CN112831532A

CN112831532A - Method for enzymatic synthesis of D-leucine

Info

Publication number: CN112831532A
Application number: CN202110333271.9A
Authority: CN
Inventors: 范文超; 高书良; 丁鹏
Original assignee: Luoyang Huarong Biotechnology Co ltd
Current assignee: Luoyang Huarong Biotechnology Co ltd
Priority date: 2021-03-29
Filing date: 2021-03-29
Publication date: 2021-05-25
Anticipated expiration: 2041-03-29
Also published as: CN112831532B

Abstract

The invention discloses a method for enzymatically synthesizing D-leucine, which takes alpha-ketoisocaproic acid as a substrate and uses D-amino acid dehydrogenase to catalyze the substrate to carry out dehydrogenation reaction and transamination reaction to obtain D-leucine, wherein the ee value of the product exceeds 99 percent, and the industrial application prospect is wide.

Description

Method for enzymatic synthesis of D-leucine

Technical Field

The invention belongs to the technical field of enzyme catalysis, and particularly relates to a method for enzymatically synthesizing D-leucine and a D-amino acid dehydrogenase mutant used in the method.

Background

D-leucine is an unnatural amino acid, also known as D-2-amino-4-methylpentanoic acid, having CAS number 328-38-1, and is a white flaky powder. It is a branched chain amino acid, and can be used as nutritional supplement, and is effective in recovering muscle after training, controlling blood sugar, providing energy to body tissue, and enhancing immunity. D-leucine also has many applications in the fields of medicine and chemical industry.

D-leucine can be obtained by fermentation (CN 110330441A); or by chiral resolution of DL-leucine racemate (CN103981248A), which has advantages and disadvantages, and has the common characteristic of higher cost. The fermentation method has long production period and complicated extraction and purification steps of the product after fermentation, and chiral separation, which is either a chemical separation method, an enzymatic method or an induced crystallization method, needs to use leucine with higher price as a raw material, so the method is not feasible economically.

Disclosure of Invention

In order to reduce the production cost of D-leucine, the invention develops a new way for preparing D-leucine by a synthesis method, and the route of the enzyme catalysis reaction is shown as follows.

The inventor carries out extensive screening on amino acid dehydrogenase which can catalyze alpha-ketoisocaproic acid to generate D-leucine, and screens out amino acid dehydrogenase which has high stereoselectivity and relatively high enzyme activity, and is NADP + cofactor-dependent D-type amino acid dehydrogenase (UTDAADH) (SEQ ID NO:1) from Bacillus sphaericus (Ureibacillus thermosphaericus). Furthermore, in order to improve the enzyme activity, the wild enzyme is modified through genetic engineering to construct a mutant capable of efficiently catalyzing the reaction of the alpha-ketoisocaproic acid, so that the mutant is used for synthesizing the D-leucine with high optical purity. Specifically, the present invention includes the following technical contents.

A method for enzymatic synthesis of D-leucine uses alpha-ketoisocaproic acid as a substrate, and uses D-amino acid dehydrogenase to catalyze the substrate to perform dehydrogenation reaction and transamination reaction to obtain D-leucine.

In the above method, glucose dehydrogenase and coenzyme NADPH may be added to the reaction system. Since D-amino acid dehydrogenase is an NADP + cofactor-dependent enzyme, a glucose dehydrogenase-dependent NADP + cofactor regeneration system, for example, to which glucose dehydrogenase and a coenzyme NADPH (i.e., NADP +) are added, is included in the reaction system. With the aid of the NADP + cofactor regeneration system, the production costs of D-leucine can be further reduced, which is economically advantageous.

Preferably, an ammonium salt or aqueous ammonia may be further added as an ammonia donor to the reaction system.

In one embodiment, the D-amino acid dehydrogenase used in the above method is a D-amino acid dehydrogenase SEQ ID NO:1 derived from Bacillus sphaericus (Ureibacillus thermosphaericus) or a mutant having 90% or more homology, preferably 95% or more homology, preferably 98% or more homology, more preferably 99% or more homology thereto.

