CN112877307A

CN112877307A - Amino acid dehydrogenase mutant and application thereof

Info

Publication number: CN112877307A
Application number: CN202110108232.9A
Authority: CN
Inventors: 范文超; 高书良; 王金刚; 丁鹏
Original assignee: Luoyang Huarong Biotechnology Co ltd
Current assignee: Luoyang Huarong Biotechnology Co ltd
Priority date: 2021-01-27
Filing date: 2021-01-27
Publication date: 2021-06-01
Anticipated expiration: 2041-01-27
Also published as: CN112877307B

Abstract

The invention constructs a D-amino acid dehydrogenase mutant SEQ ID NO 3, the enzyme activity of the mutant for catalyzing the dehydrogenation reaction of substrate trimethylpyruvic acid is obviously improved compared with that of a wild enzyme SEQ ID NO 1, the mutant is applied to the synthesis of D-tert-leucine, the substrate concentration is 30mM, the reaction time is 8 hours, the conversion rate reaches 89%, the ee value of the product exceeds 99%, and the industrial development and application values are realized.

Description

Amino acid dehydrogenase mutant and application thereof

Technical Field

The invention belongs to the technical field of enzyme catalysis, and particularly relates to a D-amino acid dehydrogenase mutant and application thereof in synthesis of D-tert-leucine.

Background

D-tert-leucine is also known as R-2-amino-3, 3-dimethylbutyric acid, CAS number 26782-71-8, is white crystalline powder, and can be used as chiral resolving agent or pharmaceutical intermediate.

At present, two main synthesis processes of D-tert-leucine are available, one is a chemical method, and the other is a biological enzyme catalysis method. The chemical method mainly uses racemic DL-tert-leucine as a raw material and adopts tartaric acid as a resolving agent to carry out chemical resolution, but the optical purity of a product prepared by the method is poor, and the functional use of the product is difficult to meet.

Two main reports of the biological enzyme catalysis method synthesis are that one is an enzymatic resolution method, and the other is D-type amino acid dehydrogenase synthesis. The enzymatic resolution mainly uses racemic tertiary leucine or derivatives thereof such as ester derivatives, nitrile derivatives, phenylacetated derivatives and the like as raw materials, and the chiral resolution is carried out by L-leucine dehydrogenase, esterase, nitrilase and penicillin G hydrolase to finally obtain the D-tertiary leucine, wherein the utilization rate of the raw materials in the method is only 50% at most, and the synthesis cost is relatively high. The D-amino acid dehydrogenase synthesis method mainly adopts trimethylpyruvic acid as a raw material, and takes NAD + or NADP + as a cofactor to catalyze and generate D-tert-leucine under the action of D-amino acid dehydrogenase.

Disclosure of Invention

In order to improve the D-amino acid dehydrogenase synthesis method, it is necessary to increase the enzymatic activity of D-amino acid dehydrogenase or to screen D-amino acid dehydrogenase having high enzymatic activity. Therefore, the inventor carries out extensive screening on the amino acid dehydrogenase capable of catalyzing trimethylpyruvic acid to generate D-tertiary leucine, screens out D-amino acid dehydrogenase (SEQ ID NO:1) (utDAADH for short) from Bacillus sphaericus (Ureibacillus thermophilus) as a target, and utilizes genetic engineering technologies such as site-directed mutagenesis to transform the D-amino acid dehydrogenase (SEQ ID NO:1), so as to construct a mutant capable of efficiently catalyzing the reaction of trimethylpyruvic acid, thereby synthesizing the D-tertiary leucine with high optical purity.

