L-glutamate dehydrogenase mutant and its application

Abstract

The present invention relates to an L-glutamate dehydrogenase mutant and its application, belonging to the field of enzyme engineering, wherein the mutant has a mutation at least at one of the following positions of the wild-type L-glutamate dehydrogenase amino acid sequence: the 96th lysine is mutated to arginine, the 97th glycine is mutated to threonine, the 170th alanine is mutated to lysine, the 199th threonine is mutated to tyrosine, and the 383rd serine is mutated to tyrosine. The amino acid sequence of the wild-type L-glutamate dehydrogenase mutant is shown in SEQ ID NO.1. The L-glutamate dehydrogenase mutant provided by the present invention has significantly improved catalytic activity for non-natural substrate oxaloacetate, and the molecular modification of L-glutamate dehydrogenase by the present invention expands its substrate specificity and improves its value in industrial applications.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to an L-glutamate dehydrogenase mutant and application thereof. Background Art

[0002] L-Aspartic acid is a commonly used organic chemical raw material, which can be used as a synthetic precursor for amino acids such as lysine, threonine, isoleucine, and methionine. As an important precursor raw material, aspartic acid amino acids are widely used in the fields of medicine, food additives, and chemical industry. For example, aspartic acid can be used as a raw material for the new polymer material polyaspartic acid; lysine is often used as a feed additive, etc., accounting for more than half of the global amino acid market demand. At present, enzyme conversion is the main method for producing aspartic acid amino acids, usually using aspartate aminotransferase and aspartate dehydrogenase as the main catalysts. However, in the current reports on aspartate aminotransferase, there are still problems such as low product conversion rate and the presence of by-products, which affect the recovery and purification of the product; and although the application of aspartate dehydrogenase can remove the influence of reaction by-products on the subsequent product extraction, its source in nature is relatively small, and the enzyme activity is generally low, making it difficult to develop.

[0003] L-glutamate dehydrogenase is widely present in bacteria, yeast, animals and plants. It is a key enzyme that is important for the balance of redox in the intracellular environment and metabolism and other life activities. Its coenzyme is NAD + or NADP + , but according to their different dependence on coenzymes, they can be divided into NAD + Dependent, NADP + Dependent, NAD + / NADP + Dual-dependence type. L-glutamate dehydrogenase is not only widely used, such as in the preparation of medical reagent blood ammonia detection kits; the preparation of enzyme electrodes in biosensors, etc., but also has great application potential and value in the field of amino acid synthesis. Although L-glutamate dehydrogenase has the advantages of strict stereoselectivity, high enzyme activity, and easy separation and purification of reaction products, it has strong substrate specificity and extremely weak catalytic activity on non-natural substrates, which limits its wider application. Therefore, the present invention improves the catalytic activity of L-glutamate dehydrogenase on non-natural substrates through molecular modification, which will help expand the application of L-glutamate dehydrogenase in industry. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide an L-glutamate dehydrogenase mutant and its application. The present invention transforms the wild-type L-glutamate dehydrogenase (LpGDH, WP_003643310.1, gene sequence as shown in SEQ ID NO.2) from Lactobacillus plantarum through random mutation, semi-rational design and other technologies to obtain an L-glutamate dehydrogenase mutant with catalytic activity of non-natural substrates, and further improves its catalytic activity.

[0005] The present invention is achieved through the following technical solutions:

[0006] One of the objects of the present invention is to provide a L-glutamate dehydrogenase mutant.

[0007] The second object of the present invention is to provide a gene encoding the L-glutamate dehydrogenase mutant.

[0008] The third object of the present invention is to provide a recombinant plasmid and an expression vector comprising the gene.

[0009] A fourth object of the present invention is to provide a recombinant cell transformed with the above-mentioned recombinant plasmid.

[0010] A fifth object of the present invention is to provide the use of the recombinant plasmid, expression vector or recombinant cell in the preparation of L-aspartic acid.

[0011] The sixth object of the present invention is to provide the use of the recombinant plasmid, expression vector or recombinant cell in the preparation of L-phosphoamino.

[0012] To achieve the above object, the technical solution of the present invention is as follows:

[0013] A L-glutamate dehydrogenase mutant, wherein the amino acid sequence of the mutant is mutated at least at one of the following positions of the wild-type L-glutamate dehydrogenase amino acid sequence: lysine at position 96 is mutated to arginine (Lys96Arg), glycine at position 97 is mutated to threonine (Gly97Thr), alanine at position 170 is mutated to lysine (Ala170Lys), threonine at position 199 is mutated to tyrosine (Thr199Tyr), and serine at position 383 is mutated to tyrosine (Ser383Tyr), and the amino acid sequence of the wild-type L-glutamate dehydrogenase is shown in SEQ ID NO.1.

[0014] Specifically, the mutants are: BL21 (DE3) / PET28a-LpGDHmK96R, BL21 (DE3) / PET28a-LpGDHmG97T, BL21 (DE3) / PET28a-LpGDHmA170K, BL21 (DE3) / PET28a-LpGDHmT199Y, BL21 (DE3) / PET28a-LpGDHmS383Y, LpGDHmT199Y-K96R, LpGDHmT199Y-G97T, Lp GDHmT199Y-A170K, LpGDHmT199Y-S383Y, LpGDHmT199Y-K96R-G97T, LpGDHmT199Y-K96R-A170K, LpGDHmT199Y-K96R-S383Y, LpGDHmT199Y-K96R-G97T-A170K, LpGDHmT199Y-K96R-G97T-S383Y or LpGDHmT199Y-K96R-G97T-A170K-S383Y.

[0015] The present invention provides a gene sequence encoding wild-type L-glutamate dehydrogenase, as shown in SEQ ID NO.2.

