Application of valine dehydrogenase and its mutants in the preparation of chiral L-amino acids

Abstract

The present invention discloses the use of valine dehydrogenase and its mutants in the preparation of chiral L-amino acids. The present invention provides a valine dehydrogenase derived from a hot spring metagenome, and transforms the valine dehydrogenase by site-directed mutagenesis to obtain a valine dehydrogenase mutant, which significantly improves the catalytic activity of the enzyme for multiple large sterically hindered substrates. The valine dehydrogenase and the valine dehydrogenase mutants of the present invention exhibit good temperature stability and can be used for the biocatalytic synthesis of high-value non-natural chiral amino acids such as L-2-aminobutyric acid, L-tert-leucine and L-phenylglycine, with a substrate conversion rate of >99%, an ee value of the obtained product of >99%, and a space-time yield of up to 251.49 g·L ‑1 ·d ‑1 , which has high practical application value. The present invention enriches the enzyme tool library for the preparation of chiral amino acids and provides a new and efficient enzyme catalyst for the biocatalytic synthesis of high-value non-natural chiral amino acids.

Description

Technical Field

[0001] The present invention belongs to the technical field of enzyme engineering, and more specifically, relates to the application of valine dehydrogenase and its mutants in the preparation of chiral L-amino acids. Background Art

[0002] Amino acid dehydrogenases (EC 1.4.1.X) are a class of oxidoreductases that use NAD(H) or NADP(H) as coenzymes and reversibly catalyze the reductive amination of α-keto acids to produce the corresponding amino acids. Amino acid dehydrogenases catalyze the reductive amination of keto acids, using NH3 as the amino group donor. The thermodynamic equilibrium of the reaction tends to the direction of amino acid production, which has great advantages from the perspective of atom economy. They have received extensive attention in the field of extracellular enzyme-catalyzed preparation or intracellular biosynthesis of non-natural chiral amino acids. At the same time, amino acid dehydrogenases can also be converted into amine dehydrogenases through molecular modification technology and applied to the synthesis of high-value chiral compounds such as chiral amines or chiral amine alcohols (Chen Feifei, Wang Donghao, Xu Jianhe, et al. Opportunities and challenges of amine dehydrogenase-catalyzed synthesis of chiral amines [J]. Bioprocess Engineering, 2020, 18(1): 10.).

[0003] Valine dehydrogenase (EC 1.4.1.8) is a member of the amino acid dehydrogenase family, with valine or the corresponding keto acid as its natural substrate. Compared with leucine dehydrogenase, phenylalanine dehydrogenase and glutamate dehydrogenase, which are also members of the Glu / Leu / Phe / Val dehydrogenase subfamily and have been studied extensively, very few valine dehydrogenases have been reported, which greatly limits the practical application of valine dehydrogenase. Valine dehydrogenase exhibits unique substrate preference and is an important material for the development of new enzyme catalysts for the synthesis of chiral amino acids and chiral amines. The discovery and modification of new valine dehydrogenases have important scientific research value and practical application prospects.

[0004] As an extreme ecological environment, hot springs have fostered many unique life forms and are an important resource for discovering new enzyme catalysts. However, most microorganisms in hot springs cannot be cultured or are difficult to culture. The traditional enzyme catalyst mining method based on microbial culture will result in the omission of most potential novel enzyme catalysts. Using metagenomic enzyme mining technology to mine the hot spring metagenome is expected to obtain new valine dehydrogenases, providing new and efficient enzyme catalysts for the biocatalytic synthesis of high-value non-natural chiral amino acids. Summary of the invention

[0005] The first object of the present invention is to provide a valine dehydrogenase

[0006] The second object of the present invention is to provide a valine dehydrogenase mutant.

[0007] The third object of the present invention is to provide the use of the valine dehydrogenase or the valine dehydrogenase mutant in the preparation of chiral L-amino acids.

[0008] The fourth object of the present invention is to provide a method for preparing chiral L-amino acids.

[0009] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0010] The present invention predicts gene functions by using metagenomic sequencing data of Yunnan hot spring (pH 3, 75.3°C) sediments, and using the existing amino acid sequence of valine dehydrogenase (NCBI ID: Q53872.1) as a probe, a new valine dehydrogenase with a 46% identity to the probe amino acid sequence is obtained from the hot spring metagenome, named HsVDH, whose amino acid sequence is shown in SEQ ID NO.3, and the nucleotide sequence of the gene encoding HsVDH is shown in SEQ ID NO.1.

[0011] Based on the obtained valine dehydrogenase HsVDH, the present invention also obtains its mutant through targeted point mutagenesis, and the mutant is obtained by site-directed mutation of isoleucine at position 294 of the valine dehydrogenase amino acid sequence shown in SEQ ID NO.3 to valine or alanine. The valine dehydrogenase or its mutant can be used to prepare chiral L-amino acids, which is of great significance for the biocatalytic synthesis of high-value non-natural chiral amino acids.

[0012] The present invention provides a valine dehydrogenase, and the amino acid sequence of the valine dehydrogenase is shown as SEQ ID NO.3.

[0013] The present invention also provides a gene encoding the valine dehydrogenase.

[0014] Specifically, the nucleotide sequence of the gene encoding valine dehydrogenase is shown in SEQ ID NO.1.

[0015] In order to facilitate the smooth functional expression of the valine dehydrogenase in the E. coli expression system, the present invention performs codon optimization on the gene shown in SEQ ID NO.1, and the optimized gene sequence is shown in SEQ ID NO.2.

