An Aspartate Dehydrogenase Mutant and Its Application

Abstract

This invention discloses an aspartate dehydrogenase mutant and its applications. The aspartate dehydrogenase can catalyze the production of aspartic acid from oxaloacetate, but its catalytic ability is low, with a molar conversion rate of only about 8.4% for the product aspartic acid. The mutant exhibits mutations at positions 155, 182, 273, and 355 of the original aspartate dehydrogenase amino acid sequence: proline at position 155 is mutated to serine, phenylalanine at position 182 is mutated to leucine, aspartate at position 273 is mutated to glycine, and valine at position 355 is mutated to glycine. The aspartate dehydrogenase mutant significantly increases the amount of aspartic acid produced from oxaloacetate, achieving a molar conversion rate of up to 95.2%, thus demonstrating high potential for industrial application.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an aspartic acid dehydrogenase mutant and its application in the production of aspartic acid. Background Technology

[0002] Aspartic acid (also known as aminosuccinic acid) is a non-essential amino acid in living organisms, possessing two optical isomers: D-form and L-form. Aspartic acid can act as an ammonia detoxifier, reducing blood ammonia levels and enhancing liver function. L-aspartic acid can be used to synthesize novel sweeteners suitable for patients with diabetes and phenylketonuria. Aspartic acid also acts as an ion carrier, improving myocardial contractility and can be used to synthesize drugs that protect the myocardium. It can also be used as an additive in some functional beverages and foods. Furthermore, in living organisms, aspartic acid is an important precursor in the synthesis of many amino acids, such as threonine, lysine, isoleucine, methionine, and homoserine, serving as a key node in the biosynthesis of these amino acids.

[0003] In living organisms, the biosynthesis of aspartic acid mainly occurs through three pathways ( Figure 1 The biosynthesis of aspartic acid consists of three pathways: one is the transamination of glutamate to oxaloacetate catalyzed by aspartate transaminase, leading to the synthesis of aspartic acid; another is the synthesis of aspartic acid from fumarate and its amino group catalyzed by aspartate taurine oxidase; and the third is the direct synthesis of aspartic acid from oxaloacetate and its amino group catalyzed by aspartate dehydrogenase. Since most biofermentation occurs under anaerobic conditions, and the first two pathways involve the synthesis of glutamate and fumarate from the tricarboxylic acid cycle, the activities of aspartate transaminase and aspartate taurine oxidase may be inhibited under anaerobic conditions. Therefore, utilizing aspartate dehydrogenase to directly catalyze the amination of oxaloacetate to form aspartic acid may be a key step in improving the biosynthesis of aspartic acid and its family of amino acids.

[0004] In 2003, the first aspartate dehydrogenase from *Thermotoga maritima* was reported. Subsequently, aspartate dehydrogenases were discovered in seven microorganisms, including *Archaeoglobus fulgidus*, *Pseudomonas aeruginosa*, *Ralstonia eutropha*, *Rhodopseudomonas palustris*, *Bradyrhizobium japonicum*, *Klebsiella pneumoniae 34618*, and *Delftia sp. Cs1-4*. However, due to the low sequence homology of aspartate dehydrogenases from different sources, the number of reported aspartate dehydrogenases is currently limited. Moreover, most reported aspartate dehydrogenases exhibit bidirectional catalytic function, which limits their use in the biosynthesis of aspartate. Therefore, it is necessary to continue screening and identifying aspartate dehydrogenases with higher activity. Based on previous work, this invention screened a possible aspartate dehydrogenase that can unidirectionally catalyze the reaction of oxaloacetate and amino groups to produce aspartic acid, but has no activity against aspartic acid. Due to the low catalytic efficiency of the enzyme, it is necessary to modify the enzyme to optimize its catalytic activity. Summary of the Invention

[0005] One of the objectives of this invention is to provide an aspartate dehydrogenase.

[0006] The second objective of this invention is to provide an aspartate dehydrogenase mutant.

[0007] A third objective of this invention is to provide a gene encoding the aforementioned aspartate dehydrogenase and its mutants.

[0008] The fourth objective of this invention is to provide recombinant plasmids and expression vectors containing the above-mentioned genes.