[0016] The present invention also provides a recombinant plasmid and an expression vector comprising the above gene. The plasmid comprises a vector for expressing the above gene, preferably a PET series, such as PET28a, but not limited thereto.

[0017] The present invention also provides a recombinant cell, which carries the above gene or the above recombinant plasmid. The recombinant cell can be used as a host to express the above L-glutamate dehydrogenase mutant.

[0018] Preferably, the recombinant cell is selected from Corynebacterium glutamicum, Bacillus, yeast, and Escherichia coli. In one embodiment of the present invention, the recombinant cell is Escherichia coli BL21 (DE3) as the host cell.

[0019] The present invention also provides the use of the above-mentioned L-glutamate dehydrogenase mutant, recombinant plasmid, expression vector or recombinant cell in the preparation of L-aspartic acid. The method is: adding the above-mentioned L-glutamate dehydrogenase mutant, recombinant plasmid, expression vector or recombinant cell to a reaction system containing oxaloacetate, glucose dehydrogenase and NADP to react to obtain.

[0020] The present invention also provides the use of the above-mentioned L-glutamate dehydrogenase mutant, recombinant plasmid, expression vector or recombinant cell in enzymatic hydrolysis of 2-carbonyl-4-(hydroxymethylphosphonyl)butyric acid (PPO), wherein the use is to carry out a catalytic reaction using PPO as a substrate and isopropanol as a hydrogen donor to obtain L-phosphoammonium; the mutant is

[0021] LpGDHmT199Y-K96R-S383Y, LpGDHmT199Y-K96R-A170K, LpGDHmT199Y-K96R-G97T-S383Y, and recombinant plasmids, expression vectors or recombinant cells containing the mutant genes.

[0022] In one embodiment of the present invention, the reaction coenzyme is reduced nicotinamide adenine dinucleotide phosphate (NADPH for short) or nicotinamide adenine dinucleotide phosphate (NADP for short). The NADPH / NADP regeneration system used in the present invention is known in the art, and the optional dehydrogenases include but are not limited to glucose dehydrogenase, formate dehydrogenase or alcohol dehydrogenase. In some embodiments, the NADPH / NADP regeneration system is: glucose dehydrogenase or alcohol dehydrogenase is used as a coenzyme regeneration enzyme, and glucose or isopropanol is used as a hydrogen donor.

[0023] As an optional embodiment, the above-mentioned recombinant cells can be added to the reaction system in the form of wet bacteria or cell fragments thereof.

[0024] The beneficial effects of the present invention compared with the prior art are as follows:

[0025] The present invention is based on the wild-type L-glutamate dehydrogenase (LpGDH, WP_003643310.1) from Lactobacillus plantarum, and through random mutation and semi-rational design molecular transformation methods, a mutant of L-glutamate dehydrogenase that can catalyze non-natural substrates and has high activity is obtained. The mutant is applied to the synthesis of L-aspartic acid, with oxaloacetate as the substrate, in the presence of inorganic ammonia and coenzymes, the conversion rate can reach up to 98.2%, which has very large industrial application and development prospects.

[0026] The mutant of the present invention can also perform a catalytic reaction with 2-carbonyl-4-(hydroxymethylphosphonyl)butyric acid (PPO) as a substrate and isopropanol as a hydrogen donor to obtain L-phosphinothion. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is a comparison of the relative enzyme activities of L-glutamate dehydrogenase and its mutants;

[0028] Figure 2 is the physical map of the pMV-Adh recombinant plasmid;

[0029] Figure 3 This is the physical map of the pET-21b+Adh recombinant plasmid. DETAILED DESCRIPTION

[0030] The present invention is further described below in conjunction with specific implementation examples, and the advantages and features of the present invention will become clearer as the description proceeds. However, the specific experimental methods involved in the following implementation examples are all conventional methods or implemented under the conditions recommended by the manufacturer's instructions unless otherwise specified.

[0031] Unless otherwise specified, the technical means used in the examples are conventional means known to those skilled in the art. The test methods in the following examples are conventional methods unless otherwise specified. Unless otherwise specified, the reagents and materials used can all be purchased from the market.

[0032] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applied to the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0033] The L-glutamate dehydrogenase mutant constructed by the present invention is derived from the wild-type L-glutamate dehydrogenase of Lactobacillus plantarum, and the amino acid sequence is shown in SEQ ID NO.1, which is a new protein formed after the amino acids in the sequence of SEQ ID NO.1 are replaced. The wild-type L-glutamate dehydrogenase encoding gene is shown in SEQ ID NO.2 in the sequence table.

[0034] In order to obtain L-glutamate dehydrogenase with catalytic activity on non-natural substrates, the present invention uses the gene sequence of wild-type L-glutamate dehydrogenase (SEQ ID NO.2) as a basis, uses error-prone PCR to perform base mutation, and obtains L-glutamate dehydrogenase mutants.

[0035] The specific experimental process is as follows:

[0036] The whole gene synthesis and primer synthesis in the following experiments were completed by BGI. The molecular biology experiments in the implementation examples, including plasmid construction, enzyme digestion, ligation, preparation of competent cells, gene transformation, and preparation of culture medium, were carried out according to the reaction conditions provided by the supplier or the instructions of the kit. Simple adjustments were made when necessary.

[0037] The materials and instruments used in the experiment are as follows:

[0038] Vector and strain: The expression vectors used were pET-28a and PET-21b, the plasmids were purchased from Novagen, and the host cells used were Escherichia coli BL21 (DE3), purchased from Quanshijin Biotechnology Co., Ltd.