MSKIRIGIVGYGNLGRGVEAAIQQNPDMELVAVFTRRDPKTVAVKSNVKVLHVDDAQSYKDEIDVMILCGGSATDLPEQGPYFAQYFNTIDSFDTHARIPDYFDAVNAAAEQSGKVAIISVGWDPGLFSLNRLLGEVVLPVGNTYTFWGKGVSQGHSDAIRRIQGVKNAVQYTIPIDEAVNRVRSGENPELSTREKHARECFVVLEEGADPAKVEHEIKTMPNYFDEYDTTVHFISEEELKQNHSGMPHGGFVIRSGKSDEGHKQIIEFSLNLESNPMFTSSALVAYARAAYRLSQNGDKGAKTVFDIPFGLLSPKSPEDLRKELL(SEQ ID NO:1)。

Preferably, the amino acid sequence of the mutant is SEQ ID NO. 3 or SEQ ID NO. 4. However, the D-amino acid dehydrogenase mutant of the present invention is not limited thereto. Among them, the D-amino acid dehydrogenase mutant SEQ ID NO 3 has been reported in patent document CN202110108232.9, and it has been found that it can catalyze the reaction of α -ketoisocaproic acid to produce D-leucine.

MSKIRIGIVGYGNLGRGVEAAIQQNPDMELVAVFTRRDPKTVAVKSNVKVLHVDDAQSYKDEIDVMILCGGSATDLPEQGPYFAQYFNTIDSFATHARIPDYFDAVNAAAEQSGKVAIISVGWDPGLFSLNRLLGEVVLPVGNTYTFWGKGVSQGHSDAIRRIQGVKNAVQYTIPIDEAVNRVRSGENPELSTREKHAVECFVVLEEGADPAKVEHEIKTMPNYFDEYDTTVHFISEEELKQNHSGMPTGGFVIRSGKSDEGHKQIIEFSLNLESNPMFTSSALVAYARAAYRLSQNGDKGAKTVFDIPFGLLSPKSPEDLRKELL(SEQ ID NO:3)。

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.

Another D-amino acid dehydrogenase mutant, SEQ ID NO. 4 differs from SEQ ID NO. 3 only in that D at position 94 of 1 is replaced with L, the amino acid sequence of which is:

MSKIRIGIVGYGNLGRGVEAAIQQNPDMELVAVFTRRDPKTVAVKSNVKVLHVDDAQSYKDEIDVMILCGGSATDLPEQGPYFAQYFNTIDSFLTHARIPDYFDAVNAAAEQSGKVAIISVGWDPGLFSLNRLLGEVVLPVGNTYTFWGKGVSQGHSDAIRRIQGVKNAVQYTIPIDEAVNRVRSGENPELSTREKHAVECFVVLEEGADPAKVEHEIKTMPNYFDEYDTTVHFISEEELKQNHSGMPTGGFVIRSGKSDEGHKQIIEFSLNLESNPMFTSSALVAYARAAYRLSQNGDKGAKTVFDIPFGLLSPKSPEDLRKELL(SEQ ID NO:4)。

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

Alternatively, the glucose dehydrogenase is derived from Bacillus cereus (Bacillus cereus), has an amino acid sequence of SEQ ID NO. 6, and can be obtained by expressing the coding gene of SEQ ID NO. 7(GenBank sequence number AE016877.1) in Escherichia coli. However, the glucose dehydrogenase used in combination with the D-amino acid dehydrogenase of the present invention or the mutant thereof is not limited thereto.

Another aspect of the present invention provides a mutant D-amino acid dehydrogenase having the amino acid sequence of SEQ ID NO. 4.

The D-amino acid dehydrogenase mutant can be obtained by fermentation expression of genetically engineered bacteria. When the expression host bacterium of the D-amino acid dehydrogenase mutant SEQ ID NO. 4 is Escherichia coli, the nucleotide sequence of the coding gene may be SEQ ID NO. 5.