[0016] The present invention also provides a valine dehydrogenase mutant, which is obtained by site-directed mutation of isoleucine at position 294 of the valine dehydrogenase amino acid sequence shown in SEQ ID NO.3 to valine or alanine.

[0017] Specifically, when the isoleucine at position 294 is site-directedly mutated to valine, the amino acid sequence of the valine dehydrogenase mutant (I294V) is shown in SEQ ID NO.4.

[0018] Specifically, when the isoleucine at position 294 is site-directedly mutated to alanine, the amino acid sequence of the valine dehydrogenase mutant (I294A) is shown in SEQ ID NO.5.

[0019] In view of the fact that the valine dehydrogenase and the valine dehydrogenase mutant of the present invention can be used to prepare chiral L-amino acids, the present invention also applies to protect the use of the valine dehydrogenase or the valine dehydrogenase mutant in preparing chiral L-amino acids.

[0020] Based on the amino acid sequence of the valine dehydrogenase or valine dehydrogenase mutant of the present invention, those skilled in the art can obtain genes encoding the valine dehydrogenase or valine dehydrogenase mutant, and then obtain recombinant valine dehydrogenase or valine dehydrogenase mutant by genetic engineering technology. Therefore, the use of genes encoding the valine dehydrogenase and valine dehydrogenase mutant of the present invention, recombinant expression vectors containing genes encoding the valine dehydrogenase and valine dehydrogenase mutant of the present invention, or genetically engineered bacteria or cells capable of expressing the valine dehydrogenase and valine dehydrogenase mutant of the present invention in the preparation of chiral L-amino acids should also be within the scope of protection of the present invention.

[0021] Specifically, the amino acid sequence of the valine dehydrogenase is shown as SEQ ID NO.3, and the amino acid sequence of the valine dehydrogenase mutant is shown as SEQ ID NO.4 or SEQ ID NO.5.

[0022] Specifically, the chiral L-amino acid is L-2-aminobutyric acid, L-tert-leucine or L-phenylglycine.

[0023] The present invention also provides a method for preparing chiral L-amino acids using the valine dehydrogenase or the valine dehydrogenase mutant of the present invention, the method comprising: preparing an enzyme solution of the valine dehydrogenase or the valine dehydrogenase mutant of the present invention, adding the prepared enzyme solution or transgenic engineering bacterial cells capable of expressing the valine dehydrogenase or the valine dehydrogenase mutant to a mixed system containing an α-keto acid substrate, an amino donor and a coenzyme regeneration system to carry out a reductive amination reaction, thereby preparing the chiral L-amino acid.

[0024] Specifically, the enzyme solution is a crude enzyme solution or a purified enzyme solution obtained by disrupting cells of a genetically engineered bacterium capable of expressing the valine dehydrogenase or the valine dehydrogenase mutant.

[0025] Specifically, the amino acid sequence of the valine dehydrogenase is shown as SEQ ID NO.3, and the amino acid sequence of the valine dehydrogenase mutant is shown as SEQ ID NO.4 or SEQ ID NO.5.

[0026] Specifically, the α-keto acid substrate is 2-ketobutyric acid, 3,3-dimethyl-2-oxobutyric acid, phenylacetoacetic acid or their respective salt forms.

[0027] Specifically, the chiral L-amino acid is L-2-aminobutyric acid, L-tert-leucine or L-phenylglycine.

[0028] Specifically, the coenzyme regeneration system uses formate dehydrogenase as the coenzyme regeneration enzyme and ammonium formate as the coenzyme regeneration substrate, and contains NADH and NAD + A formate dehydrogenase coenzyme regeneration system; or a system using glucose dehydrogenase as a coenzyme regeneration enzyme and glucose as a coenzyme regeneration substrate, including NADH and NAD + Glucose dehydrogenase coenzyme regeneration system; or alcohol dehydrogenase as coenzyme regeneration enzyme, isopropanol as coenzyme regeneration substrate, including NADH and NAD + Alcohol dehydrogenase coenzyme regeneration system.

[0029] Specifically, the temperature of the reductive amination reaction is 25-50° C., and the pH value of the reaction system is 6-10.

[0030] Specifically, the substrate concentration of the reductive amination reaction is 0.2M to 0.4M.

[0031] The present invention has the following beneficial effects:

[0032] The present invention provides a valine dehydrogenase derived from a hot spring metagenome, and transforms the valine dehydrogenase by site-directed mutagenesis, significantly improving the catalytic activity of the enzyme for multiple large steric hindrance substrates, revealing the potential evolutionary path of amino acid dehydrogenases, and expanding the application scenarios of the valine dehydrogenase.

[0033] The valine dehydrogenase and the valine dehydrogenase mutants of the present invention exhibit good temperature stability and can be used for the biocatalytic synthesis of multiple high-value non-natural chiral amino acids such as L-2-aminobutyric acid, L-tert-leucine and L-phenylglycine. The substrate conversion rate is >99%, the ee value of the obtained product is >99%, and the space-time yield can reach up to 251.49 g·L -1 ·d -1 , which has high practical application value. The present invention enriches the enzyme tool library for biocatalytic synthesis of chiral amino acids and provides a new and efficient enzyme catalyst for the biocatalytic synthesis of high-value non-natural chiral amino acids. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Shown are the inactivation curves of valine dehydrogenase HsVDH at different temperatures.

[0035] Figure 2 This is the reaction formula for preparing chiral L-amino acids by the valine dehydrogenase HsVDH derived from the hot spring metagenome described in the present invention.