[0039] Glucose dehydrogenase: Glucose dehydrogenase from Anoxybacillus flavithermus synthesized by BGI (NCBI accession number NC_011567.1) was recombinantly expressed using pET-28a as the expression vector and Escherichia coli BL21 (DE3) as the host cell.

[0040] Alcohol dehydrogenase: pMV-Adh recombinant plasmid synthesized by BGI (such as Figure 2 As shown), pET-21b was used as the expression vector (as Figure 3 As shown in the figure, Escherichia coli BL21 (DE3) was used as the host cell for recombinant expression, and the synthesized alcohol dehydrogenase (ADH) gene sequence was SEQ ID NO.3.

[0041] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, adjusted to pH 7.0 with 1M sodium hydroxide, and sterilized at 121°C for 20 min. (LB solid medium is supplemented with 15 g / L agar powder).

[0042] TB medium: tryptone 12g / L, yeast extract 24g / L, K2HPO4·3H2O 16.43g / L, KH2PO42.31g / L, glycerol 5g / L, pH 7.0-7.5, high temperature sterilization at 121℃ for 20min. (TB solid medium is added with 15g / L agar powder on this basis).

[0043] Reverse phase high performance liquid chromatography (HPLC) analysis method of the product L-aspartic acid: After the L-aspartic acid in the reaction solution is derivatized using the OPA derivatization method of amino acids, it is analyzed using HPLC. The specific implementation steps are as follows:

[0044] Derivatization step: Centrifuge the conversion solution at 12000 rpm for 1 min, take the supernatant, dilute the supernatant 5-10 times and take 1 ml, add 1.5 ml OPA derivatization reagent, mix well, and derivatize at room temperature in a dark environment for 10 min. After the reaction is completed, perform HPLC detection.

[0045] HPLC conditions were as follows: chromatographic column: C18 column, mobile phase A: 50 mmol / L ammonium acetate solution (pH 6.0); mobile phase B: methanol, mobile phase A:B = 93:7; detection wavelength: 345 nm; column temperature: 25 °C; flow rate: 1 ml / min; injection volume: 20 ul.

[0046] Example 1 Construction of wild-type L-glutamate dehydrogenase gene recombinant Escherichia coli

[0047] 1. For L-glutamate dehydrogenase LpGDH from Lactobacillus plantarum, according to the literature ( r,et al., Complete genome sequence of Lactobacillusplantarum WCFS1. 2003 Feb 18; 100(4): 1990–1995. andRoland J. Siezen,et al.,Complete Resequencing and Reannotation of theLactobacillus plantarum WCFS1 Genome.Journal of Bacteriology. 2012 Jan; 194(1): 195–196.) Based on the gene sequence published by BGI, namely SEQ ID NO.2, the whole gene was synthesized by BGI, and restriction endonuclease sites BamH I and Xho I were introduced at both ends of the gene. The restriction fragment of L-glutamate dehydrogenase LpGDH was ligated with the restriction fragment of vector pET28a to obtain the recombinant plasmid pET28a-LpGDH.

[0048] 2. The plasmid pET28a-LpGDH was transformed into Escherichia coli BL21 (DE3) competent cells by heat shock transformation to obtain recombinant Escherichia coli expressing wild-type L-glutamate dehydrogenase LpGDH.

[0049] Example 2 Construction of a random mutant library of wild-type L-glutamate dehydrogenase LpGDH by error-prone PCR

[0050] 1. Using wild-type L-glutamate dehydrogenase recombinant plasmid pET28a-LpGDH as template, random mutant library was constructed by error-prone PCR technology. Error-prone PCR kit was purchased from Clontech (PT3396-1); single point mutation kit was purchased from Nanjing Novezan Biotechnology Co., Ltd. (Novezan, C214).

[0051] 2. Error-prone PCR amplification was performed using the Takara Biotech Error-prone PCR Kit (Clontech, PT3396-1), plasmid pET28a-LpGDH as template, and LpGDH-F and LpGDH-R as primers.

[0052] The primer sequences used are as follows:

[0053] LpGDH-F: acagcaaatgggtcgcggatcc ATGATCATATTGAGTCAAGCAACCG (SEQ ID NO.4),

[0054] LpGDH-R: gtggtggtggtggtggtgctcgag AACTAAGCCTTGCGCTAACATG (SEQ ID NO.5);

[0055] The underlined letters are homology arm sequences.

[0056] The PCR reaction system is 50 uL: template DNA 1uL (final concentration is about 1ng / uL), forward primer (10 nM) and reverse primer (10 nM) 1uL each, 50× Diversify dNTP Mix 1uL, dGTP (2mM) 1uL, MnSO4 (8mM) 2uL, 10× TITANIUM Taq Buffer 5uL, TITANIUM Taq Polym 1uL, ddH2O 37uL.

[0057] The error-prone PCR program was: 97℃ pre-denaturation for 30 sec, followed by 25 cycles of 97℃ 30 sec, 68℃ 2 min. The reaction was terminated by a final extension at 68℃ for 1 min. Result: An amplified band of approximately 1356 bp was successfully obtained.

[0058] The amplified band and the double-enzyme (BamH I and Xho I) fragment of vector pET28a were recovered using a DNA gel recovery kit, and homologous recombination was performed using the Novizan single mutation kit. 20 μL of the reaction system was directly used for heat shock transformation of Escherichia coli BL21 (DE3) competent cells to obtain the L-glutamate dehydrogenase mutant library.