The coding gene can be cloned on a proper vector, and then the vector is transformed into an escherichia coli competent cell to obtain a transformant expressing the D-amino acid dehydrogenase mutant SEQ ID NO. 4. Preferably, the vector is a PET series plasmid, such as, but not limited to, PET24a or PET28 a.

The D-amino acid dehydrogenase mutant SEQ ID NO. 4 can be obtained by fermentation of the genetic engineering bacteria. 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.

Another aspect of the present invention provides the use of the above D-amino acid dehydrogenase mutant of SEQ ID NO. 4 or SEQ ID NO. 3 for the preparation of D-leucine.

The invention provides a new idea for preparing high-optical-purity D-leucine, which adopts D-amino acid dehydrogenase such as SEQ ID NO. 4, takes 100mM alpha-ketoisocaproic acid as a substrate, can achieve the conversion rate of 95 percent within 8 hours, and the ee value of the product D-leucine is over 99 percent, thereby showing better industrial development and application prospects.

Detailed Description

When the enzymatic synthesis of D-type amino acid such as D-tert-leucine, D-leucine and the like is explored, wild type D-amino acid dehydrogenase is screened, wherein the wild type D-amino acid dehydrogenase is derived from microorganism Bacillus sphaericus (Ureibacillus thermosphaericus), the GenBank sequence number is BAK86217.1, and the amino acid sequence is SEQ ID NO 1. When it is expressed in E.coli, the gene encoding it may be SEQ ID NO 2.

Experiments show that the catalytic activity is low, and the requirements for industrial production of D-tert-leucine, D-leucine and the like are difficult to meet. Then, the wild enzyme SEQ ID NO 1 is analyzed by bioinformatics technology, some sites in the amino acid sequence of the wild enzyme are judged to play key roles in the aspects of the structure and the function of the enzyme, and then the wild enzyme is modified by technologies such as site-specific saturation mutation and the like, and the key point is to replace active site amino acids (including 94 th aspartic acid site, 199 th arginine site and 249 th histidine site) which catalyze the substrate to generate dehydrogenation reaction and amino transfer reaction so as to obtain a mutant with improved enzyme activity. A series of mutants with improved enzyme activity, such as mutants SEQ ID NO:3 (D94A, R199V and H249T) and mutants SEQ ID NO:4 (D94L, R199V and H249T), are screened out through screening a plurality of mutants.

It was found that these mutants are more selective for the substrate, for example mutant SEQ ID NO. 3, which is highly efficient in catalyzing the reaction of trimethylpyruvic acid to D-tert-leucine, as reported in patent document CN202110108232.9, the contents of which are incorporated herein by reference. But when the substrate is changed to alpha-ketoisocaproic acid, the catalytic activity is obviously reduced. While the mutant SEQ ID NO. 4 with only one site difference (the difference between alanine A and leucine L at position 94) of another amino acid shows higher enzyme activity and high stereoselectivity to the substrate alpha-ketoisocaproic acid, and the reason for the difference between the two mutants on the substrate selectivity is needed to be analyzed and researched.

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 mutants SEQ ID NO 3 and SEQ ID NO 4 may also be referred to simply as "mutants" or "mutases".

The D-amino acid dehydrogenase mutant of the present invention has only 326 amino acids in SEQ ID NO. 4 and a definite structure, so that those skilled in the art can easily obtain the 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.

Based on the same considerations of catalytic function, the wild-type D-amino acid dehydrogenase SEQ ID NO 1, its mutants SEQ ID NO 3 and SEQ ID NO 4 are collectively referred to herein as D-amino acid dehydrogenase.

The glucose dehydrogenase used in combination with the D-amino acid dehydrogenase of the present invention may be expressed by Escherichia coli.