Therefore, the first objective of the invention is to provide a D-amino acid dehydrogenase mutant, the amino acid sequence of which is SEQ ID NO. 3:

MSKIRIGIVGYGNLGRGVEAAIQQNPDMELVAVFTRRDPKTVAVKSNVKVLHVDDAQSYKDEIDVMILCGGSATDLPEQGPYFAQYFNTIDSFATHARIPDYFDAVNAAAEQSGKVAIISVGWDPGLFSLNRLLGEVVLPVGNTYTFWGKGVSQGHSDAIRRIQGVKNAVQYTIPIDEAVNRVRSGENPELSTREKHAVECFVVLEEGADPAKVEHEIKTMPNYFDEYDTTVHFISEEELKQNHSGMPTGGFVIRSGKSDEGHKQIIEFSLNLESNPMFTSSALVAYARAAYRLSQNGDKGAKTVFDIPFGLLSPKSPEDLRKELL

the mutant is a wild-type D-amino acid dehydrogenase SEQ ID NO 1 in which D at position 94 is replaced by A, R at position 199 is replaced by V, and H at position 249 is replaced by T.

The second object of the present invention is to provide a gene encoding the D-amino acid dehydrogenase mutant.

Preferably, the nucleotide sequence of the gene is SEQ ID NO: 4:

ATGTCTAAAATCCGTATCGGTATCGTTGGTTACGGTAACCTGGGTCGTGGTGTTGAAGCTGCTATCCAGCAGAACCCGGACATGGAACTGGTTGCTGTTTTCACCCGTCGTGACCCGAAAACCGTTGCTGTTAAATCTAACGTTAAAGTTCTGCACGTTGACGACGCTCAGTCTTACAAAGACGAAATCGACGTTATGATCCTGTGCGGTGGTTCTGCTACCGACCTGCCGGAACAGGGTCCGTACTTCGCTCAGTACTTCAACACCATCGACTCTTTCGCCACCCACGCTCGTATCCCGGACTACTTCGACGCTGTTAACGCTGCTGCTGAACAGTCTGGTAAAGTTGCTATCATCTCTGTTGGTTGGGACCCGGGTCTGTTCTCTCTGAACCGTCTGCTGGGTGAAGTTGTTCTGCCGGTTGGTAACACCTACACCTTCTGGGGCAAGGGTGTAAGCCAGGGTCACTCTGACGCTATCCGTCGTATCCAGGGTGTTAAAAACGCTGTTCAGTACACCATCCCGATCGACGAAGCTGTTAACCGTGTTCGTTCTGGTGAAAACCCGGAACTGTCTACCCGTGAAAAACACGCTGTTGAATGCTTCGTTGTTCTGGAAGAAGGTGCTGACCCGGCTAAAGTTGAACACGAAATCAAAACCATGCCGAACTACTTCGACGAATACGACACCACCGTTCACTTCATCTCTGAAGAAGAACTGAAACAGAACCACTCTGGTATGCCCACCGGCGGCTTCGTTATCCGTTCGGGTAAATCTGACGAAGGTCACAAACAGATCATCGAATTCTCTCTGAACCTGGAATCTAACCCGATGTTCACCTCTTCTGCTCTGGTTGCTTACGCTCGTGCTGCTTACCGTCTGTCTCAGAACGGTGACAAAGGTGCTAAAACCGTTTTCGACATCCCGTTCGGTCTGCTGTCTCCGAAATCTCCGGAAGACCTGCGTAAAGAACTGCTGTAA(SEQ ID NO:4)。

the third object of the present invention is to provide a plasmid containing the above gene. The plasmid contains a vector for expressing the above gene, and preferably the vector is PET series, such as PET24a or PET28a, but not limited thereto.

The fourth object of the present invention is to provide a microorganism expressing the D-amino acid dehydrogenase mutant SEQ ID NO. 3. The microorganism is, for example, a microorganism transformed with the above-mentioned plasmid.

Preferably, the above microorganisms are selected from Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, preferably Escherichia coli, more preferably Escherichia coli BL21(DE 3).

A D-amino acid dehydrogenase mutant can be obtained by fermentation of the microorganism. For example, after microbial fermentation, the cells are resuspended in buffer solution, sonicated, centrifuged, the supernatant is collected and passed through column chromatography, and the target protein is eluted, thus obtaining the purified D-amino acid dehydrogenase mutant.