[0009] The fifth objective of this invention is to provide microorganisms transformed with the above-mentioned recombinant plasmids.

[0010] The sixth objective of this invention is to provide the application of the above-mentioned aspartate dehydrogenase and its mutant in the production of aspartic acid.

[0011] To achieve the above objectives, the technical solution of the present invention is summarized as follows:

[0012] This invention screened an aspartate dehydrogenase (BmAspDH, CP063276.1, nucleotide sequence as shown in SEQ ID NO.3, amino acid sequence as shown in SEQ ID NO.1) derived from Bacillus mojavensis LDFZ001. To improve the activity of this enzyme in catalyzing the transamination of oxaloacetate, the gene was randomly mutated. After enzyme activity screening, a mutant of aspartate dehydrogenase that unidirectionally catalyzes the transamination of oxaloacetate was obtained, laying the foundation for the enzyme-catalyzed production of aspartic acid and its family of amino acids.

[0013] The mutant of aspartate dehydrogenase BmASpDH is named BmAspDHmSLGG. The mutant has mutations at the following amino acid positions in the naïve aspartate dehydrogenase BmAspDH: proline at position 155 is mutated to serine, phenylalanine at position 182 is mutated to leucine, aspartate at position 273 is mutated to glycine, and valine at position 355 is mutated to glycine. The amino acid sequence of the BmAspDH mutant is shown in SEQ ID NO.2.

[0014] The gene of the aspartate dehydrogenase mutant BmAspDHmSLGG, wherein the nucleotide sequence of the mutant has the following mutations: base C at position 463 of the original aspartate dehydrogenase BmAspDH is mutated to T, base C at position 546 is mutated to G, base A at position 818 is mutated to G, and base T at position 1064 is mutated to G. The nucleic acid sequence of the aspartate dehydrogenase mutant BmAspDHmSLGG is shown in SEQ ID NO.4.

[0015] The aspartate dehydrogenase can generate aspartic acid using NADH as a coenzyme and oxaloacetic acid and amino groups as substrates.

[0016] The enzyme used in the experiments of this invention is aspartate dehydrogenase and its mutant derived from Bacillus mojave LDFZ001.

[0017] Specifically, the enzyme is:

[0018] In the aspartate dehydrogenase BmAspDH of Bacillus mojave LDFZ001, position 155 is mutated from proline to serine, position 182 from phenylalanine to leucine, position 273 from aspartic acid to glycine, and position 355 from valine to glycine. The mutant aspartate dehydrogenase containing these four mutation sites is designated BmAspDHmSLGG.

[0019] The DNA molecule encoding the enzyme described in this invention was cloned using PCR. In a specific embodiment, seven possible aspartate dehydrogenase genes were screened from the genome sequence of Bacillus mojave LDFZ001 using homology alignment. After cloning the corresponding genes using specific primers, heterologous expression was performed in Escherichia coli, and enzyme activity was identified. One gene was screened to have the activity of catalyzing the amination of oxaloacetate to aspartic acid. Then, error-prone PCR was used to introduce a mutation point into this gene, and mutants with significantly increased enzyme activity were obtained.

[0020] The present invention also provides recombinant plasmids, expression vectors, and recombinant cells containing the recombinant plasmids for the aforementioned DNA molecules, all of which fall within the scope of protection of the present invention. The plasmid contains a vector for expressing the aforementioned DNA molecules, preferably pET28a, but is not limited thereto.

[0021] It also includes microorganisms transformed with the above-mentioned recombinant plasmids, which can serve as hosts for expressing the above-mentioned aspartate dehydrogenase and its mutants. Preferably, the above-mentioned microorganisms are Escherichia coli BL21(DE3), but are not limited thereto.

[0022] The above-mentioned aspartic acid dehydrogenase and its mutants or microorganisms can also be used to produce aspartic acid family amino acids.

[0023] In the production of aspartic acid, oxaloacetic acid and ammonium chloride are used as substrates. The above-mentioned aspartic acid dehydrogenase mutant catalyzes the production of aspartic acid from oxaloacetic acid and ammonium chloride, with NADH as a coenzyme.