The method adopts a coupling reaction mode of D-amino acid dehydrogenase and glucose dehydrogenase, takes alpha-ketoisocaproic acid and glucose as substrates and NADP + as a cofactor, and prepares the D-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-leucine, the D-amino acid dehydrogenase and glucose dehydrogenase used in the catalytic synthesis of the present invention may be in the form of enzymes or in the form of expressed microbial fermentation 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.

Expression strains of wild-type D-amino acid dehydrogenase SEQ ID NO:1, glucose dehydrogenase SEQ ID NO:6, and D-amino acid dehydrogenase (D94A, R199V, H249T) mutants of SEQ ID NO:3 were constructed according to the methods reported in examples 1 to 3 of patent document CN202110108232.9, respectively.

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

A wild D-amino acid dehydrogenase SEQ ID NO:1 from Bacillus sphaericus (Ureibacillus thermosphaericus) is subjected to codon optimization, a coding gene sequence SEQ ID NO:2 is synthesized by a whole gene, restriction enzyme sites Nde I and XhoI are designed at two ends of the gene, and the gene is subcloned to a corresponding site of a vector pET24a (Novagen) to obtain a recombinant plasmid pET24 a-utDAADH. 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:6, the whole gene is synthesized to encode the gene sequence SEQ ID NO:7, 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 construction of (D94A, R199V, H249T) mutant strains

The construction of (D94A, R199V, H249T) mutant SEQ ID NO 3 expression strain according to the method reported in example 3 of patent document CN202110108232.9, comprising the following steps:

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 competent cells of Escherichia coli E.coli BL21(DE3), performing 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. The strain can express mutant enzyme SEQ ID NO. 3.

Example 4 construction of (D94L, R199V, H249T) mutant strains

In analogy to example 3, another (D94L, R199V, H249T) mutant, SEQ ID No. 4 expression strain was constructed comprising the following steps:

a mutant strain utDAADH-M437 containing three mutations is constructed by using a plasmid pET24a-utDAADH of the utDAADH strain as a template and modifying three sites of 94 site, 199 site and 249 site into D94L site, R199V site and H249T site by a site-directed mutagenesis technology. The primers used in the construction process were as follows: utDAADH-94 LF: 5' -CAACACCATCGACTCTTTCCTGACCCACGCTCGTATC-3’utDAADH-199F：5’-CTACCCGTGAAAAACACGCTGTTGAATGCTTCGTTGTTC-3’utDAADH-199R：5’-GAACAACGAAGCATTCAACAGCGTGTTTTTCACGGGTAG-3’utDAADH-249R：5’-GATAACGAAGCCGCCGGTGGGCATACCAGAGTG-3’

Using pET24a-utDAADH plasmid as a template, using utDAADH-94LF and utDAADH-199R as primer pairs to directly amplify P3 fragment, using 2utDAADH-199F and utDAADH-249R as primer pairs to amplify P4 fragment, using utDAADH-94LF and utDAADH-249R as primer pairs to amplify large fragment P-1 through over-lapping PCR of P3 and P4 fragments, and using P-1 large fragment as a primer to construct a site-directed mutant strain utDAADH-M437.

P3 and P4 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 P3 and P4 fragments: 1min at 95 ℃; 10s at 98 ℃, 30s at 57 ℃ and 1min/kbp at 68 ℃; 30 cycles; 10min at 68 ℃.

After the PCR is finished, the PCR product of the P3 fragment is about 353bp, the PCR product of the P4 fragment is about 188bp, and the PCR products of the P3 and the P4 fragment are respectively cut and recovered.

And (3) performing over-lapping PCR by using the fragment P3 and the fragment P4 after gel cutting recovery as templates and the utDAADH-94LF and the utDAADH-249R as primers to obtain a fragment P-1 with the length of about 502bp, and performing gel cutting recovery.

The Over-plating PCR reaction system comprises the following steps: 5 μ L P3 fragment, 5 μ L P4 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 ℃ and 1min/kbp at 68 ℃; 25 cycles; 10min at 68 ℃.