The fifth aspect of the present invention is to provide a method for producing D-tert-leucine. The method takes trimethylpyruvic acid as a substrate, and the D-amino acid dehydrogenase mutant or the expression microorganism thereof as a catalyst to catalyze the substrate to carry out dehydrogenation reaction and amino transfer reaction to obtain the D-tert-leucine.

Since D-amino acid dehydrogenase (UTDAADH) is an NADP + cofactor-dependent enzyme, a glucose dehydrogenase-dependent NADP + cofactor regeneration system, for example, to which glucose dehydrogenase and coenzyme NADPH (i.e., NADP +) are added, is included in the reaction system. By means of an NADP + cofactor regeneration system, the synthesis cost of the D-tert-leucine is further reduced, and the industrial production is favorably realized.

Ammonium salt or ammonia water is also added into the reaction system as an ammonia donor.

In a preferred embodiment, the glucose dehydrogenase is derived from Bacillus cereus (Bacillus cereus), has an amino acid sequence of SEQ ID NO:5, and the coding gene can be SEQ ID NO:6(GenBank SEQ ID NO: AE016877.1), and can be obtained by expression in Escherichia coli. However, the glucose dehydrogenase used in combination with the D-amino acid dehydrogenase mutant of the present invention is not limited thereto.

The enzyme activity of the D-amino acid dehydrogenase mutant SEQ ID NO 3 constructed by the invention is 83 times higher than that of the wild enzyme SEQ ID NO 1, and the stereoselectivity is high, the mutant is applied to the synthesis of D-tert-leucine, the reaction is carried out for 8 hours at the substrate concentration of 30mM, the conversion rate reaches 89%, the ee value of the product exceeds 99%, and the industrial development and application values are realized.

Detailed Description

The D-amino acid dehydrogenase selected by the invention is derived from the microorganism Bacillus sphaericus (Ureibacillus thermosphaericus), the GenBank sequence number is BAK86217.1, and the amino acid sequence is SEQ ID NO: 1:

MSKIRIGIVGYGNLGRGVEAAIQQNPDMELVAVFTRRDPKTVAVKSNVKVLHVDDAQSYKDEIDVMILCGGSATDLPEQGPYFAQYFNTIDSFDTHARIPDYFDAVNAAAEQSGKVAIISVGWDPGLFSLNRLLGEVVLPVGNTYTFWGKGVSQGHSDAIRRIQGVKNAVQYTIPIDEAVNRVRSGENPELSTREKHARECFVVLEEGADPAKVEHEIKTMPNYFDEYDTTVHFISEEELKQNHSGMPHGGFVIRSGKSDEGHKQIIEFSLNLESNPMFTSSALVAYARAAYRLSQNGDKGAKTVFDIPFGLLSPKSPEDLRKELL(SEQ ID NO:1)。

the inventor analyzes the wild enzyme SEQ ID NO:1 by using bioinformatics technology, judges that some sites in the amino acid sequence play key roles in the aspects of the structure and the function of the enzyme, and then modifies the wild enzyme by using technologies such as site-directed mutagenesis and the like, and focuses on replacing active site amino acids (including 94-site aspartic acid, 199-site arginine and 249-site histidine) for catalyzing substrate to perform dehydrogenation reaction and amino transfer reaction so as to obtain a mutant with improved enzyme activity. The mutants (D94A, R199V, H249T) were selected by screening a large number of mutants.

In the present invention, the terms "wild type", "wild enzyme" and "wild-type enzyme" have the same meaning and refer to the wild sequence of the D-amino acid dehydrogenase SEQ ID NO: 1. Correspondingly, the D-amino acid dehydrogenase mutant may also be referred to simply as "mutant" or "mutant enzyme".