[0059] Construction of recombinant Escherichia coli: Thaw the competent E. coli BL21 (DE3) cells on ice, then add 20 μL of the above fusion reaction system, mix gently and place on ice for 30 min, heat shock in a 42°C water bath for 30 sec, immediately place on ice for 2 min, add 800 μL of liquid LB medium, and culture at 37°C, 180 rpm for 1 h; spread on LB solid medium plates containing 50 mg / L kanamycin, and culture inverted at 37°C for 14-16 h to obtain L-glutamate dehydrogenase mutant engineered bacteria containing the recombinant plasmid.

[0060] Example 3 High-throughput screening of mutant library

[0061] 1. Preparation of mutant crude enzyme liquid wet cells

[0062] The mutant recombinant E. coli monoclone obtained in Example 2 was picked into a sterile 96-deep-well plate, 1 ml of LB liquid culture medium containing 50 mg / L kanamycin was added to each well, and the plate was placed in a shaker at 37°C and 180 rpm for 8 hours, and then 500 ul of bacterial solution was transferred to another 96-deep-well plate containing 500 ul of LB liquid culture medium with 50 mg / L kanamycin and a final concentration of 0.2 M IPTG per well at a ratio of 1:1, and cultured overnight at 20°C and 180 rpm in a shaker for 16 hours. After the culture was completed, the cells were collected in a 96-deep-well plate by centrifugation at 8000 rpm for 5 minutes at room temperature to obtain a large number of wet cells of recombinant E. coli containing mutant genes, i.e., wet cells of mutants.

[0063] 2. Preliminary screening

[0064] Detection of L-glutamate dehydrogenase activity: The enzyme activity was detected using the HPLC method as described above. The specific steps are as follows: 1 ml of a reaction solution consisting of oxaloacetate (20 mmol / L), NADPH (0.12 mM), and NH4Cl-NH4OH (200 mM, pH 8.0) buffer was added to a 96-well plate, the reaction solution was preheated at 50°C for 10 min before detection, and then 0.1 g of wet bacteria was added and immediately mixed to start the reaction. After 30 min at 50°C, the reaction solution was centrifuged and the supernatant was aspirated and diluted 5-10 times, and derivatized using the OPA derivatization method, and the L-aspartic acid production was detected by HPLC. According to the above enzyme activity detection method, the L-aspartic acid production of each single clone in the L-glutamate dehydrogenase mutant library was determined, and mutant strains with L-aspartic acid production greater than the wild-type L-glutamate dehydrogenase LpGDH original strain were screened.

[0065] 3. Rescreening

[0066] The wet cells of the strains obtained in the initial screening were used as catalysts for rescreening. The rescreening method was the same as the initial screening except for the addition of the reaction system. The amount of L-aspartic acid produced by the original recombinant strain of wild-type L-glutamate dehydrogenase LpGDH was defined as the relative enzyme activity of 100%.

[0067] Through the above steps, about 2000 mutant strains were screened, and 9 dominant mutant strains with catalytic activity were obtained, which were named LpGDHm-1, LpGDHm-2, LpGDHm-3, LpGDHm-4, LpGDHm-5, LpGDHm-6, LpGDHm-7, LpGDHm-8, and LpGDHm-9. The results are as follows Figure 1 shown.

[0068] 4. Nucleotide sequence determination of L-glutamate dehydrogenase mutant

[0069] The L-glutamate dehydrogenase mutants LpGDHm-1, LpGDHm-2, LpGDHm-3, LpGDHm-4, LpGDHm-5, LpGDHm-6, LpGDHm-7, LpGDHm-8, and LpGDHm-9 strains obtained by the above screening were inoculated into 3 ml of LB liquid culture medium containing 50 mg / L kanamycin, and cultured in a shaker at 37°C and 180 rpm for 14-16 hours. At room temperature, centrifuge at 12000 rpm for 1 min to collect the bacteria, and extract the plasmid according to the method described in the instructions of the plasmid extraction kit. This experiment uses the plasmid purification kit (AG21001) of Acori Biotechnology, but it is not limited to the plasmid purification kit of the biological company, and any commercial plasmid extraction kit can be used. The extracted plasmid was sent to a sequencing company for sequence determination.

[0070] The sequencing results show that the amino acid sequences of the L-glutamate dehydrogenase mutants LpGDHm-1, LpGDHm-2, LpGDHm-3, LpGDHm-4, LpGDHm-5, LpGDHm-6, LpGDHm-7, LpGDHm-8 and LpGDHm-9 of the present invention are mutated at different positions, among which the mutation sites of LpGDHm-1, LpGDHm-5 and LpGDHm-8 are consistent, and the 96th lysine is mutated to arginine, the mutation sites of LpGDHm-3 and LpGDHm-7 are consistent, and the 97th glycine is mutated to threonine, the mutation site of LpGDHm-2 is the 170th alanine is mutated to lysine, the mutation site of LpGDHm-9 is the 199th threonine is mutated to tyrosine, and the mutation sites of LpGDHm-4 and LpGDHm-6 are consistent, and the 383rd serine is mutated to tyrosine. That is, BL21(DE3) / PET28a-LpGDHmK96R, BL21(DE3) / PET28a-LpGDHmG97T, BL21(DE3) / PET28a-LpGDHmA170K, BL21(DE3) / PET28a-LpGDHmT199Y, and BL21(DE3) / PET28a-LpGDHmS383Y were obtained.

[0071] Example 4: Fusion of multi-site mutants of L-glutamate dehydrogenase and construction of engineered bacteria

[0072] Since the L-glutamate dehydrogenase mutant LpGDHm-9, namely LpGDHmT199Y, has the highest activity in catalyzing the synthesis of L-aspartate from oxaloacetate, multi-site mutations were further constructed based on LpGDHm-9, namely LpGDHmT199Y.