[0024] As an alternative implementation method, for example, the concentration of oxaloacetic acid and ammonium chloride in the reaction system can be selected as 1% (mass / volume ratio), the concentration of coenzyme NADH is 0.1 g / L, the reaction temperature is 30-37℃, preferably 37℃, and the pH of the reaction system is 8.0.

[0025] More preferably, the method for producing aspartic acid specifically comprises:

[0026] (1) Preparation of wet bacterial cells of aspartate dehydrogenase mutant;

[0027] First, a single colony of recombinant Escherichia coli with aspartate dehydrogenase mutant was inoculated into 3 mL of LB medium containing 50 μg / mL kanamycin sulfate and cultured overnight at 37°C and 180 rpm. Then, 1% of the colony was inoculated into 150 mL of TB medium and cultured at 30°C and 180 rpm until the OD600 reached 0.4-0.6. 0.2 mM IPTG was then added and the culture was induced for another 6 hours. The wet cells were then collected by centrifugation at 10,000 rpm for 10 min and stored at -80°C.

[0028] (2) The obtained wet bacterial cells were added to a 1L reaction system at a final concentration of 20g / L. The substrates oxaloacetic acid and ammonium chloride were added at a mass / volume ratio of 1%. The final concentration of coenzyme NADH was 0.1g / L. 50mM pH8.0 phosphate buffer was added. The reaction temperature was 37℃ and the catalytic period was 5h. After the reaction was completed, 1mL of the reaction solution was taken for HPLC detection.

[0029] The method for generating recombinant bacteria with aspartate dehydrogenase mutants is as follows: using the recombinant plasmid pET28a-BmAspDH containing the initial type of aspartate dehydrogenase gene as a template, error-prone PCR amplification is performed. The PCR fragment is fused with the BamHI and XhoI double enzyme digestion fragments of pET28a and then transformed into BL21(DE3) competent cells. The obtained positive clones are screened for enzyme activity.

[0030] The aspartate dehydrogenase mutant provided by this invention can be applied to the regulation of metabolic pathways of aspartate family amino acids, but it is not limited to this. Other metabolic pathway modification processes using aspartate as a precursor can also be applied.

[0031] Advantages of this invention:

[0032] This invention provides a novel aspartate dehydrogenase mutant, which exhibits significantly enhanced enzyme activity in catalyzing the amination of oxaloacetate. In the production of aspartate, using oxaloacetate and ammonium chloride as substrates and NADH as a coenzyme, the aspartate dehydrogenase mutant catalyzes the synthesis of aspartate. During the catalytic process, this aspartate dehydrogenase mutant is inactive for aspartate and cannot catalyze the reaction from aspartate to oxaloacetate, thus reducing the loss of aspartate. The system containing the aspartate dehydrogenase mutant provided by this invention exhibits minimal substrate residue and a significantly improved product molar conversion rate, reaching up to 95.2%. In contrast, the system containing the initial aspartate dehydrogenase BmAspDH contains a large amount of residual oxaloacetate, with a product molar conversion rate of only 8.4%. Therefore, the aspartate dehydrogenase mutant provided by this invention has high prospects for industrialization and application. Attached Figure Description

[0033] Figure 1 It is the biosynthetic pathway of aspartic acid in organisms.

[0034] Figure 2 This is a comparison of the enzyme activities of BmAspDH and its various mutants.

[0035] Figure 3 This is an HPLC analysis of the catalytic products of BmAspDH and the mutant BmAspDHmSLGG. Detailed Implementation

[0036] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, unless otherwise specified, the specific experimental methods involved in the following embodiments are conventional methods or implemented according to the conditions recommended in the manufacturer's instructions.

[0037] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the experimental methods in the following embodiments are all conventional methods. Unless otherwise specified, the reagents and materials used can be purchased commercially.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be used in this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0039] This invention utilizes amino acid sequence homology alignment, using the amino acid sequences of BjaAspDH (WP_014492994), DelAspDH (WP_239973385.1), KpnAspDH (AGT22664.1), ReuAspDH (WP_041680386.1), MplAspDH (ACL16745.1), AfAspDH (AF18388), and PaeAspDH (PA3505) as bait, to search for possible aspartate dehydrogenases in the sequencing library of Bacillus mojave LDFZ001 (CP063276.1), obtaining a total of 7 possible aspartate dehydrogenases.