The D-amino acid dehydrogenase mutant of the present invention has a definite structure and a number of amino acids of only 326, and thus, those skilled in the art can easily obtain genes encoding the same, expression cassettes and plasmids containing the genes, and transformants containing the plasmids.

These genes, expression cassettes, plasmids, and transformants can be obtained by genetic engineering construction means well known to those skilled in the art.

In order to achieve optimal expression of the protein SEQ ID NO. 3 in different microorganisms, codon optimization can be performed for specific microorganisms such as E.coli, Pichia pastoris, or Bacillus subtilis. Codon optimization is one technique that can be used to maximize protein expression in an organism by increasing the translation efficiency of a gene of interest. Different organisms often show a special preference for one of several codons encoding the same amino acid due to mutation tendencies and natural selection. For example, in rapidly growing microorganisms such as E.coli, the optimized codons reflect the composition of their respective pools of genomic tRNA's. Thus, in a fast growing microorganism, low frequency codons for an amino acid can be replaced by codons for the same amino acid but with a high frequency. Thus, expression of optimized DNA sequences is improved in fast growing microorganisms. For example, when a wild-type D-amino acid dehydrogenase is expressed in E.coli, the gene SEQ ID NO 2; when expressing the mutant SEQ ID NO 3 in E.coli, the gene SEQ ID NO 4 can be selected.

The above-mentioned transformant host may be any microorganism suitable for expressing the D-amino acid dehydrogenase, including bacteria and fungi. Preferably the microorganism is Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae, or Escherichia coli, preferably Escherichia coli, more preferably Escherichia coli BL21(DE 3).

The glucose dehydrogenase used in combination with the D-amino acid dehydrogenase mutant of the present invention may be, but is not limited to, a glucose dehydrogenase derived from Bacillus cereus (Bacillus cereus) SEQ ID NO: 5. The coding gene of the enzyme can be SEQ ID NO. 6(GenBank sequence number is AE016877.1), and can be expressed by Escherichia coli.

The method can adopt a coupling reaction mode of D-amino acid dehydrogenase and glucose dehydrogenase, takes trimethylpyruvic acid and glucose as substrates, takes NADP + as a cofactor, and prepares the D-tert-leucine by a one-pot method. Wherein glucose is a substrate for glucose dehydrogenase, and during the reaction, the glucose dehydrogenase catalyzes the oxidation of glucose and simultaneously carries out NADP (N-terminal dehydrogenase)⁺Reduced to NADPH.

When used as a biocatalyst for the production of D-tert-leucine, the D-amino acid dehydrogenase mutants and glucose dehydrogenase used in the catalytic synthesis of the present invention may be in the form of enzymes or in the form of bacterial cells. The enzyme forms comprise free enzyme and immobilized enzyme, including purified enzyme, crude enzyme, fermentation liquor, carrier-immobilized enzyme and the like.

The present invention will be described in further detail with reference to specific examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.

The addition amount, content and concentration of various substances are referred to herein, wherein the percentage refers to the mass percentage unless otherwise specified.

Examples

Materials and methods

The whole gene synthesis, primer synthesis and sequencing in the examples were performed by Jinzhi Biotechnology, Inc., Suzhou.

The molecular biological experiments in the examples include plasmid construction, digestion, ligation, competent cell preparation, transformation, culture medium preparation, and the like, and are mainly performed with reference to "molecular cloning experimental manual" (third edition), sambrook, d.w. rasel (american), translation of huang peitang et al, scientific press, beijing, 2002). The specific experimental conditions can be determined by simple experiments if necessary.

PCR amplification experiments were performed according to the reaction conditions or kit instructions provided by the supplier of the plasmid or DNA template. If necessary, it can be adjusted by simple experiments.

LB culture medium: 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride, pH 7.2. (20 g/L agar powder was additionally added to LB solid medium.)