[0073] The multi-site mutagenesis of L-glutamate dehydrogenase was completed using the single-site mutagenesis kit (NoviZan, C214) from Novezan. The primers were designed as shown in Table 1:

[0074] Table 1 Primers for site-directed mutagenesis of L-glutamate dehydrogenase

[0075] .

[0076] Using the vector PET28a-LpGDHmT199Y as a template and K96R-F / K96R-R in the table as primers, the 96th lysine in the amino acid sequence of the L-glutamate dehydrogenase mutant LpGDHmT199Y was mutated to arginine by site-directed mutagenesis to obtain PET28a-LpGDHmT199Y-K96R.

[0077] Using the vector PET28a-LpGDHmT199Y as a template and G97T-F / G97T-R in the table as primers, the glycine at position 97 in the amino acid sequence of the L-glutamate dehydrogenase mutant LpGDHmT199Y was mutated to threonine by site-directed mutagenesis to obtain PET28a-LpGDHmT199Y-G97T.

[0078] Using the vector PET28a-LpGDHmT199Y as a template and A170K-F / A170K-R in the table as primers, the 170th alanine in the amino acid sequence of the L-glutamate dehydrogenase mutant LpGDHmT199Y was mutated to lysine by site-directed mutagenesis to obtain PET28a-LpGDHmT199Y-A170K.

[0079] Using the vector PET28a-LpGDHmT199Y as a template and S383Y-F / S383Y-R in the table as primers, the serine at position 383 of the amino acid sequence of the L-glutamate dehydrogenase mutant LpGDHmT199Y was mutated to tyrosine by site-directed mutagenesis to obtain PET28a-LpGDHmT199Y-S383Y.

[0080] Using the vector PET28a-LpGDHmT199Y-K96R as a template and G97T-F / G97T-R in the table as primers, the glycine at position 97 in the amino acid sequence of the L-glutamate dehydrogenase mutant LpGDHmT199Y-K96R was mutated to threonine by site-directed mutagenesis to obtain PET28a-LpGDHmT199Y-K96R-G97T.

[0081] Using the vector PET28a-LpGDHmT199Y-K96R as a template and A170K-F / A170K-R in the table as primers, the 170th alanine in the amino acid sequence of the L-glutamate dehydrogenase mutant LpGDHmT199Y-K96R was mutated to lysine by site-directed mutagenesis to obtain PET28a-LpGDHmT199Y-K96R-A170K.

[0082] Using the vector PET28a-LpGDHmT199Y-K96R as a template and S383Y-F / S383Y-R in the table as primers, the serine at position 383 of the amino acid sequence of the L-glutamate dehydrogenase mutant LpGDHmT199Y-K96R was mutated to tyrosine by site-directed mutagenesis to obtain PET28a-LpGDHmT199Y-K96R-S383Y.

[0083] Using the vector PET28a-LpGDHmT199Y-K96R-G97T as a template and A170K-F / A170K-R in the table as primers, the 170th alanine in the amino acid sequence of the L-glutamate dehydrogenase mutant LpGDHmT199Y-K96R-G97T was mutated to lysine by site-directed mutagenesis to obtain PET28a-LpGDHmT199Y-K96R-G97T-A170K.

[0084] Using the vector PET28a-LpGDHmT199Y-K96R-G97T as a template and S383Y-F / S383Y-R in the table as primers, the serine at position 383 of the amino acid sequence of the L-glutamate dehydrogenase mutant LpGDHmT199Y-K96R-G97T was mutated to tyrosine by site-directed mutagenesis to obtain PET28a-LpGDHmT199Y-K96R-G97T-S383Y.

[0085] Using the vector PET28a-LpGDHmT199Y-K96R-G97T-A170K as a template and S383Y-F / S383Y-R in the table as primers, the serine at position 383 of the amino acid sequence of the L-glutamate dehydrogenase mutant LpGDHmT199Y-K96R-G97T-A170K was mutated to tyrosine by site-directed mutagenesis to obtain PET28a-LpGDHmT199Y-K96R-G97T-A170K-S383Y.

[0086] The mutation PCR system (50 μL) was: 25 μL 2×Max buffer, 1 μL dNTP, a final concentration of 1 ng / μL template plasmid, 2 μL each of forward primer and reverse primer (10 mM each), 1 μL Phanta Max Super-Fidelity DNA polymerase, and finally filled to 50 μL with ddH2O.

[0087] PCR reaction program: pre-denaturation at 95°C for 30 sec; 30 cycles of denaturation at 95°C for 15 sec, annealing at 60°C for 15 sec, and extension at 72°C for 6 min; and finally total extension at 72°C for 5 min.

[0088] The amplified fragment was directly added with 1 μL Dpn I, digested at 37℃ for 1h, and then subjected to agarose gel electrophoresis. After the fragment was recovered, the recombination reaction was carried out. The reaction system was: 50-400ng of the above Dpn I digestion product, 4 μL 5×CE II Buffer, 2μLExnase II, and filled to 20μL with ddH2O. After mixing, it was fused at 37℃ for 30min, immediately placed on ice for 10min, and then transformed into Escherichia coli BL21 (DE3) by heat shock method. It was spread on LB solid culture medium plates containing 50 mg / L kanamycin and inverted in a 37℃ incubator for 14-16h. Recombinant bacteria containing multiple site mutations were obtained.

[0089] The correct recombinant bacteria verified by sequencing were preserved and used to further detect the activity of catalyzing oxaloacetate.