[0036] Figure 3 Figure 1 is the pre-column derivatization HPLC detection spectrum of the amino acid standard sample; wherein, Figure A is the analysis spectrum of the D / L-2-amino acid butyric acid standard sample, Figure B is the analysis spectrum of the D / L-tertiary leucine standard sample, and Figure C is the analysis spectrum of the D / L-phenylglycine standard sample.

[0037] Figure 4 Figure A is the detection spectrum of the amino acid product generated in Example 4, Figure B is the detection spectrum of the amino acid product generated in Example 5, Figure C is the detection spectrum of the amino acid product generated in Example 7, Figure D is the detection spectrum of the amino acid product generated in Example 8, and Figure E is the detection spectrum of the amino acid product generated in Example 9.

[0038] Figure 5 The reaction process of valine dehydrogenase HsVDH in preparing L-2-aminobutyric acid.

[0039] Figure 6 Reaction progress for the production of L-tert-leucine by valine dehydrogenase HsVDH (0.2 M substrate).

[0040] Figure 7 Reaction progress of preparing L-tert-leucine for valine dehydrogenase HsVDH (0.4 M substrate)

[0041] Figure 8 The reaction process of valine dehydrogenase HsVDH in producing L-phenylglycine.

[0042] Fig. 9 Reaction progress for the preparation of L-phenylglycine from a valine dehydrogenase mutant (I294V).

[0043] Fig.10 Reaction progress for the preparation of L-phenylglycine from a valine dehydrogenase mutant (I294A). DETAILED DESCRIPTION

[0044] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0045] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0046] The experimental methods described in the present invention are all conventional methods unless otherwise specified. For details of gene cloning operations, please refer to "Molecular Cloning Experiment Guide" edited by J. Sambrook et al.

[0047] The DNA polymerase (2×Phanta Max Master Mix), Dpn I enzyme, recombination cloning kit and plasmid extraction kit used in the examples of the present invention were purchased from Nanjing Novozymes Biotech Co., Ltd.; the synthesis of genes and primers and gene sequencing were completed by Qingke Zixi Biotechnology (Guangzhou) Co., Ltd., and the use of the above reagents refers to the product instructions.

[0048] The expression vector involved in the present invention is pET-28a(+), and the host used is Escherichia coli BL21(DE3), both of which are preserved in the Marine Natural Products Laboratory of the School of Marine Sciences of Sun Yat-sen University where the inventor works.

[0049] All α-keto acids, amino acid standards, NAD + and NADH were purchased from Sigma-Aldrich; other commonly used reagents were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0050] The three-letter or one-letter representation of amino acids used in the present invention adopts the amino acid code specified by IUPAC (Eur. J. Biochem., 138: 9-37, 1984).

[0051] Example 1

[0052] 1. Discovery of valine dehydrogenase gene and construction of recombinant engineering bacteria

[0053] The online tool MetaGeneMark (http: / / exon.gatech.edu / meta_gmhmmp.cgi) was used to predict gene function from the metagenomic sequencing data of the Yunnan hot spring (pH 3, 75.3℃) sediments. The amino acid sequence of the previously reported valine dehydrogenase (NCBI ID: Q53872.1) was used as a probe, and the predicted gene sequence encoding the amino acid dehydrogenase was searched using the local BLAST tool. A valine dehydrogenase with a 46% identity to the probe amino acid sequence was obtained, named HsVDH. The amino acid sequence of the valine dehydrogenase is shown in SEQ ID NO.3, and the gene sequence is shown in SEQ ID NO.1.

[0054] The gene sequence of the obtained valine dehydrogenase HsVDH was codon-optimized for the E. coli expression system and handed over to the company for full synthesis. The optimized gene sequence is shown in SEQ ID NO. 2. The synthesized gene was connected to the pET-28a(+) plasmid (with a His-tag purification tag at the N-terminus) by a one-step cloning kit (ClonExpress II One Step Cloning Kit) to construct a recombinant plasmid pET-28a-HsVDH. The recombinant plasmid was transformed into E. coli BL21(DE3) competent cells, positive clones were screened and detected, and recombinant engineered bacteria containing the HsVDH gene were obtained.

[0055] 2. Expression and purification of recombinant valine dehydrogenase HsVDH

[0056] The recombinant engineering bacteria successfully constructed above were streaked and activated on LB solid medium containing 50 μg / mL Kan; a single colony was picked and inoculated into 5 mL LB liquid medium containing 50 μg / mL Kan, and cultured at 37°C, 200 rpm, and shaken for 10-12 h; 2% of the inoculum was transferred to 250 mL of fresh LB liquid medium also containing 50 μg / mL Kan, and cultured at 37°C, 200 rpm, and shaken until OD 600 When the p-value reached 0.8, IPTG was added at a final concentration of 0.5 mM and the culture was induced at 18°C ​​and 200 rpm for 16 h. After the culture was completed, the bacteria were collected by centrifugation at 9000 rpm and 4°C, and the bacteria were washed twice with physiological saline.