TB culture medium: 24g/L yeast extract, 12g/L tryptone, 16.43g/L K₂HPO₄.3H₂O、2.31g/L KH₂PO₄5g/L of glycerol, and the pH value is 7.0-7.5. (20 g/L agar powder was additionally added to TB solid medium.)

For convenience of description, the number of a certain enzyme protein, the number of its gene, and the number of its expression strain are sometimes mixed/applied in the examples, and those skilled in the art will readily understand that they refer to different organisms in different environments.

EXAMPLE 1 construction of wild-type D-amino acid dehydrogenase-expressing Strain

For wild D-amino acid dehydrogenation SEQ ID NO:1 from Bacillus sphaericus (Ureibacillus thermosphaericus), codon optimization is carried out, a coding gene sequence SEQ ID NO:2 is synthesized by whole genes, restriction enzyme sites Nde I and XhoI are designed at two ends of the genes and are subcloned to corresponding sites of a vector pET24a (Novagen), and a recombinant plasmid pET24a-utDAADH is obtained. The recombinant plasmid pET24a-utDAADH is transformed into expression host Escherichia coli BL21(DE3) to obtain the recombinant Escherichia coli utDAADH for expressing wild enzyme.

Example 2 construction of glucose dehydrogenase-expressing Strain

For Bacillus cereus (Bacillus cereus) derived glucose dehydrogenase SEQ ID NO:5, the whole gene is synthesized to encode the gene sequence SEQ ID NO:6, restriction enzyme sites Nde I and XhoI are designed at both ends of the gene, and the restriction enzyme sites Nde I and XhoI are subcloned into corresponding sites of a vector pET24a (Novagen), so as to obtain a recombinant plasmid pET24 a-bcGDH. The recombinant plasmid pET24a-bcGDH was transformed into expression host E.coli BL21(DE3) to obtain recombinant E.coli bcGDH expressing glucose dehydrogenase.

Example 3 site-directed mutagenesis for mutant Strain construction

3.1 using plasmid pET24a-utDAADH plasmid of the utDAADH strain as a template, aiming at three sites of 94 th, 199 th and 249 th in wild type D-amino acid dehydrogenation SEQ ID NO. 1, modifying the wild type D-amino acid dehydrogenation SEQ ID NO. into D94A, R199V and H249T by using a gene site-directed mutagenesis technology, and then constructing a mutant expression strain utDAADH-M containing the three mutations according to the method in the example 1. The primers used in the construction process were as follows:

utDAADH-94F：5’-CAACACCATCGACTCTTTCGCCACCCACGCTCGTATC-3’，

utDAADH-199F：5’-CTACCCGTGAAAAACACGCTGTTGAATGCTTCGTTGTTC-3’，

utDAADH-199R：5’-GAACAACGAAGCATTCAACAGCGTGTTTTTCACGGGTAG-3’，

utDAADH-249R：5’-GATAACGAAGCCGCCGGTGGGCATACCAGAGTG-3’。

directly amplifying a P1 fragment by taking a pET24a-utDAADH plasmid as a template and utDAADH-94F and utDAADH-199R as a primer pair, amplifying a P2 fragment by taking 2utDAADH-199F and utDAADH-249R as a primer pair, amplifying a large fragment P by using over-lapping PCR of P1 and P2 fragments by taking utDAADH-94F and utDAADH-249R as a primer pair, and then performing Megaprimer PCR by taking the large fragment P as a primer to construct a site-directed mutant expression plasmid.

P1 and P2 fragments 50 μ L PCR reaction: 10ng of plasmid template, 10pmol of primer set, 1 XKOD plus buffer, 0.2mM dNTP, 1.5mM MgSO₄5 units of KOD-plus DNA polymerase.

PCR conditions for P1 and P2 fragments: 1min at 95 ℃; 10s at 98 ℃, 30s at 57 ℃, 1min/kbp at 68 ℃ and 30 cycles; 10min at 68 ℃.

After the PCR is finished, the PCR product of the P1 fragment is about 353bp, the PCR product of the P2 fragment is about 188bp, and the PCR products of the P1 and the P2 fragment are respectively cut and recovered.