[0090] Example 5 Determination of enzyme activity and kinetic parameters of wild-type L-glutamate dehydrogenase and its mutants

[0091] 1. Purification of target protein

[0092] The advantageous L-glutamate dehydrogenase mutant and wild-type L-glutamate dehydrogenase LpGDH obtained in Example 4 were washed twice with 50 mM pH 8.0 phosphate buffer, and then resuspended in 50 mM pH 8.0 phosphate buffer containing 0.3 M NaCl and 30 mM imidazole, ultrasonically disrupted for 10 min, centrifuged at 12,000 rpm for 10 min at 4°C, and the supernatant was collected. The protein was purified using a His tag purification column (Roche, COHISC-RO, 6781535001).

[0093] The L-glutamate dehydrogenase mutants PET28a-LpGDHmT199Y, PET28a-LpGDHmT199Y-K96R, PET28a-LpGDHmT199Y-G97T, PET28a-LpGDHmT199Y-A170K, PET28a-LpGDHmT199Y-S383Y, PET28a-LpGDHmT199Y-K96R-G97T, PET28a-LpGDHmT199Y-A170K, PET28a-LpGDHmT199Y-S383Y The concentrations of pure enzyme solutions of S383Y, PET28a-LpGDHmT199Y-K96R-G97T-A170K, PET28a-LpGDHmT199Y-K96R-G97T-S383Y, PET28a-LpGDHmT199Y-K96R-G97T-A170K-S383Y, and wild-type L-glutamate dehydrogenase pET28a-LpGDH were determined using the modified Bradford protein concentration determination kit of Bioengineering (Bioengineering, C503041). For specific methods, please refer to the website of Bioengineering, and all proteins were diluted to the same concentration with 50 mM pH 8.0 phosphate buffer.

[0094] 2. Enzyme activity detection:

[0095] The catalytic system was: 50 mM oxaloacetate, 0.12 mM NADPH, 200 mM NH4Cl-NH4OH (pH 8.0), the reaction was carried out at 50°C, and the enzyme activity was determined by HPLC.

[0096] Enzyme activity (U) definition: The amount of enzyme required to generate 1umol of L-aspartic acid per minute is one enzyme activity unit. Specific enzyme activity: The activity units contained in each milligram of enzyme protein. The test results are shown in Table 2:

[0097] Table 2 Specific enzyme activities of L-glutamate dehydrogenase and its mutants

[0098] .

[0099] 3. Analysis of the kinetic parameters of L-glutamate dehydrogenase and its dominant mutants

[0100] Using the wild-type L-glutamate dehydrogenase and its mutants purified in 1 above, kinetic analysis was performed at a fixed concentration of NADPH (0.12mM) at 30°C and pH 8.0 to measure the enzyme activity under different substrate (oxaloacetic acid) concentrations of 2-20mM. Based on the obtained data, a relationship curve between the maximum reaction rate (Vmax) and the substrate concentration was plotted, and the Michaelis-Menten equation was used to obtain the Km value. The Kcat value was calculated by Vmax and the enzyme concentration. The results are shown in Table 3 below:

[0101] Table 3 Comparison of kinetic parameters of L-glutamate dehydrogenase and its mutants

[0102] .

[0103] “—” indicates that data was not detected.

[0104] Comparison of the Km value of the mutant with that of the wild type shows that the replacement of amino acids in the L-glutamate dehydrogenase mutant significantly increases the affinity of the enzyme to the substrate, and its ability to catalyze non-natural substrates is greatly improved compared with the wild-type L-glutamate dehydrogenase.

[0105] Example 6 Enzyme thermal stability test

[0106] Optimal reaction temperature determination: The purified mutant LpGDHmT199Y-K96R-S383Y, LpGDHmT199Y-K96R-A170K, LpGDHmT199Y-K96R-G97T-S383Y enzyme solutions were placed in the same assay system and reacted at different temperatures of 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, and 80°C to determine their enzyme activities. The highest enzyme activity was taken as 100%. The results are shown in Table 4:

[0107] Table 4 Optimal catalytic temperature of mutants

[0108] .

[0109] It can be seen from Table 4 that the optimal reaction temperature of the L-glutamate dehydrogenase mutant is 50°C.

[0110] Thermal stability test: The enzyme solutions of the purified wild-type L-glutamate dehydrogenase LpGDH and the L-glutamate dehydrogenase mutants LpGDHmT199Y-K96R-S383Y, LpGDHmT199Y-K96R-A170K, and LpGDHmT199Y-K96R-G97T-S383Y were heated at 40°C, 50°C, 60°C, and 70°C for 6 hours, and reacted at 50°C for 30 minutes to determine their relative enzyme activities. The untreated enzyme activity was taken as 100%.

[0111] The catalytic system for determining the activity of wild-type L-glutamate dehydrogenase LpGDH was: 50 mM α-ketoglutarate, 0.12 mM NADPH, 200 mM NH4Cl-NH4OH (pH 8.0). The results are shown in Table 5:

[0112] Table 5 Relative enzyme activities of L-glutamate dehydrogenase and its mutants after treatment at different temperatures

[0113] .

[0114] As can be seen from Table 5, the relative enzyme activity of the L-glutamate dehydrogenase mutants gradually decreased with the increase of the incubation temperature, but the relative enzyme activities of the mutants LpGDHmT199Y-K96R-S383Y, LpGDHmT199Y-K96R-A170K, and LpGDHmT199Y-K96R-G97T-S383Y were all higher than those of the wild-type L-glutamate dehydrogenase LpGDH under the same treatment conditions, proving that the thermal stability of the L-glutamate dehydrogenase mutants was higher than that of the wild-type L-glutamate dehydrogenase, among which the mutant LpGDHmT199Y-K96R-S383Y had the highest thermal stability.

