcgaga acgacattaa gggaactgtc 420

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gctgaccctg agcagcgagc tgacgtcgag tctatgattc ctatgggtta cattggagag 660

cctgaggaga ttgctgctgt cgctgcttgg ctggcttctt ctgaggcttc ttacgtcact 720

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ggatag 786

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Met Tyr Lys Asp Leu Glu Gly Lys Val Val Val Ile Thr Gly Ser Ser

1 5 10 15

Thr Gly Leu Gly Lys Ser Met Ala Ile Arg Phe Ala Thr Glu Lys Ala

20 25 30

Lys Val Val Val Asn Tyr Arg Ser Lys Glu Asp Glu Ala Asn Ser Val

35 40 45

Leu Glu Glu Ile Lys Lys Val Gly Gly Glu Ala Ile Ala Val Lys Gly

50 55 60

Asp Val Thr Val Glu Ser Asp Val Ile Asn Leu Val Gln Ser Ala Ile

65 70 75 80

Lys Glu Phe Gly Lys Leu Asp Val Met Ile Asn Asn Ala Gly Leu Glu

85 90 95

Asn Pro Val Ser Ser His Glu Met Ser Leu Ser Asp Trp Asn Lys Val

100 105 110

Ile Asp Thr Asn Leu Thr Gly Ala Phe Leu Gly Ser Arg Glu Ala Ile

115 120 125

Lys Tyr Phe Val Glu Asn Asp Ile Lys Gly Thr Val Ile Asn Met Ser

130 135 140

Ser Val His Glu Lys Ile Pro Trp Pro Leu Phe Val His Tyr Ala Ala

145 150 155 160

Ser Lys Gly Gly Met Lys Leu Met Thr Lys Thr Leu Ala Leu Glu Tyr

165 170 175

Ala Pro Lys Gly Ile Arg Val Asn Asn Ile Gly Pro Gly Ala Ile Asn

180 185 190

Thr Pro Ile Asn Ala Glu Lys Phe Ala Asp Pro Glu Gln Arg Ala Asp

195 200 205

Val Glu Ser Met Ile Pro Met Gly Tyr Ile Gly Glu Pro Glu Glu Ile

210 215 220

Ala Ala Val Ala Ala Trp Leu Ala Ser Ser Glu Ala Ser Tyr Val Thr

225 230 235 240

Gly Ile Thr Leu Phe Ala Asp Gly Gly Met Thr Leu Tyr Pro Ser Phe

245 250 255

Gln Ala Gly Arg Gly

260

Claims

1. A leucine dehydrogenase mutant, which is obtained by mutating 362 th phenylalanine of an amino acid sequence shown in SEQ ID No.2 into leucine.

2. A recombinant genetically engineered bacterium comprising a gene encoding the leucine dehydrogenase mutant of claim 1.

3. Use of a leucine dehydrogenase mutant according to claim 1 for the preparation of (S) -o-chlorophenylglycine by asymmetric catalytic amination of o-chlorobenzoic acid.

4. The use according to claim 3, wherein the application method is: mixing wet thalli obtained by induction culture of engineering bacteria containing leucine enzyme mutant genes and wet thalli obtained by induction culture of engineering bacteria containing glucose dehydrogenase genes, taking the mixed thalli as a catalyst, o-chlorobenzoyl formic acid as a substrate, glucose as an auxiliary substrate, ammonium sulfate as an amino donor, and adding NAD ⁺ The buffer solution with pH value of 7-10 is used as reaction medium to form conversion system, and the reaction is carried out at 30-50 deg.c and 500-800rpm, and the reaction liquid is separated and purified to obtain (S) -o-chlorobenzeneglycine.

5. The use according to claim 4, wherein in the conversion system the final substrate concentration is 20-500mM, the final glucose concentration is 24-600mM, NAD ⁺ The final concentration is 0.2-5mM, the final concentration of ammonium sulfate is 30-750mM, the catalyst dosage is 1-10g DCW/L based on the dry weight of the total amount of the mixed bacterial cells, the wet bacterial cells obtained by the induction culture of the engineering bacteria containing leucine dehydrogenase mutant genes in the mixed bacterial cells and the engineering bacteria containing glucose dehydrogenase genes are inducedThe dry weight ratio of the wet thalli obtained by culture is 1-5:1.

6. The use according to claim 4, wherein the wet cells are prepared as follows: inoculating engineering bacteria containing leucine dehydrogenase mutant genes into LB liquid medium containing 50 mug/mL kanamycin at a final concentration, culturing for 10 hours at 37 ℃, inoculating fresh LB liquid medium containing 50 mug/mL kanamycin at a final concentration of 1.5% by volume, culturing for 2 hours at 37 ℃ at 180rpm, adding 0.10mM isopropyl thiogalactoside at a final concentration into the culture solution, culturing for 12 hours at 28 ℃, and centrifuging for 10 minutes at 4 ℃ at 8000rpm to obtain wet bacterial bodies containing leucine dehydrogenase mutants; the preparation method of the wet bacterial body obtained by induced culture of the engineering bacteria containing the glucose dehydrogenase gene is the same as that of the wet bacterial body containing the leucine dehydrogenase gene.

7. Use of the leucine dehydrogenase mutant according to claim 1 for chemo-enzymatic synthesis of (S) -o-chlorophenylglycine, characterized in that the method of use is: (1) Using acetophenone as substrate, selenium dioxide as oxidant, anhydrous pyridine as solvent, and adding the mixture into N ₂ After refluxing at 100 ℃ for 15 hours under protection, the solvent was removed under reduced pressure, and the reaction solution was adjusted to ph=12 with 2M aqueous sodium hydroxide solution and stirred for 0.5 hour; extracting with water for 2 times, and adjusting pH to=2 with 40% hydrochloric acid aqueous solution; extracting with ethyl acetate for 4 times, mixing organic phases, drying with anhydrous sodium sulfate, and removing solvent under reduced pressure to obtain o-chlorobenzoyl formic acid; (2) Mixing wet thalli obtained by induction culture of engineering bacteria containing leucine enzyme mutant genes and wet thalli obtained by induction culture of engineering bacteria containing glucose dehydrogenase genes, taking the mixed thalli as a catalyst, taking o-chlorobenzoyl formic acid prepared in the step (1) as a substrate, taking glucose as an auxiliary substrate, and adding NAD ⁺ Ammonium sulfate is used as an amino donor, buffer solution with pH of 7-10 is used as a reaction medium to form a conversion system, the reaction is carried out at the temperature of 30-50 ℃ and the speed of 500-800rpm, and after the reaction is finished, the reaction solution is separated and purified to obtain the (S) -o-chlorophenylglycine.

8. The use according to claim 7, wherein the amount ratio of acetophenone to selenium dioxide charge material of step (1) is 1:2; the volume of the anhydrous pyridine was 1.25mL/mmol based on the amount of acetophenone species.

9. The use according to claim 7, wherein in the conversion system according to step (2) the final substrate concentration is 20-500mM, the final glucose concentration is 24-600mM, NAD ⁺ The final concentration is 0.2-5mM, the final concentration of ammonium sulfate is 30-750mM, the catalyst dosage is 1-10g DCW/L based on the dry weight of the total amount of the mixed bacteria, and the dry weight ratio of wet bacteria obtained by the induction culture of the engineering bacteria containing leucine dehydrogenase mutant genes to wet bacteria obtained by the induction culture of the engineering bacteria containing glucose dehydrogenase genes in the mixed bacteria is 1-5:1.