And (3) performing over-lapping PCR by using the fragment P1 and the fragment P2 after gel cutting recovery as templates and the utDAADH-94F and the utDAADH-249R as primers to obtain a fragment P with the length of about 502bp, and performing gel cutting recovery.

The Over-plating PCR reaction system comprises the following steps: 5 μ l P1 fragment, 5 μ l P2 fragment, 10pmol primer set, 1 XKOD plus buffer, 0.2mM dNTP, 1.5mM MgSO₄5 units of KOD-plus DNA polymerase.

Over-loading PCR reaction conditions: 3min at 95 ℃; 10s at 98 ℃, 30s at 60 ℃, 1min/kbp at 68 ℃ and 25 cycles; 10min at 68 ℃.

Using fragment P as a large primer, pET24a-utDAADH plasmid as a template, and KOD-plus DNA polymerase as Megaprimer PCR, wherein the reaction system comprises the following steps: 250ng of P fragment, 10ng of plasmid template, 1 XKOD plus buffer, 0.2mM dNTP, 1.5mM MgSO₄5 units of KOD-plus DNA polymerase.

MegaPrimer PCR reaction conditions: 5min at 94 ℃; 10s at 98 ℃, 30s at 60 ℃, 2min/kbp at 68 ℃ and 25 cycles; 10min at 68 ℃.

Digesting a plasmid template by DpnI, chemically transforming Escherichia coli E.coli BL21(DE3), carrying out test tube culture on a positive clone strain, extracting a plasmid, and determining the success construction of a mutant strain utDAADH-M by plasmid sequencing.

3.2 Shake flask fermentation

Single colonies were picked from LB plates containing the strains utDAADH and utDAADH-M, respectively, and inoculated into LB liquid medium containing 50. mu.g/mL kanamycin sulfate, respectively, and cultured at 37 ℃ and 230rpm overnight. The overnight cultures were transferred to 1L TB medium containing 50. mu.g/mL kanamycin sulfate at 1% v/v, respectively, and cultured at 37 ℃ and 230rpm to OD₆₀₀When the concentration was 0.6 to 0.8, IPTG was added to the mixture at a final concentration of 0.1mM, and the mixture was cultured overnight at 25 ℃ and 200 rpm. Then, the cells were centrifuged at 8000rpm for 10min at 4 ℃ to collect the cells.

3.3 extraction of pure enzyme UTDAADH and tDAADH-M

The cells were resuspended in 50mL of an equilibration buffer (20mM potassium phosphate buffer, 200mM NaCl, pH7.8), then disrupted by sonication, and the disrupted cells were centrifuged at 12000rpm at 4 ℃ for 20min to collect the supernatant. The supernatant was applied to an affinity column containing 10mL of Ni-NAT matrix at a rate of 1mL/min, and the column was then washed with an equilibration buffer containing 50mM imidazole to elute impurities. Finally, the target protein is removed by washing with an equilibrium buffer containing 500mM imidazole, and the peak eluent is collected.

Desalting the eluent by an ultrafiltration tube with the molecular weight cutoff of 10kDa to obtain pure enzyme.

3.4 determination of specific Activity of enzymes

Mu.l of the reaction mother liquor (200mM glycine-KOH buffer (pH 10.5), 200mM ammonium chloride ((pH 10.5), 20mM trimethylpyruvic acid, 5mM NADPH), 50. mu.l of the desalted pure enzyme obtained in step 3.3, 450. mu.l of pure water, water bath at 45 ℃ and reaction for 20min, and the magnitude of the activity was judged by measuring the change in the absorbance at 340 nm.

Meanwhile, the Protein concentration of the pure enzyme is measured by adopting a BCA Protein Assay Kit of Thermo Scientific company, so that the specific activity of the pure enzyme is obtained.