[0040] Primer synthesis in the following experiments was performed by BGI Genomics. The molecular biology experiments in the examples, including plasmid construction, enzyme digestion, ligation, preparation of competent cells, gene transformation, and culture medium preparation, were conducted according to the reaction conditions provided by the supplier or the kit instructions. Simple adjustments can be made as needed.

[0041] The materials and instruments used in the experiment are as follows:

[0042] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, adjusted to pH 7.0 with 1 M sodium hydroxide, sterilized at 121°C for 20 min. (LB solid medium is prepared with 15 g / L agar powder added).

[0043] TB medium: 12 g / L tryptone, 24 g / L yeast extract, 16.43 g / L K2HPO4·3H2O, 2.31 g / L KH2PO4, 5 g / L glycerol, pH 7.0-7.5, sterilized at 121℃ for 20 min (TB solid medium is prepared with 15 g / L agar powder added).

[0044] Example 1: Screening of potential aspartate dehydrogenases

[0045] 1. Using the amino acid sequences of BjaAspDH (WP_014492994), DelAspDH (WP_239973385.1), KpnAspDH (AGT22664.1), ReuAspDH (WP_041680386.1), MplAspDH (ACL16745.1), AfAspDH (AF18388), and PaeAspDH (PA3505) as bait, possible aspartate dehydrogenases were searched in the sequencing library of Bacillus mojave LDFZ001 (CP063276.1). A total of 7 possible aspartate dehydrogenases were obtained and named BmAspDH1, BmAspDH2, BmAspDH3, BmAspDH4, BmAspDH5, BmAspDH6, and BmAspDH7, respectively. Specific primers were designed (see Table 1). Using Bacillus mojave LDFZ001 genomic DNA as a template, seven aspartate dehydrogenase genes were cloned and ligated into the prokaryotic expression vector pET28a to construct recombinant plasmids pET28a-BmAspDH1, pET28a-BmAspDH2, pET28a-BmAspDH3, pET28a-BmAspDH4, pET28a-BmAspDH5, pET28a-BmAspDH6, and pET28a-BmAspDH7. After confirming the correctness of the recombinant plasmids through sequencing, they were transformed into BL21(DE3) competent cells to induce protein expression. The enzyme activity was then identified using the expressed crude enzyme wet cells.

[0046] Table 1 Primers required for cloning aspartate dehydrogenase

[0047]

[0048] Note: Bold text represents homologous arms.

[0049] 2. Method for identifying the activity of aspartate dehydrogenase: The reduction reaction system consisted of 1 mL of 10 mg oxaloacetic acid, 10 mg ammonium chloride, and a final concentration of NADH of 0.1 g / L. 50 mM pH 8.0 phosphate buffer was added, and the reaction temperature was 37 °C. The decrease in NADH was measured at 340 nm. The enzyme activity unit was defined as the amount of enzyme required to consume 1 μmol of NADH per minute.

[0050] The oxidation reaction system consisted of 1 mL of 50 mM L-aspartic acid, with a final NAD(+) concentration of 0.1 g / L, added to 50 mM pH 8.0 phosphate buffer. The reaction temperature was 37 °C. The increase in NADH was measured at 340 nm. Enzyme activity was defined as the amount of enzyme required to generate 1 μmol of NADH per minute.

[0051] Table 2 Comparison of possible aspartate dehydrogenase activities

[0052]

[0053] Note: "-" indicates no activity.

[0054] The results are shown in Table 2. Only BmAspDH-4 exhibited aspartate dehydrogenase activity, while the others did not have the activity to catalyze the reaction of oxaloacetic acid and ammonium chloride to produce aspartic acid. BmAspDH-4 was renamed BmAspDH, and this name will be used to refer to the gene thereafter. Its recombinant plasmid was renamed pET28a-BmAspDH.

[0055] Example 2: Error-prone PCR construction of a random mutant library of BmAspDH

[0056] Error-prone PCR amplification was performed using the recombinant plasmid pET28a-BmAspDH as a template. The error-prone PCR kit was purchased from Takara Bio Inc. (Clontech, PT3393-1); the homologous recombinase was purchased from Nanjing Novizan Biotechnology Co., Ltd. (Novozymes, C214). The error-prone PCR amplification primers are shown below:

[0057] DH-F:TGGTGGACAGCAAATGGGTCGCggatcc

[0058] DH-R: TCATGGTGGGTGGTGGTGGTGctcgag;

[0059] The uppercase letters represent homologous arms, and the italics represent enzyme cleavage sites.