With fragment P-1 as largePrimer pET24a-utDAADH plasmid is used as a template, KOD-plus DNA polymerase is used for Megaprimer PCR, and the reaction system is as follows: 250ng of P-1 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 competent cells of Escherichia coli E.coli BL21(DE3), performing test tube culture on a positive clone strain, extracting a plasmid, and determining the success construction of a mutant strain utDAADH-M437 by plasmid sequencing. The strain can express mutant enzyme SEQ ID NO. 4.

Example 5 comparison of enzyme activities

5.1 Shake flask fermentation

Single colonies were picked from LB medium plates of utDAADH, utDAADH-M, and utDAADH-M437, respectively, and inoculated into LB medium liquid containing 50. mu.g/mL kanamycin sulfate, respectively, and cultured at 37 ℃ and 230rpm overnight. According to the volume ratio of 1: 100 ratio, the overnight cultures were individually transferred to 1L TB medium containing 50. mu.g/mL kanamycin sulfate 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.

5.2 extraction of D-amino acid dehydrogenase-pure enzyme expressed by UTDAADH, UTDAADH-M and tDAADH-M437

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.

5.3 according to the steps 5.1 and 5.2 similar method, preparation of glucose dehydrogenase SEQ ID NO 6 pure enzyme.

5.4 determination of the specific Activity of D-amino acid dehydrogenases

A500. mu.l reaction was used: 200mM glycine-KOH buffer (pH 10.5), 200mM ammonium chloride, 20mM α -ketoisocaproic acid, 5mM NADPH, 50 μ l of the desalted pure enzyme in step 5.2, 450 μ l of pure water, a water bath at 45 ℃ and reacted for 20min, and the magnitude of activity was judged by measuring the change in 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.

Test results show that the unit enzyme activity of the mutant SEQ ID NO. 4 is improved by 211 times compared with that of a wild enzyme SEQ ID NO. 1 in the reaction of catalyzing the substrate alpha-ketoisocaproic acid to be converted into D-leucine; the unit enzyme activity of the mutant SEQ ID NO. 3 is increased by 55 times compared with that of the wild enzyme SEQ ID NO. 1.

EXAMPLE 6 mutant SEQ ID NO 4 for the Synthesis of D-leucine

The catalytic reaction of the substrate, alpha-ketoisocaproic acid, was carried out using pure D-amino acid dehydrogenase SEQ ID NO 4 and glucose dehydrogenase SEQ ID NO 6 as follows.

50ml reaction system: Glycine-KOH buffer (20mM, pH 10), 100mM of α -ketoisocaproic acid, 0.3M glucose, 1mM of coenzyme NADP +, 150mM of ammonium chloride, 0.1mg/ml of glucose dehydrogenase, and 10U/ml of D-amino acid dehydrogenase pure enzyme, and the pH of the reaction system was adjusted with ammonia water to 10.0. After 4 hours of reaction at 45 ℃, adding 10U/ml D-amino acid dehydrogenase pure enzyme, continuing the reaction for 6 to 10 hours, sampling and centrifuging, directly carrying out HPLC analysis after a supernatant passes through a 0.22 mu m membrane, and finally determining that the reaction is carried out for 8 hours, the substrate conversion rate is over 95 percent, and the ee value of the product is over 99 percent.

HPLC detection method: agilent 1260; kromasil 100C18 column (250X 4mm, 5 μm); mobile phase A: 10mM sodium acetate, pH 6.00; mobile phase B: 85% acetonitrile in water; 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 mu l of the solution; the column temperature is 30 ℃; flow rate: 1 ml/min; detection wavelength: 334 nm.

In conclusion, the D-amino acid dehydrogenase SEQ ID NO:1 and the mutant SEQ ID NOs:3-4 screened by the invention can catalyze and synthesize D-leucine by using alpha-ketoisocaproic acid as a substrate, for example, the conversion rate of the substrate is over 95 percent and the ee value of the product D-leucine is over 99 percent within 8 hours by adopting the substrate concentration of 100mM, so that the D-amino acid dehydrogenase has industrial development and application potential.