[0057] Recombinant valine dehydrogenase HsVDH was purified by nickel column affinity chromatography:

[0058] 1) Sample pretreatment: Resuspend the cells in loading buffer (20 mM pH 7.5 phosphate buffer, 25 mM imidazole, 500 mM NaCl), disrupt by ultrasonication in an ice bath, centrifuge at 4°C, 12000 rpm to remove cell debris, and filter the supernatant with a 0.22 μm filter membrane;

[0059] 2) Loading: Wash the nickel column with 10 column volumes of loading buffer, and slowly load the pretreated sample in (1) onto the column to allow the protein to fully bind to the nickel column;

[0060] 3) Washing: Wash the nickel column with 10 column volumes of washing buffer (20 mM pH 7.5 phosphate buffer, 50 mM imidazole, 500 mM NaCl);

[0061] 4) Elution of target protein: Elution of target protein was performed with elution buffer (20 mM pH 7.5 phosphate buffer, 250 mM imidazole, 500 mM NaCl);

[0062] 5) Ultrafiltration and storage: The collected eluate was transferred to an ultrafiltration tube, and the protein sample was desalted and concentrated by centrifugal ultrafiltration at 4°C; glycerol with a final concentration of 20% was added to the ultrafiltered protein sample, and the sample was quickly frozen in liquid nitrogen and stored at -80°C.

[0063] 3. Determination of amino acid oxidative deamination activity of purified recombinant valine dehydrogenase HsVDH

[0064] The amino acid oxidation deamination activity detection system (1 mL) is: an appropriate amount of enzyme (0.01-1 mg recombinant HsVDH), 10 mM L-amino acid, 0.5 mM NAD + The reaction medium was 0.1 M Tris-HCl buffer (pH 9.0) and the reaction temperature was 35°C. The reaction was triggered by adding enzyme solution and the OD within 1 min was detected by spectrophotometer. 340 Changes in nm.

[0065] Definition of oxidative deaminase activity unit (U): the amount of enzyme required to generate 1 μmol NADH per minute under standard reaction conditions.

[0066] According to the above enzyme activity determination method, the present invention determines the specific activity of the purified recombinant valine dehydrogenase HsVDH to 9 L-amino acids, and the results are shown in Table 1. From the results shown in Table 1, it can be seen that the valine dehydrogenase HsVDH can catalyze the oxidative deamination of various L-amino acids, among which the enzyme has the highest catalytic activity for valine, with a specific enzyme activity of 4839.4 U / g, indicating that HsVDH belongs to valine dehydrogenase.

[0067] Table 1 Catalytic activity of HsVDH towards different amino acids

[0068]

[0069]

[0070] Note: ND means no catalytic activity detected

[0071] Example 2 Thermal stability of HsVDH

[0072] The pure HsVDH protein purified in Example 1 was diluted to an appropriate concentration with 0.1 M Tris-HCl buffer (pH 9.0), and kept warm in a water bath at 40°C, 45°C and 50°C, respectively. Samples were taken at time intervals of 24 h (40°C), 12 h (45°C) or 1 h (50°C), and the residual enzyme activity was determined by the amino acid oxidation deamination activity detection method described in Example 1. The inactivation curves of valine dehydrogenase HsVDH at different temperatures were plotted to characterize its thermal stability.

[0073] The inactivation curves of valine dehydrogenase HsVDH at different temperatures are shown in Figure 2. Figure 1 As shown by Figure 1 The results show that HsVDH still has 75.4% enzyme activity after being stored at 40°C for 14 days, and the half-life calculated by fitting reaches 832.9h, indicating that HsVDH has very good temperature stability.

[0074] Example 3 Rational Design and Modification of HsVDH

[0075] The invention obtains a HsVDH mutant by site-directed mutation of isoleucine (Ile) at position 294 of the HsVDH amino acid sequence into valine (Val) and alanine (Ala), thereby broadening the substrate spectrum of HsVDH.

[0076] Specifically, when the isoleucine at position 294 is site-directed mutated to valine, the amino acid sequence of the valine dehydrogenase mutant (I294V) is shown in SEQ ID NO.4; when the isoleucine at position 294 is site-directed mutated to alanine, the amino acid sequence of the valine dehydrogenase mutant (I294A) is shown in SEQ ID NO.5.

[0077] 1. Construction of I294V and I294A mutants

[0078] The present invention designed specific primers (see Table 2) and introduced mutations using the Quickchange technology (An efficient one-step site-directed and site-saturation mutagenesis protocol [J]. Nucleic Acids Research, 2004, 32 (14): e115) to construct I294V and I294A mutants.

[0079] Table 2 HsVDH site-directed mutagenesis primers

[0080]

[0081] Note: The underlined codons encode the mutated amino acid.

[0082] The PCR reaction system and reaction conditions are as follows:

[0083] PCR amplification system:

[0084]

[0085] The plasmid template is the recombinant expression plasmid pET-28a-HsVDH constructed in Example 1.

[0086] PCR amplification conditions:

[0087] 1) Pre-denaturation: 95°C for 2 min;

[0088] 2) Denaturation: 95°C for 15 seconds; annealing: 56°C for 15 seconds; extension: 72°C for 3 minutes; 30 cycles in total;

[0089] 3) Post-extension: 72°C for 8 min;

[0090] 4) Store at 4℃.

[0091] After PCR amplification, the amplified product was digested with DpnⅠ digestion enzyme for 3 hours to remove the template plasmid, and the digestion product was transformed into E.coli BL21 (DE3) competent cells, plated, and positive single colonies were picked to LB liquid culture. The correctness of the mutation was verified by sequencing, and the positive mutant single colonies that showed correct mutation after verification were stored at -80℃ for future use.

[0092] 2. Determination of amino acid oxidative deamination activity of HsVDH mutants

[0093] The obtained mutants I294V and I294A single colonies were streaked and activated on LB solid culture medium containing 50 μg / mL Kan; the recombinant HsVDH mutants I294V and I294V were cultured and collected according to the expression culture method described in Example 1, and the proteins were purified by nickel column affinity chromatography, and the purification process was the same as in Example 1.