[0060] The PCR reaction system consisted of 50 μL of the following components: 1 μL template DNA (final concentration approximately 1 ng / μL), 1 μL each of forward and reverse primers (10 nM), 1 μL 50× Diversify dNTP Mix, 1 μL dGTP (2 mM), 2 μL MnSO4 (8 mM), 5 μL 10× TITANIUM Taq Buffer, 1 μL TITANIUM Taq Polym., and 37 μL ddH2O.

[0061] The error-prone PCR program was as follows: pre-denaturation at 94℃ for 30 seconds, followed by 25 cycles of the following program: 94℃ for 30 seconds, 68℃ for 2 minutes. The final extension was performed at 68℃ for 1 minute to terminate the reaction. Result: Amplified band of approximately 1330 bp was successfully obtained.

[0062] The amplified band and the pET28a double-digested (BamHI and XhoI) fragment of the vector were recovered using a DNA gel recovery kit. Homologous recombination was performed using novizan mono-homologous recombination enzyme. The reaction system was directly heat-shocked and transformed into Escherichia coli BL21(DE3) competent cells in 20 μL to obtain an aspartate dehydrogenase mutant library, with a total of 1324 positive clones.

[0063] Example 3: High-throughput screening of mutant libraries

[0064] 1. Preparation of wet culture of mutant crude enzyme solution

[0065] The 1324 positive clones obtained in Example 2 were picked and placed into sterile 96-well plates, with 1 mL of LB liquid medium containing 50 μg / mL kanamycin in each well. The plates were incubated overnight at 37°C and 180 rpm for 8 h. Then, 500 μL of the bacterial culture was transferred to another LB liquid medium containing 500 μL of 50 μg / mL kanamycin and 0.2 mM IPTG in each well. The plates were incubated at 20°C and 180 rpm for 16 h. The cells were then collected by centrifugation at 8000 rpm for 5 min at room temperature, yielding recombinant Escherichia coli wet cells containing the mutant gene, i.e., mutant wet bacteria.

[0066] 2. Initial screening

[0067] Method for identifying aspartate dehydrogenase activity: The reduction reaction system consisted of 1 mL of 10 mg oxaloacetic acid, 10 mg ammonium chloride, and a final concentration of NADH of 0.1 g / L. 50 mM pH 8.0 phosphate buffer was added, and the reaction temperature was 37 °C. The reduction in NADH was measured at 340 nm. Enzyme activity was defined as the amount of enzyme required to consume 1 μmol of NADH per minute. The reaction was performed using 96-well plates. Initial screening was conducted using a microplate reader, with the initially obtained BmAspDH strain serving as a control. Mutant strains with higher enzyme activity than the original BmAspDH strain were screened. A total of 20 mutant strains were screened.

[0068] 3. Secondary screening

[0069] The crude enzyme solutions of the 20 strains obtained from the initial screening were used as catalysts for secondary screening. The secondary screening method was the same as the initial screening method, except that the reaction system was increased. The enzyme activity of the original strain of aspartate dehydrogenase BmAspDH was defined as 1. The standard for screening was that the enzyme activity was more than twice that of the original strain, and that the strain did not have the oxidative activity of catalyzing the production of oxaloacetate from aspartate. A total of 4 dominant mutants were screened, and their relative enzyme activities were all more than twice that of the original strain. They were named BmAspDHm-1, BmAspDHm-2, BmAspDHm-3, and BmAspDHm-4, respectively. The results are as follows. Figure 2 As shown.