[0115] Example 7 Fermentation and application of L-glutamate dehydrogenase mutant

[0116] 1. Pick the strains containing LpGDHmT199Y-K96R-S383Y, LpGDHmT199Y-K96R-A170K, and LpGDHmT199Y-K96R-G97T-S383Y recombinant plasmids, inoculate them into 3 mL LB liquid medium containing 50 mg / L kanamycin, and culture them at 37°C, 180 rpm overnight. Inoculate them into 150 mL TB medium at a volume ratio of 1% (V / V), and culture them at 30°C, 180 rpm until OD600 is 0.4-0.6. Then add 0.2 mM IPTG and continue to induce the culture for 6 hours. Then, collect the bacteria by centrifugation at 10,000 rpm for 10 min and store them in a -80°C refrigerator.

[0117] 2. According to the method in step 1 of Example 7, the wild-type L-glutamate dehydrogenase LpGDH strain was fermented in a shake flask, the cells were collected, and stored in a -80°C refrigerator.

[0118] 3. Add the wet cells obtained in the above steps 1 and 2 to 50 mL of the catalytic reaction system at a final concentration of 10 g / L. The substrates include 50 mM oxaloacetate, 0.12 mM NADP, and 10 g / L glucose dehydrogenase.

[0119] Specifically, the reaction catalyst and substrate were added to a 100 ml shake flask, the reaction system was 50 ml, the rotation speed was 200 rpm / min, the pH was controlled at 8.0 with ammonia water, the temperature was controlled at 50°C, the reaction was carried out for 30 minutes, samples were taken at regular intervals, the supernatant was collected by centrifugation at 6000 rpm, the reaction supernatant was treated with the OPA derivatization method, and the content of L-aspartic acid was detected by HPLC. The results are shown in Table 6:

[0120] Table 6 L-glutamate dehydrogenase and its mutants synthesize L-aspartate

[0121] .

[0122] “—” indicates that no product was detected.

[0123] As shown in Table 6, the wild-type L-glutamate dehydrogenase LpGDH cannot synthesize detectable L-aspartate, while the mutant LpGDHmT199Y-K96R-S383Y can catalyze the synthesis of L-aspartate from oxaloacetate, with a yield of 49.1 mmol / L and a substrate molar conversion rate of 98.2%.

[0124] Example 8 Application of L-glutamate dehydrogenase mutants in other directions

[0125] The cells of the wild-type L-glutamate dehydrogenase LpGDH strain stored in a -80°C refrigerator, and the cells of the strains containing the recombinant plasmids LpGDHmT199Y-K96R-S383Y, LpGDHmT199Y-K96R-A170K, and LpGDHmT199Y-K96R-G97T-S383Y were combined with alcohol dehydrogenase, and 2-carbonyl-4-(hydroxymethylphosphono)butyric acid (PPO) was used as a substrate and isopropanol as a hydrogen donor to carry out the catalytic reaction.

[0126] Specifically, the obtained bacteria were added to 50 mL of the catalytic reaction system at a final concentration of 5 g / L. The substrates included 250 mM 2-carbonyl-4-(hydroxymethylphosphonyl)butyric acid (PPO), 0.5 M isopropanol, 0.15 mM NADP, and 5 g / L alcohol dehydrogenase.

[0127] The reaction catalyst and substrate were added to a 500 ml shake flask, the reaction system was 50 ml, the rotation speed was 200 rpm / min, the pH was controlled at 7.5-8.5 with ammonia water, the temperature was controlled at 30-50°C, the reaction was carried out for 10-30 minutes, samples were taken at regular intervals, the supernatant was collected by centrifugation at 6000 rpm, the reaction supernatant was treated with the OPA derivatization method, and the content of L-phosphochloridite was detected by HPLC. The results are shown in Table 7:

[0128] Table 7 L-glutamate dehydrogenase and its mutants synthesizing L-phosphoammonium

[0129] .

[0130] A novel L-glutamate dehydrogenase mutant was obtained by mutation screening of different amino acid sites of the wild-type L-glutamate dehydrogenase. Its substrate specificity was expanded, enabling it to catalyze the non-natural substrate oxaloacetate, and it also had good specificity for another substrate 2-carbonyl-4-(hydroxymethylphosphonyl)butyrate (PPO). This modification expanded the application range of L-glutamate dehydrogenase in the synthesis or detection of amino acids and other related products, and improved its industrial application value.

[0131] The embodiments described above are only preferred embodiments of the present invention and are only used to explain the present invention, not to limit the scope of implementation of the present invention. For those skilled in the art, other implementation methods can certainly be easily made by replacement or modification based on the technical contents disclosed in this specification. Therefore, all changes and improvements made on the principles of the present invention should be included in the scope of the patent application of the present invention.

[0132] Amino acid sequence of wild-type L-glutamate dehydrogenase: (SEQ ID NO.1)

[0133] MIILSQATDYVQHVYQVIEHRDPNQTEFLEAINDVFKTITPVLEQHPEYIEANILERLTEPERIIQFRVPWLDDAGHARVNRGFRVQFNSAIGPYKGGLRLHPSVNLSIVKFL GFEQIFKNALTGLPIGGGKGGSDFDPKGKSDNEIMRFCQSFMTELSKYIGLDTDVPAGDIGVGGREIGFLYGQYKRLRGADRGVLTGKGLNYGGSLARTEATGYGLAYYTNEM LKANQLSFPGQRVAISGAGNVAIYAIQKVEELGGKVITCSDSNGYVIDENGIDFKIVKQIKEVERGRIKDYADRVASASYYEGSVWDAQVAYDIALPCATQNEISGDQAKNLI ANGAKVVAEGANMPSSPEAIATYQAASLLYGPAKAANAGGVAVSALEMSQNSMRLSWTFEEVDNRLKQIMQDIFAHSVAAADEYHVSGDYLSGANIAGFTKVADAMLAQGLV.