[0094] Determination of the catalytic activity of HsVDH mutants I294V and I294A on different keto acid substrates: The activity detection system is (1 mL): appropriate amount of enzyme (0.0001-0.01 mg recombinant HsVDH mutant I294V or I294A), 10 mM keto acid, 0.15 mM NADH, the reaction medium is 1 M NH4Cl / NH3·H2O buffer (pH 9.0), the reaction temperature is 40 ° C, the reaction is triggered by adding enzyme solution, and the OD within 1 min is detected by spectrophotometer 340 Changes in nm.

[0095] Definition of reductive amination enzyme activity unit (U): the amount of enzyme required to consume 1 μmol NADH per minute under standard reaction conditions.

[0096] The results are shown in Table 3. It can be seen from the results shown in Table 3 that the HsVDH mutant exhibits different substrate specificity from the wild type. Compared with the wild type, the catalytic activity of I294V and I294A for large steric hindrance substrates such as 4-methyl-2-oxopentanoic acid, 3-methyl-2-oxopentanoic acid, phenylacetoic acid, 2-oxo-4-phenylbutyric acid, etc. is significantly improved, indicating that the present invention has successfully changed the substrate preference of HsVDH through site-directed mutagenesis and broadened the practical application scenarios of the enzyme.

[0097] Table 3 Catalytic activity of wild-type and mutant HsVDH on different ketoacid substrates

[0098]

[0099] Example 4 Biocatalytic Preparation of L-2-Aminobutyric Acid

[0100] According to the method shown in Example 1, the gene encoding formate dehydrogenase (the amino acid sequence of the enzyme is shown in SEQ ID NO.6) was connected to the pET-28a (+) plasmid (with a His-tag purification tag at the N-terminus), and the ligation product was transformed into E. coli BL21 (DE3) competent cells, and positive clones were screened and detected to obtain recombinant engineered bacteria containing the formate dehydrogenase gene. The obtained recombinant engineered bacteria were subjected to expression culture to obtain recombinant formate dehydrogenase and purified.

[0101] Biocatalytic preparation of L-2-aminobutyric acid: The total reaction system is 30 mL: weigh 0.2 M sodium 2-butyrate, 0.25 M ammonium formate and 0.2 M NH4Cl, dissolve them in deionized water, add ammonia water to adjust the pH to 9.0, add 0.2 mM NAD + , 0.5 mg valine dehydrogenase HsVDH and 31 mg formate dehydrogenase were used to start the reaction. The reaction device was a magnetic stirrer, the reaction temperature was controlled at 40°C by a water bath, and the pH was controlled at 9.0 by dropping ammonia water during the reaction.

[0102] The residual concentration of the substrate 2-ketobutyric acid was detected by non-chiral high performance liquid chromatography to monitor the reaction progress; the chromatographic column model used was Athena C18, 5μm, 4.6×250mm; 2-ketobutyric acid analysis mobile phase: 50mM pH 7.5 phosphate buffer: acetonitrile = 0.8:0.05 (mL / min); detection wavelength: 210nm; column temperature: 30°C; 2-ketobutyric acid peaks at 5.8min under the mobile phase conditions.

[0103] After the reaction is completed, the concentration and ee value of L-2-aminobutyric acid are detected by pre-column derivatization HPLC. The specific analysis method is as follows:

[0104] (1) Chromatographic conditions: Chromatographic column model: Athena C18, 5μm, 4.6×250mm; detection wavelength: 338nm; column temperature: 30℃; L-2-aminobutyric acid mobile phase: 0~15min mobile phase A (50mM pH 5.7 sodium acetate solution): mobile phase B (acetonitrile / water=10 / 1.5)=0.9:01 (mL / min), 15~40min mobile phase A: mobile phase B concentration gradient decreased to 0.75:0.25 (mL / min);

[0105] (2) Derivatization reagent: Weigh 0.03 g of o-phthalaldehyde and 0.1 g of N-acetyl-L-cysteine ​​respectively, dissolve them in 400 μL of ethanol, add 4 mL of 0.1 mol / L boric acid buffer (pH 9.8), shake to fully dissolve, and store in a refrigerator at 4°C until used;

[0106] (3) Derivatization reaction and determination: Take 100 μL of sample and add 150 μL of derivatization reagent. Mix well and keep at 25°C for 5 min. Add 1 mL of ddH2O for dilution. After passing through the membrane, inject 20 μL of the sample.

[0107] The reaction formula for preparing chiral L-amino acids by the valine dehydrogenase HsVDH derived from the hot spring metagenome of the present invention is as follows: Figure 2 As shown by Figure 2 It can be seen that the valine dehydrogenase HsVDH of the present invention can be added to a mixed system containing an α-keto acid substrate, an amino donor and a coenzyme regeneration system for a reductive amination reaction to obtain a chiral L-amino acid. The coenzyme regeneration system can be a system that uses formate dehydrogenase as a coenzyme regeneration enzyme, ammonium formate as a coenzyme regeneration substrate, and contains NADH and NAD + A formate dehydrogenase coenzyme regeneration system; or a system using glucose dehydrogenase as a coenzyme regeneration enzyme and glucose as a coenzyme regeneration substrate, including NADH and NAD + Glucose dehydrogenase coenzyme regeneration system; or alcohol dehydrogenase as coenzyme regeneration enzyme, isopropanol as coenzyme regeneration substrate, including NADH and NAD + Alcohol dehydrogenase coenzyme regeneration system.