[0070] 4. Determination of nucleotide sequence of aspartate dehydrogenase mutant

[0071] The plasmids containing the aspartate dehydrogenase mutants BmAspDHm-1, BmAspDHm-2, BmAspDHm-3, and BmAspDHm-4 obtained above were extracted and sent to a sequencing company for sequencing. Sequencing results showed that in BmAspDHm-1, a C-to-T mutation occurred at position 463 of the naïve BmAspDH nucleotide sequence, resulting in a proline-to-serine mutation at position 155 of the naïve BmAspDH amino acid sequence; in BmAspDHm-2, a C-to-G mutation occurred at position 546 of the naïve BmAspDH nucleotide sequence, resulting in a phenylalanine-to-leucine mutation at position 182 of the naïve BmAspDH amino acid sequence; in BmAspDHm-3, an A-to-G mutation occurred at position 818 of the naïve BmAspDH nucleotide sequence, resulting in an aspartic acid-to-glycine mutation at position 273 of the naïve BmAspDH gene; and in BmAspDHm-4, a T-to-G mutation occurred at position 1064 of the naïve BmAspDH gene sequence, resulting in a valine-to-glycine mutation at position 355 of the naïve BmAspDH gene.

[0072] Example 4: Fusion of multi-site mutants of aspartate dehydrogenase and construction of engineered bacteria

[0073] Multiple site-directed mutagenesis of aspartate dehydrogenase was performed using a single-point mutagenesis kit (Novozymes, C214) from Novizumab, and the primer design is shown in Table 3. PCR amplification was performed based on the sequence of the mutant pET28-BmAspDHm-1 to introduce the corresponding mutation sites.

[0074] Table 3 Primers for site-directed mutagenesis of aspartate dehydrogenase

[0075]

[0076] Note: Bold uppercase letters indicate mutant bases.

[0077] The mutant PCR system (20 μL) consisted of: 10 μL 2×Max buffer, 1 μL dNTP (10 mM each), template plasmid final concentration of 1 ng / μL, 1 μL forward primer, 1 μL reverse primer, 1 μL L Hanta Max Super-Fidelity DNA polymerase, and finally ddH2O to make up to 20 μL.

[0078] PCR reaction program: 95℃ pre-denaturation for 30 seconds; 30 cycles, 95℃ denaturation for 15 seconds, 60℃ annealing for 15 seconds, 72℃ extension for 6 minutes; final extension at 72℃ for 5 minutes.

[0079] The amplified fragment was directly added to 1 μL of Dpn I, digested at 37°C for 1 h, and then subjected to agarose gel electrophoresis. After fragment recovery, a recombinant reaction was performed. The reaction system was: 10 μL, 400 ng of the Dpn I digestion product, 2 μL of 5×CE II Buffer, 1 μL of LExase II, and ddH2O to make up to 10 μL. After mixing, the mixture was incubated at 37°C for 30 min, immediately cooled on ice for 10 min, and then transformed into E. coli BL21(DE3) competent cells. The cells were plated on LB agar plates containing 50 μg / nL kanamycin and incubated upside down at 37°C for 14-16 h. A recombinant bacterium containing multiple mutations was obtained and named BmAspDHmSLGG. The recombinant bacterium, verified by sequencing, was preserved, and its aspartate dehydrogenase activity was measured. The measurement method was as described in Example 3. The mutant BmAspDHmSLGG showed a significantly increased activity in catalyzing the conversion of oxaloacetate to aspartic acid, as shown in the results. Figure 2 As shown, it also lacks the catalytic activity for the oxidation of aspartic acid.

[0080] Example 5: Fermentation and Application of Aspartate Dehydrogenase Mutant

[0081] 1. Select strains containing the BmAspDHmSLGG recombinant plasmid, inoculate them into 3 mL of LB liquid medium containing 50 μg / mL kanamycin sulfate, and incubate overnight at 37°C and 180 rpm. Inoculate them into 150 mL of TB medium at a volume ratio of 1% (V / V), and incubate at 30°C and 180 rpm until the OD600 reaches 0.4-0.6. Add 0.2 mM IPTG and continue induction culture for 6 hours. Then, collect the bacterial cells by centrifugation at 10,000 rpm for 10 min and store them in a -80°C refrigerator.

[0082] 2. Following the method in step 1 of Example 3, the mutant strain BmAspDHmSLGG was subjected to shake-flask fermentation, the bacterial cells were collected, and stored in a -80°C refrigerator.