[0134] The nucleotide sequence of wild-type L-glutamate dehydrogenase: (SEQ ID NO.2)

[0135]

[0136] Alcohol dehydrogenase gene: (SEQ ID NO.3)

[0137] atgaaagcagcaatagtttccgcagcaggcgaactgccggtatacggcgatttccctgaacctcaagccgatgaccaccaggttgtggtcacagtcaaagccgccgccattagccagttggcgaaggcgcgtgccgcaggttcccattacagctcggccacgcactatccgtttattaccgggatcgatggcaccggttatctcaacaatggcgacccggtgtacttcctggcatttaacgccccctggggaagtatggcggaaaaaacacgggtgcccaccgaggctatcgtcccgctaccgaaaacgctggatccggtgctggctgctgcacttgccaatccgggtatgtcctcgtgggcggctttaacgcgtcgcgcacgtttactacaaggcgaaaccgtgctaatcaatggcgcaaccgggacatcaggtggactggcagtgcgtatcgcgcgtcatcttggtgccggaaaaattattgccaccggacgcaaccgtgcggtgctggaacaacttcacacccagggggcagatattacgctgacccttgatgcgttaccgaccgctctgcccgcgctgatggctgacggcattgatgtggtgctggattatctgtggggccagagcgcgctcgatatcatgcaggcggcggtggccggtggtgagaaggtggtccgttttgtgcaaattggttccctcagcggccaggagatctcattgcacagcaagttactgcgctcctctgggctgaccctaatgggcagcggcctcggtagcgtatccaatagcgaactggtggcttgtatcggcgagctactgacagccgcagcgcaaagcgatttttcgattcccttccagatgcgaccactgagtgaagtccatagcgcctggggtgaagacgatagccgttgccgtacggtatttacgctctga 。

Claims

1. A L-glutamate dehydrogenase mutant, characterized in that: The mutant is a wild-type L-glutamate dehydrogenase amino acid sequence mutated, the wild-type L-glutamate dehydrogenase amino acid sequence is shown in SEQ ID NO.1; the mutant is: the 96th lysine mutates to arginine--BL21 (DE3) / PET28a-LpGDHmK96R, the 97th glycine mutates to threonine--BL21 (DE3) / PET28a-LpGDHmG97T, the 170th alanine mutates to lysine--BL21 (DE3) / PET28a-LpGDHmA170K, the 199th threonine mutates to tyrosine--BL21 (DE3) / PET28a-LpGDHmT199Y, the 383rd serine mutates to tyrosine--BL21 (DE3) / PET28a-LpG DHmS383Y, threonine at position 199 mutated to tyrosine and lysine at position 96 mutated to arginine--LpGDHmT199Y-K96R, threonine at position 199 mutated to tyrosine and glycine at position 97 mutated to threonine--LpGDHmT199Y-G97T, threonine at position 199 mutated to tyrosine and alanine at position 170 mutated to lysine--LpGDHmT199Y-A170K, threonine at position 199 mutated to tyrosine and serine at position 383 mutated to tyrosine--LpGDHmT199Y-S383Y, threonine at position 199 mutated to tyrosine and lysine at position 96 mutated to arginine The 199th threonine was mutated to tyrosine, the 96th lysine was mutated to arginine and the 170th alanine was mutated to lysine - LpGDHmT199Y-K96R-A170K, the 199th threonine was mutated to tyrosine, the 96th lysine was mutated to arginine and the 383rd serine was mutated to tyrosine - LpGDHmT199Y-K96R-S383Y, the 199th threonine was mutated to tyrosine, the 96th lysine was mutated to arginine, the 97th glycine was mutated to threonine and the 170th alanine was mutated to lysine - LpGDHmT199Y-K96R-S383Y The 199th threonine mutated to tyrosine, the 96th lysine mutated to arginine, the 97th glycine mutated to threonine and the 383rd serine mutated to tyrosine -- LpGDHmT199Y-K96R-G97T-S383Y or the 199th threonine mutated to tyrosine, the 96th lysine mutated to arginine, the 97th glycine mutated to threonine, the 170th alanine mutated to lysine and the 383rd serine mutated to tyrosine -- LpGDHmT199Y-K96R-G97T-A170K-S383Y.

2. A recombinant plasmid, characterized in that: The recombinant plasmid comprises the gene of the mutant according to claim 1.

3. An expression vector, characterized in that: The expression vector comprises the gene of the mutant according to claim 1.

4. A recombinant cell, characterized in that The recombinant cell carries the gene of the mutant according to claim 1.

5. Use of the L-glutamate dehydrogenase mutant according to claim 1, the recombinant plasmid according to claim 2, the expression vector according to claim 3 or the recombinant cell according to claim 4 in the preparation of L-aspartic acid, characterized in that: The application method comprises the following steps: performing a reaction in a reaction system containing oxaloacetic acid, glucose dehydrogenase and NADP to obtain L-aspartic acid.

6. Use of an L-glutamate dehydrogenase mutant, a recombinant plasmid containing the mutant, or a recombinant cell containing the mutant in enzymatic hydrolysis of 2-carbonyl-4-(hydroxymethylphosphonyl)butyric acid, characterized in that: The application is to use 2-carbonyl-4-(hydroxymethylphosphonyl)butyric acid as a substrate and isopropanol as a hydrogen donor to carry out a catalytic reaction to obtain L-phosphoammonium; the mutant is LpGDHmT199Y-K96R-S383Y, LpGDHmT199Y-K96R-A170K or LpGDHmT199Y-K96R-G97T-S383Y in the L-glutamate dehydrogenase mutant according to claim 1.

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