[0108] The HPLC analysis spectrum of D / L-2-amino acid butyric acid standard sample after pre-column derivatization is shown in Figure 3 As shown in A, the detection spectrum of the amino acid product generated in this example is as follows Figure 4 As shown in A, Figure 3 A and 4A show that the retention time of the product prepared by valine dehydrogenase HsVDH in this example is the same as that of the L-2-aminobutyric acid standard, indicating that the product is L-2-aminobutyric acid. The reaction process of L-2-aminobutyric acid is as follows Figure 5 As shown by Figure 5 It can be seen that the conversion rate of 0.2M sodium 2-butyric acid is greater than 99.0% in 2 hours. The results of pre-column derivatization HPLC showed that the concentration of L-2-aminobutyric acid was 19.02 g / L, and the calculated space-time yield was 234.72 g·L -1 ·d -1 The ee value of the product L-2-aminobutyric acid is >99.0%, indicating that the L-aminobutyric acid prepared using the HsVDH of the present invention has a very high optical purity.

[0109] Example 5 Biocatalytic Preparation of L-tert-Leucine (0.2 M Substrate)

[0110] The formate dehydrogenase used in this example is the same as that in Example 4.

[0111] Preparation of biocatalytic reaction of L-tert-leucine: The total reaction system is 30 mL: weigh 0.2 M 3,3-dimethyl-2-oxobutyric acid and 0.25 M ammonium formate, dissolve them in deionized water, add ammonia water to adjust the pH to 9.0, add 0.2 mM NAD + , 1 mg of valine dehydrogenase HsVDH and 31 mg of formate dehydrogenase were used to start the reaction. The reaction device was a magnetic stirrer, the reaction temperature was controlled at 40°C by a water bath, and the pH was controlled at 9.0 by dropping ammonia water during the reaction.

[0112] The residual concentration of the substrate 3,3-dimethyl-2-oxobutanoic acid was detected by non-chiral high performance liquid chromatography to monitor the reaction progress; the chromatographic column model used was Athena C18, 5μm, 4.6×250mm; 3,3-dimethyl-2-oxobutyric acid analysis mobile phase: 50mM pH 7.5 phosphate buffer: acetonitrile = 0.9:0.1 (mL / min); detection wavelength: 210nm; column temperature: 30°C; 3,3-dimethyl-2-oxobutyric acid peaked at 6.7min under the mobile phase conditions.

[0113] After the reaction is completed, the concentration and ee value of L-tert-leucine are detected by pre-column derivatization HPLC. The specific analysis method is as follows:

[0114] (1) Chromatographic conditions: Chromatographic column model: Athena C18, 5μm, 4.6×250mm; detection wavelength: 338nm, column temperature: 30°C; L-tert-leucine mobile phase: mobile phase A (23mM, pH 6.0 sodium acetate solution): mobile phase B (acetonitrile / water=10 / 1.5)=0.8:0.2 (mL / min);

[0115] (2) Derivatization reagent: Weigh 0.03 g of o-phthalaldehyde and 0.1 g of N-acetyl-L-cysteine ​​respectively, dissolve them in 400 μL of ethanol, add 4 mL of 0.1 mol / L boric acid buffer (pH 9.8), shake to fully dissolve, and store in a refrigerator at 4°C until used;

[0116] (3) Derivatization reaction and determination: Take 100 μL of sample and add 150 μL of derivatization reagent. Mix well and keep at 25°C for 5 min. Add 1 mL of ddH2O for dilution. After passing through the membrane, inject 20 μL of the sample.

[0117] The chromatogram of the pre-column derivatization HPLC analysis of D / L-tert-leucine standard is as follows: Figure 3 As shown in B, the detection spectrum of the amino acid product generated in this example is as follows Figure 4 As shown in B, Figure 3 B and 4B show that the retention time of the product prepared by valine dehydrogenase HsVDH in this example is the same as that of the L-tert-leucine standard sample, indicating that the product is L-tert-leucine. The reaction process of preparing L-tert-leucine by valine dehydrogenase HsVDH is as follows: Figure 6 As shown by Figure 6 It can be seen that the conversion rate of 0.2M 3,3-dimethyl-2-oxobutyric acid is greater than 99.0% at 3.5h. The test results show that the concentration of L-tert-leucine produced is 27.76g / L, and the calculated space-time yield is 179.89g·L -1 ·d -1 The ee value of the product L-tert-leucine is >99.0%, indicating that the L-tert-leucine prepared by the HsVDH of the present invention has a very high optical purity.

[0118] Example 6 Biocatalytic Preparation of L-tert-Leucine (0.4 M Substrate)

[0119] The formate dehydrogenase used in this example is the same as that in Example 4.

[0120] The total system of the biocatalytic reaction of L-tert-leucine is 30mL: weigh 0.4M 3,3-dimethyl-2-oxobutyric acid and 0.45M ammonium formate, dissolve them with deionized water, add ammonia water to adjust the pH to 9.0, add 0.2mM NAD+, 1mg valine dehydrogenase HsVDH and 31mg formate dehydrogenase to start the reaction. The reaction device is a magnetic stirrer, and the reaction temperature is controlled to 40°C by a water bath. The pH is controlled to 9.0 by adding ammonia water during the reaction. The residual concentration of the substrate 3,3-dimethyl-2-oxobutyric acid is detected by non-chiral high-performance liquid phase to monitor the reaction progress. After the reaction, the formation concentration and ee value of L-tert-leucine are detected by pre-column derivatization high-performance liquid phase. The specific method is the same as Example 5.