[0083] The wet bacterial cells obtained in steps 1 and 2 above were added to a 1L fermentation system at a final concentration of 10 g / L. The substrates were 0.1M oxaloacetic acid (purchased from Sangon Biotech), 0.1M ammonium chloride, and 0.1 g / L NADH. Specifically, the reaction catalyst and substrate were added to a 2L fermenter, the reaction system was 1L, the stirring speed was 500 rpm, the pH was controlled at 8.0 with ammonia, and the temperature was controlled at 37℃ for 5 hours. The supernatant was collected by centrifugation at 6000 rpm, and the aspartic acid content was determined by HPLC. The results are as follows. Figure 3 As shown, the molar conversion rate of aspartic acid produced by the fusion mutant BmAspDHmSFGG was 95.2%, significantly higher than that of the initial aspartic acid dehydrogenase, indicating a very high application prospect in actual production.

[0084] The embodiments described above are merely preferred embodiments of the present invention and are only used to explain the present invention. They are not intended to limit the scope of the present invention. For those skilled in the art, other implementation methods can be easily made by substitution or modification based on the technical content disclosed in this specification. Therefore, all changes and improvements made on the principle of the present invention should be included within the scope of the patent application of the present invention.

Claims

1. An aspartate dehydrogenase mutant characterized in that, The mutant is mutated at positions of the following amino acid sequence of the initial-type aspartate dehydrogenase BmAspDH derived from Bacillus mojavensis (GenBank No. AAD 1 1 1 1 1.1 ) : the proline at position 155 is mutated to serine, the phenylalanine at position 182 is mutated to leucine, the aspartate at position 273 is mutated to glycine, and the valine at position 355 is mutated to glycine. Bacillus mojavensis LDFZ001, the amino acid sequence of which is shown as SEQ ID NO. 1, and the amino acid sequence of the BmAspDH mutant is shown as SEQ ID NO.

2.

2. A gene encoding the mutant aspartate dehydrogenase of claim 1, characterized in that, The nucleic acid sequence of the mutant is obtained by mutating the base C at position 463, the base C at position 546, the base A at position 818 and the base T at position 1064 in the gene sequence of the initial aspartate dehydrogenase BmAspDH, wherein the nucleotide sequence of the initial aspartate dehydrogenase BmAspDH is shown as SEQ ID NO. 3 and the nucleotide sequence of the BmAspDH mutant is shown as SEQ ID NO.

4.

3. A recombinant plasmid comprising the gene of claim 2, an expression vector comprising the gene of claim 2, and a recombinant cell comprising the recombinant plasmid.

4. A microorganism obtained by transforming the recombinant plasmid of claim 3.

5. The microorganism of claim 4, wherein, The microorganism is Escherichia coli (E. coli) Escherichia coli ) BL21.

6. Use of the aspartate dehydrogenase mutant of claim 1 or the microorganism of claim 4 or 5 in the production of aspartic acid.

7. A method for producing aspartate using the aspartate dehydrogenase mutant according to claim 1, characterized by, The method is to use the aspartate dehydrogenase mutant to catalyze the production of aspartic acid from oxaloacetic acid and ammonium chloride as substrates and NADH as coenzyme; specifically: (1) preparing aspartate dehydrogenase mutant wet bacteria; The obtained wet bacteria are added into a 1 L reaction system at a final concentration of 20 g / L, the substrates oxaloacetic acid and ammonium chloride are added at a mass / volume ratio of 1%, the final concentration of coenzyme NADH is 0.1 g / L, 50 mM pH 8.0 phosphate buffer is added, the reaction temperature is 37°C, and the catalytic cycle is 5 h.

8. The aspartate production method according to claim 7, characterized by, The preparation method of the aspartate mutant wet bacteria is as follows: first, the aspartate dehydrogenase mutant recombinant E. coli monoclonal is inoculated into 3 mL of LB medium containing 50 μg / mL kanamycin sulfate, and cultured at 37°C and 180 rpm overnight, then inoculated into 150 mL of TB medium at a volume ratio of 1%, and cultured at 30°C and 180 rpm until OD600 is 0.4-0.6, then 0.2 mM IPTG is added, and the induction culture is continued for 2 hours, then the wet bacteria are collected by centrifugation at 10,000 rpm for 10 min, and stored in a-80°C refrigerator.

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