Through tests, compared with the wild enzyme SEQ ID NO. 1, the unit enzyme activity of the mutant SEQ ID NO. 4 in catalyzing the reaction of substrate trimethylpyruvic acid is improved by 83 times.

EXAMPLE 4 Synthesis of D-Tertiary leucine by mutants

Pure enzymes of utDAADH-M and bcGDH were prepared according to the procedures described in step 3.2 and step 3.3 of example 3, respectively, and then reactions for catalyzing the conversion of trimethylpyruvic acid were carried out as follows.

50ml reaction system: Glycine-KOH buffer (20mM, pH10), 30mM trimethylpyruvic acid, 0.3M glucose, 1mM coenzyme NADP +, 150mM ammonium chloride, 0.1mg/ml bcGDH pure enzyme, 5U/ml utDAADH-M mutant enzyme, and pH-corrected reaction system with ammonia water were kept at 10.0. After 4 hours of reaction at 45 ℃, 5U/ml of utDAADH-M pure enzyme is supplemented, the reaction is continued for 6 to 10 hours, after sampling and centrifugation, the supernatant is directly subjected to HPLC analysis after passing through a 0.22 mu M membrane, and finally the reaction is determined for 8 hours, the substrate conversion rate reaches 89 percent, and the ee value of the product is more than 99 percent, which indicates that the mutant enzyme utDAADH-M has high stereospecificity.

HPLC detection method: agilent 1260; kromasil 100C 18 column (250X 4mm, 5 μm); mobile phase A10 mM sodium acetate, pH 6.00; the mobile phase B is 85% acetonitrile water solution; a derivatizing agent: 0.1372g of o-phthalaldehyde and 0.0589g N-isobutyryl-L-cysteine, and the volume is adjusted to 10ml by 0.1M boric acid buffer solution (pH 10.4); sample introduction amount: 5 ul; the column temperature is 30 ℃; flow rate: 1 ml/min; detection wavelength: 334 nm.

In conclusion, compared with wild D-amino acid dehydrogenase, the mutant SEQ ID NO 3 constructed by the invention has obviously improved catalytic activity on a trimethylpyruvic acid substrate, high stereoselectivity and better industrial development and application prospect.

Sequence listing

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<213> Artificial sequence ()