[0121] The retention time of the product prepared by valine dehydrogenase HsVDH in this example is similar to that of the L-tert-leucine standard sample ( Figure 3 B) is the same, indicating that the obtained product is L-tert-leucine. The reaction process of valine dehydrogenase HsVDH preparing L-tert-leucine is as follows Figure 7 As shown by Figure 7 It can be seen that the conversion rate of 0.4M 3,3-dimethyl-2-oxobutyric acid is greater than 99.0% in 5 hours. The test results show that the concentration of L-tert-leucine produced is 52.39 g / L, and the calculated space-time yield is 251.49 g·L -1 ·d -1 , indicating that under high substrate concentration conditions, HsVDH can still achieve efficient preparation of L-tert-leucine and maintain the strict stereoselectivity of its catalytic reaction; the ee value of the product L-tert-leucine is >99.0%, indicating that the prepared L-tert-leucine has a very high optical purity.

[0122] Example 7 Biocatalytic Preparation of L-Phenylglycine

[0123] The formate dehydrogenase used in this example is the same as that in Example 4.

[0124] The total system of L-phenylglycine biocatalytic reaction is 30mL: weigh 0.2M phenylacetoic acid, 0.25M ammonium formate and 5% (v / v) glycerol, dissolve them in deionized water, add ammonia water to adjust the pH to 9.0, add 0.2mM NAD+, 1mg valine dehydrogenase HsVDH and 31mg formate dehydrogenase to start the reaction. The reaction device is a magnetic stirrer, the reaction temperature is controlled at 40°C by a water bath, and the pH is controlled at 9.0 by adding ammonia water during the reaction.

[0125] The residual concentration of the substrate phenylacetoic acid was detected by non-chiral high performance liquid chromatography to monitor the reaction progress; the chromatographic column model used was Athena C18, 5μm, 4.6×250mm; mobile phase for phenylacetoic acid analysis: 50mM pH 7.5 phosphate buffer: acetonitrile = 0.9:0.1 (mL / min); detection wavelength: 220nm; column temperature: 30°C; phenylacetoic acid peaks at 7.7min under the mobile phase conditions.

[0126] After the reaction is completed, the concentration and ee value of L-phenylglycine are detected by pre-column derivatization HPLC; the specific analysis method is as follows:

[0127] (1) Chromatographic conditions: Chromatographic column model: Athena C18, 5μm, 4.6×250mm; detection wavelength: 338nm; column temperature: 30°C; L-phenylglycine mobile phase: mobile phase A (50mM, sodium acetate solution): mobile phase B (methanol) = 0.6:0.4 (mL / min);

[0128] (2) Derivatization reagent: Weigh 0.03 g of o-phthalaldehyde and 0.1 g of N-acetyl-L-cysteine ​​respectively, dissolve them in 400 μL of ethanol, add 4 mL of 0.1 mol / L boric acid buffer (pH 9.8), shake to fully dissolve, and store in a refrigerator at 4°C until used;

[0129] (3) Derivatization reaction and determination: Take 100 μL of sample and add 150 μL of derivatization reagent. Mix well and keep at 25°C for 5 min. Add 1 mL of ddH2O for dilution. After passing through the membrane, inject 20 μL of the sample.

[0130] The HPLC analysis spectrum of D / L-phenylglycine standard sample after pre-column derivatization is shown in Figure 3 C, the detection spectrum of the amino acid product generated in this example is as shown in Figure 4 As shown in C, Figure 3 C and 4C show that the retention time of the product prepared by valine dehydrogenase HsVDH in this example is the same as that of L-phenylglycine, indicating that the product is L-phenylglycine. The reaction process of valine dehydrogenase HsVDH preparing L-phenylglycine is shown in FIG. Figure 8 As shown by Figure 8 It can be seen that the conversion rate of 0.2M phenylacetoic acid is greater than 99.0% in 12 hours. The test results show that the concentration of L-phenylglycine produced is 28.68g / L, and the calculated space-time yield is 57.35g·L -1 ·d -1 The ee value of the product L-phenylglycine was >99.0%, indicating that the prepared L-phenylglycine had a very high optical purity.

[0131] Example 8 Preparation of L-phenylglycine by valine dehydrogenase mutant (I294V)

[0132] The formate dehydrogenase used in this example is the same as that in Example 4.

[0133] The total reaction system for the biocatalytic preparation of L-phenylglycine by the valine dehydrogenase mutant (I294V) was 30 mL: 0.2 M phenylacetoic acid and 0.2 M ammonium formate were weighed and dissolved in deionized water, 5% (v / v) glycerol was added, ammonia was added dropwise to adjust the pH to 9.0, and 0.2 mM NAD +, 1 mg of valine dehydrogenase mutant I294V and 31 mg of formate dehydrogenase were used to start the reaction. The reaction device was a magnetic stirrer, and the reaction temperature was controlled to 40°C by a water bath. During the reaction, the pH was controlled to 9.0 by dropping ammonia water. The residual concentration of the substrate phenylacetoic acid was detected by non-chiral high performance liquid chromatography to monitor the reaction progress. After the reaction was completed, the generated concentration and ee value of L-phenylglycine were detected by pre-column derivatization high performance liquid chromatography, and the specific method was the same as in Example 7.