Sequence listing

<110> Luoyang Huarong Biotechnology Co., Ltd

<120> a process for enzymatically synthesizing D-leucine

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<400> 2

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 acacccacgc 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 cgctcgtgaa 600

tgcttcgttg ttctggaaga aggtgctgac ccggctaaag ttgaacacga aatcaaaacc 660

atgccgaact acttcgacga atacgacacc accgttcact tcatctctga agaagaactg 720

aaacagaacc actctggtat gccccacggc 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> 3

<211> 326

<212> PRT

<213> Artificial sequence ()

<400> 3

Met Ser Lys Ile Arg Ile Gly Ile Val Gly Tyr Gly Asn Leu Gly Arg

1 5 10 15

Gly Val Glu Ala Ala Ile Gln Gln Asn Pro Asp Met Glu Leu Val Ala

20 25 30

Val Phe Thr Arg Arg Asp Pro Lys Thr Val Ala Val Lys Ser Asn Val

35 40 45

Lys Val Leu His Val Asp Asp Ala Gln Ser Tyr Lys Asp Glu Ile Asp

50 55 60

Val Met Ile Leu Cys Gly Gly Ser Ala Thr Asp Leu Pro Glu Gln Gly

65 70 75 80

Pro Tyr Phe Ala Gln Tyr Phe Asn Thr Ile Asp Ser Phe Ala Thr His

85 90 95

Ala Arg Ile Pro Asp Tyr Phe Asp Ala Val Asn Ala Ala Ala Glu Gln

100 105 110

Ser Gly Lys Val Ala Ile Ile Ser Val Gly Trp Asp Pro Gly Leu Phe

115 120 125

Ser Leu Asn Arg Leu Leu Gly Glu Val Val Leu Pro Val Gly Asn Thr

130 135 140

Tyr Thr Phe Trp Gly Lys Gly Val Ser Gln Gly His Ser Asp Ala Ile

145 150 155 160

Arg Arg Ile Gln Gly Val Lys Asn Ala Val Gln Tyr Thr Ile Pro Ile

165 170 175

Asp Glu Ala Val Asn Arg Val Arg Ser Gly Glu Asn Pro Glu Leu Ser

180 185 190

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

195 200 205

Ala Asp Pro Ala Lys Val Glu His Glu Ile Lys Thr Met Pro Asn Tyr

210 215 220

Phe Asp Glu Tyr Asp Thr Thr Val His Phe Ile Ser Glu Glu Glu Leu

225 230 235 240

Lys Gln Asn His Ser Gly Met Pro Thr Gly Gly Phe Val Ile Arg Ser

245 250 255

Gly Lys Ser Asp Glu Gly His Lys Gln Ile Ile Glu Phe Ser Leu Asn

260 265 270

Leu Glu Ser Asn Pro Met Phe Thr Ser Ser Ala Leu Val Ala Tyr Ala

275 280 285

Arg Ala Ala Tyr Arg Leu Ser Gln Asn Gly Asp Lys Gly Ala Lys Thr

290 295 300

Val Phe Asp Ile Pro Phe Gly Leu Leu Ser Pro Lys Ser Pro Glu Asp

305 310 315 320

Leu Arg Lys Glu Leu Leu

325

<210> 4

<211> 326

<212> PRT

<213> Artificial sequence ()