<400> 4

atgtctaaaa tccgtatcgg tatcgttggt tacggtaacc tgggtcgtgg tgttgaagct 60

gctatccagc agaacccgga catggaactg gttgctgttt tcacccgtcg tgacccgaaa 120

accgttgctg ttaaatctaa cgttaaagtt ctgcacgttg acgacgctca gtcttacaaa 180

gacgaaatcg acgttatgat cctgtgcggt ggttctgcta ccgacctgcc ggaacagggt 240

ccgtacttcg ctcagtactt caacaccatc gactctttcg ccacccacgc tcgtatcccg 300

gactacttcg acgctgttaa cgctgctgct gaacagtctg gtaaagttgc tatcatctct 360

gttggttggg acccgggtct gttctctctg aaccgtctgc tgggtgaagt tgttctgccg 420

gttggtaaca cctacacctt ctggggcaag ggtgtaagcc agggtcactc tgacgctatc 480

cgtcgtatcc agggtgttaa aaacgctgtt cagtacacca tcccgatcga cgaagctgtt 540

aaccgtgttc gttctggtga aaacccggaa ctgtctaccc gtgaaaaaca cgctgttgaa 600

tgcttcgttg ttctggaaga aggtgctgac ccggctaaag ttgaacacga aatcaaaacc 660

atgccgaact acttcgacga atacgacacc accgttcact tcatctctga agaagaactg 720

aaacagaacc actctggtat gcccaccggc ggcttcgtta tccgttcggg taaatctgac 780

gaaggtcaca aacagatcat cgaattctct ctgaacctgg aatctaaccc gatgttcacc 840

tcttctgctc tggttgctta cgctcgtgct gcttaccgtc tgtctcagaa cggtgacaaa 900

ggtgctaaaa ccgttttcga catcccgttc ggtctgctgt ctccgaaatc tccggaagac 960

ctgcgtaaag aactgctgta a 981

<210> 5

<211> 261

<212> PRT

<213> Bacillus cereus

<400> 5

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

1 5 10 15

Thr Gly Leu Gly Arg Ala Met Gly Val Arg Phe Ala Lys Glu Lys Ala

20 25 30

Lys Val Val Ile Asn Tyr Arg Ser Arg Glu Ser Glu Ala Asn Asp 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 Val Asn Leu Ile Gln Ser Ala Val

65 70 75 80

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

85 90 95

Asn Ala Val Pro Ser His Glu Met Pro Leu Glu Asp Trp Asn Arg Val

100 105 110

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

115 120 125

Lys Tyr Phe Val Glu His Asp Ile Lys Gly Ser 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 Ile Lys Leu Met Thr Glu 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 Lys Lys Arg Ala Asp

195 200 205

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

210 215 220

Ala Ala Val Ala Thr 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

<210> 6

<211> 786

<212> DNA

<213> Bacillus cereus

<400> 6

atgtatagtg atttagcagg gaaagttgtc gttattacag gatcagcaac tggtcttgga 60

agagcgatgg gagtgaggtt tgctaaggaa aaagcgaaag tggttattaa ttatcgctca 120

cgagaatcag aagcgaatga tgtgttagaa gaaattaaaa aggtaggcgg cgaagcgatt 180

gctgtaaaag gtgatgtaac cgtcgaatca gatgttgtga atctcattca atctgctgtg 240

aaagagtttg gtacgcttga cgttatgatt aataatgcag ggatagaaaa cgcggtaccg 300

tcgcatgaaa tgccgcttga agattggaat agggtaatta atacaaattt aacaggtgct 360

tttttaggaa gtcgtgaagc gattaaatat tttgtagaac atgatattaa aggttctgtc 420

attaatatgt ctagtgttca tgagaaaatt ccgtggccac tatttgtgca ctatgcagcg 480

agtaagggtg gtattaaact gatgacagaa acgttagcgc tagaatatgc gccaaaaggt 540

attcgagtaa ataatattgg accaggtgca attaataccc cgattaatgc agaaaagttt 600

gctgatccta aaaaacgtgc tgacgtagaa agtatgatac cgatgggcta tattggaaac 660

cctgaagaaa ttgcagcagt agcaacttgg ctcgcttctt cagaggcgag ttatgtaacg 720

ggcattacgc tatttgcaga tggtggaatg acgttatatc catcgtttca agctgggcgt 780

gggtaa 786

Claims

1. A D-amino acid dehydrogenase mutant has an amino acid sequence of SEQ ID NO 3.

2. A gene encoding the D-amino acid dehydrogenase mutant according to claim 1.

3. The gene of claim 2 wherein the nucleotide sequence is SEQ ID NO 4.

4. A plasmid comprising the gene of claim 3.

5. A microorganism expressing the D-amino acid dehydrogenase mutant of claim 1.

6. The microorganism of claim 5, wherein the microorganism is selected from the group consisting of E.coli, Pichia pastoris, and Bacillus subtilis.

7. The microorganism according to claim 6, wherein the microorganism is Escherichia coli BL21(DE 3).

8. A method for producing D-tert-leucine, characterized in that D-tert-leucine is obtained by using trimethylpyruvic acid as a substrate and using a D-amino acid dehydrogenase mutant as defined in claim 1 or a microorganism as defined in any one of claims 5 to 7 to catalyze the substrate to undergo dehydrogenation and transamination reactions.

9. The method according to claim 8, wherein glucose dehydrogenase and coenzyme NADPH are added to the reaction system.

10. The method of claim 9, wherein the amino acid sequence of the glucose dehydrogenase is SEQ ID No. 5.