[0134] The detection spectrum of the amino acid product generated in this example is as follows Figure 4 As shown in D, the retention time of the pre-column derivatization HPLC analysis of the product prepared by the valine dehydrogenase HsVDH mutant (I294V) in this example is the same as that of the L-phenylglycine standard sample, indicating that the product is L-phenylglycine. The reaction process of preparing L-phenylglycine by the valine dehydrogenase mutant (I294V) is shown in FIG. Fig. 9 As shown by Fig. 9 It can be seen that the conversion rate of 0.2M phenylacetoic acid is greater than 99.0% in 6 hours. The test results show that the concentration of L-phenylglycine produced is 29.96g / L, and the calculated space-time yield is 119.86g·L -1 ·d -1 , indicating that the valine dehydrogenase mutant (I294V) has a higher catalytic efficiency than the wild-type HsVDH while maintaining the same excellent stereoselectivity as the wild-type, and the ee value of the product L-phenylglycine is >99.0%, indicating that the prepared L-phenylglycine has a very high optical purity.

[0135] Example 9 Preparation of L-phenylglycine by valine dehydrogenase mutant (I294A)

[0136] The formate dehydrogenase used in this example is the same as that in Example 4.

[0137] The total reaction system for the biocatalytic preparation of L-phenylglycine by the valine dehydrogenase mutant (I294A) was 30 mL: 0.2 M phenylacetoic acid and 0.2 M ammonium formate were weighed and dissolved in deionized water, 5% (v / v) glycerol was added, ammonia was added dropwise to adjust the pH to 9.0, and 0.2 mM NAD + , 1 mg of valine dehydrogenase mutant I294A and 31 mg of formate dehydrogenase were used to start the reaction. The reaction device was a magnetic stirrer, and the reaction temperature was controlled at 40°C by a water bath. During the reaction, the pH was controlled at 9.0 by dropping ammonia water. The residual concentration of the substrate phenylacetoic acid was detected by non-chiral high performance liquid chromatography to monitor the reaction progress. After the reaction was completed, the generated concentration and ee value of L-phenylglycine were detected by pre-column derivatization high performance liquid chromatography, and the specific method was the same as in Example 7.

[0138] The detection spectrum of the amino acid product generated in this example is as follows Figure 4As shown in Figure E, the retention time of the product prepared by pre-column derivatization HPLC analysis using the valine dehydrogenase HsVDH mutant (I294A) in this example is the same as that of the L-phenylglycine standard sample, indicating that the product is L-phenylglycine. The reaction process of preparing L-phenylglycine by the valine dehydrogenase mutant (I294A) is shown in Figure 5. Fig.10 As shown by Fig.10 It can be seen that the conversion rate of 0.2M phenylacetoic acid is greater than 99.0% in 10 hours. The test results show that the concentration of L-phenylglycine produced is 29.31 g / L, and the calculated space-time yield is 70.34 g·L -1 ·d -1 , indicating that the valine dehydrogenase mutant (I294A) also has a higher catalytic efficiency than the wild-type HsVDH, and the ee value of the product L-phenylglycine is >99.0%, indicating that the prepared L-phenylglycine has a very high optical purity.

[0139] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A valine dehydrogenase, characterized in that The amino acid sequence of the valine dehydrogenase is shown in SEQ ID NO.

3.

2. A valine dehydrogenase mutant, characterized in that: The mutant is obtained by site-directed mutation of isoleucine at position 294 of the valine dehydrogenase amino acid sequence shown in SEQ ID NO.3 to valine or alanine.

3. Use of the valine dehydrogenase according to claim 1 or the valine dehydrogenase mutant according to claim 2 in the preparation of chiral L-amino acids, characterized in that: The chiral L-amino acid is L-2-aminobutyric acid, L-tert-leucine or L-phenylglycine.

4. A method for preparing a chiral L-amino acid, characterized in that: The method comprises: preparing an enzyme solution of the valine dehydrogenase according to claim 1 or the valine dehydrogenase mutant according to claim 2, adding the prepared enzyme solution or the transgenic engineering bacterial cells capable of expressing the valine dehydrogenase according to claim 1 or the valine dehydrogenase mutant according to claim 2 to a mixed system containing an α-keto acid substrate, an amino donor and a coenzyme regeneration system for reductive amination reaction to obtain a chiral L-amino acid; the chiral L-amino acid is L-2-aminobutyric acid, L-tert-leucine or L-phenylglycine.

5. The method according to claim 4, characterized in that: The enzyme solution is a crude enzyme solution or a purified enzyme solution of a genetically engineered bacterium capable of expressing the valine dehydrogenase according to claim 1 or the valine dehydrogenase mutant according to claim 2.

6. The method according to claim 4, characterized in that: The α-keto acid substrate is 2-ketobutyric acid, 3,3-dimethyl-2-oxobutyric acid, phenylacetoacetic acid or their respective salt forms.

7. The method according to claim 4, characterized in that: The coenzyme regeneration system uses formate dehydrogenase as the coenzyme regeneration enzyme and ammonium formate as the coenzyme regeneration substrate, including NADH and NAD + A formate dehydrogenase coenzyme regeneration system; or a system using glucose dehydrogenase as a coenzyme regeneration enzyme and glucose as a coenzyme regeneration substrate, including NADH and NAD + Glucose dehydrogenase coenzyme regeneration system; or alcohol dehydrogenase as coenzyme regeneration enzyme, isopropanol as coenzyme regeneration substrate, including NADH and NAD + Alcohol dehydrogenase coenzyme regeneration system.

8. The method according to claim 4, characterized in that The substrate concentration of the reductive amination reaction is 0.2 M to 0.4 M.

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