<400> 4

Met Ser Lys Ile Arg Ile Gly Ile Val Gly Tyr Gly Asn Leu Gly Arg

1 5 10 15

Gly Val Glu Ala Ala Ile Gln Gln Asn Pro Asp Met Glu Leu Val Ala

20 25 30

Val Phe Thr Arg Arg Asp Pro Lys Thr Val Ala Val Lys Ser Asn Val

35 40 45

Lys Val Leu His Val Asp Asp Ala Gln Ser Tyr Lys Asp Glu Ile Asp

50 55 60

Val Met Ile Leu Cys Gly Gly Ser Ala Thr Asp Leu Pro Glu Gln Gly

65 70 75 80

Pro Tyr Phe Ala Gln Tyr Phe Asn Thr Ile Asp Ser Phe Leu Thr His

85 90 95

Ala Arg Ile Pro Asp Tyr Phe Asp Ala Val Asn Ala Ala Ala Glu Gln

100 105 110

Ser Gly Lys Val Ala Ile Ile Ser Val Gly Trp Asp Pro Gly Leu Phe

115 120 125

Ser Leu Asn Arg Leu Leu Gly Glu Val Val Leu Pro Val Gly Asn Thr

130 135 140

Tyr Thr Phe Trp Gly Lys Gly Val Ser Gln Gly His Ser Asp Ala Ile

145 150 155 160

Arg Arg Ile Gln Gly Val Lys Asn Ala Val Gln Tyr Thr Ile Pro Ile

165 170 175

Asp Glu Ala Val Asn Arg Val Arg Ser Gly Glu Asn Pro Glu Leu Ser

180 185 190

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

195 200 205

Ala Asp Pro Ala Lys Val Glu His Glu Ile Lys Thr Met Pro Asn Tyr

210 215 220

Phe Asp Glu Tyr Asp Thr Thr Val His Phe Ile Ser Glu Glu Glu Leu

225 230 235 240

Lys Gln Asn His Ser Gly Met Pro Thr Gly Gly Phe Val Ile Arg Ser

245 250 255

Gly Lys Ser Asp Glu Gly His Lys Gln Ile Ile Glu Phe Ser Leu Asn

260 265 270

Leu Glu Ser Asn Pro Met Phe Thr Ser Ser Ala Leu Val Ala Tyr Ala

275 280 285

Arg Ala Ala Tyr Arg Leu Ser Gln Asn Gly Asp Lys Gly Ala Lys Thr

290 295 300

Val Phe Asp Ile Pro Phe Gly Leu Leu Ser Pro Lys Ser Pro Glu Asp

305 310 315 320

Leu Arg Lys Glu Leu Leu

325

<210> 5

<211> 981

<212> DNA

<213> Artificial sequence ()

<400> 5

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 gactctttcc tgacccacgc 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> 6

<211> 261

<212> PRT

<213> Bacillus cereus

<400> 6

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> 7

<211> 786

<212> DNA

<213> Bacillus cereus

<400> 7

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 method for enzymatic synthesis of D-leucine is characterized in that alpha-ketoisocaproic acid is used as a substrate, and the substrate is catalyzed by D-amino acid dehydrogenase to perform dehydrogenation reaction and transamination reaction to obtain D-leucine.

2. The method according to claim 1, wherein glucose dehydrogenase and coenzyme NADPH are added to the reaction system.

3. The method according to claim 1, wherein an ammonium salt or aqueous ammonia is further added as an ammonia donor to the reaction system.

4. The method according to claim 1, wherein the D-amino acid dehydrogenase is a Bacillus sphaericus-derived D-amino acid dehydrogenase SEQ ID NO 1 or a mutant thereof having 90% or more homology thereto.

5. The method of claim 4, wherein the amino acid sequence of the mutant is SEQ ID NO 3 or SEQ ID NO 4.

6. The method of claim 2, wherein the amino acid sequence of the glucose dehydrogenase is SEQ ID NO 6.

7. A D-amino acid dehydrogenase mutant is characterized in that the amino acid sequence is SEQ ID NO. 4.

8. A gene encoding the D-amino acid dehydrogenase mutant according to claim 7.

9. The gene of claim 8 wherein the nucleotide sequence is SEQ ID NO 5.

10. Use of the D-amino acid dehydrogenase mutant of SEQ ID NO. 4 as described in claim 7 for the preparation of D-leucine.