An L-amino acid dehydrogenase mutant, its encoding gene, a genetically engineered bacterium, and their applications in the preparation of L-glufosinate

Abstract

The present invention discloses an L - amino acid dehydrogenase mutant, its coding gene, a genetically engineered bacterium and their applications in the preparation of L - glufosinate. The L - amino acid dehydrogenase mutant includes: a mutant obtained by replacing V at the 354th position with G using the wild - type L - amino acid dehydrogenase shown in SEQ ID NO.1 as a template; a mutant obtained by replacing A at the 152nd position with G; a mutant formed by the combined mutation at the 152nd and 354th positions; a mutant formed by the combined mutation of N at the 156th position mutated to A with the 152nd and 354th positions. Through molecular modification, the present invention obtains a class of L - amino acid dehydrogenase mutants with high enzyme activity in the process of catalyzing 2 - oxo - 4 - (hydroxymethylphosphinyl) butyric acid to prepare L - glufosinate. When using whole cells to convert 500 mM substrate PPO, the substrate conversion rate can reach more than 99% within 4 hours, showing strong industrial application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of enzyme engineering, and particularly relates to an L-amino acid dehydrogenase mutant, a coding gene thereof, a genetically engineered bacterium, and an application thereof in the preparation of L-glufosinate. Background Art

[0002] Glufosinate (D,L-Glufosinate) is a kind of low-toxic and non-selective highly efficient organophosphorus herbicide. At present, the world's three major non-selective herbicides include glyphosate, paraquat, and glufosinate, and the sales volume of the former two reaches hundreds of thousands of tons. Among them, paraquat has been banned due to its toxicity to humans, and after years of use of glyphosate, the resistance of crops to it has gradually emerged. Compared with the other two herbicides, L-glufosinate, as a broad-spectrum and highly efficient organophosphorus herbicide, has become an ideal substitute for paraquat and glyphosate due to its high herbicidal efficiency, low toxicity, and good environmental compatibility.

[0003] As a chiral compound, only the L-form (L-PPT) of glufosinate has herbicidal activity, and the D-form (D-PPT) is inactive. The structural formulas of the two are as Figure 1 shown. However, the currently commercially available glufosinate is mostly a racemate. When used as a pesticide, a large amount of D-form glufosinate enters the soil, causing serious environmental pollution. With the proposal of the action plan of "zero growth in the use of pesticides and fertilizers" by the Ministry of Agriculture in recent years, the production of green, efficient, and low-toxic pesticides has become a new trend in agricultural development.

[0004] Therefore, it is imperative to develop a green production route for L-glufosinate. At present, only the chemical synthesis process of L-glufosinate developed in Japan can achieve industrial production. However, due to the monopoly of key technologies, and the reaction conditions of this chemical synthesis route are harsh, the process is complex, and the intermediate products are unstable, resulting in low yield and high price of the final product. Compared with chemical synthesis, biocatalytic methods are generally carried out under mild conditions and do not require the additional addition of toxic compounds, thus avoiding the generation of by-products that are difficult to separate, providing a favorable option for the production of L-glufosinate. Among them, the asymmetric reductive amination reaction catalyzed by L-amino acid dehydrogenase (LADH) is a very effective production strategy for L-glufosinate, which has the advantages of directly using cheap ammonia as an amino donor, high optical purity of the product (usually exceeding 99%), and high atom utilization rate.

[0005] L-Amino acid dehydrogenase (L-AADH, EC 1.4.1.X) is a class of redox enzymes widely existing in nature, using nicotinamide adenine dinucleotide (NAD + ) or nicotinamide adenine dinucleotide phosphate (NADP +) As a cofactor, it can catalyze two reversible reactions: the reductive amination of keto acids or the oxidative deamination of amino acids, and has important physiological functions in organisms. L-amino acid dehydrogenases can be classified into L-glutamate dehydrogenase (L-GluDH), L-alanine dehydrogenase (L-AlaDH), L-leucine dehydrogenase (L-LeuDH), L-phenylalanine dehydrogenase (L-PheDH), etc. according to their different substrate preferences. Among them, L-GluDH prefers glutamate as a substrate more than all other amino acids; L-LeuDH is specific only for short aliphatic amino acids; L-PheDH prefers aromatic amino acids as substrates.

[0006] L-Glutamate dehydrogenase (L-Glu DH, EC 1.4.1.2 - 4) is widely present in various organisms such as animals, insects, plants, and microorganisms, and plays an important physiological role in the metabolism of glutamate. This enzyme can catalyze the reversible reaction of L-glutamate and α-ketoglutaric acid with the participation of coenzymes. L-Glutamate dehydrogenase can be classified according to coenzyme specificity into: NADP specificity, participating in ammonia assimilation (NADP-GluDH, EC 1.4.1.4); NAD specificity, participating in glutamate catabolism (NAD-GluDH, EC 1.4.1.2), and NAD, NADP dual coenzyme dependence (NAD(P)-GluDH, EC 1.4.1.3), which is generally present in vertebrates and uses NAD and NADP with similar efficiency. Most L-glutamate dehydrogenases are composed of six subunits with a molecular weight between 42,000 and 63,000. The substrate specificity of L-GluDH is quite high, and L-amino acids other than L-glutamate and keto acids other than α-ketoglutaric acid are utilized very slowly. Therefore, to improve the reaction catalytic efficiency, it is necessary to perform molecular modification on L-amino acid dehydrogenase to improve its utilization efficiency of the substrate PPO. Summary of the Invention

[0007] The object of the present invention is to provide an L-amino acid dehydrogenase mutant, its encoding gene, a genetically engineered bacterium, and their application in the preparation of L-glufosinate, so as to solve the problem that the existing L-glutamate dehydrogenase has low asymmetric amination reduction activity for 2-oxo-4-(hydroxymethylphosphinyl)butyric acid.

[0008] To solve the above problems, the technical solution adopted by the present invention is as follows:

[0009] According to the first aspect of the present invention, there is provided an L-amino acid dehydrogenase mutant, which includes: using the wild-type L-amino acid dehydrogenase AADH22 shown in SEQ ID NO.1 as a template, the L-amino acid dehydrogenase mutant AADH22-V354G in which valine V at the 354th position is mutated to glycine G, and its amino acid sequence is shown in SEQ ID NO.2; using the wild-type L-amino acid dehydrogenase AADH22 shown in SEQ ID NO.1 as a template, the L-amino acid dehydrogenase mutant AADH22-A152G in which alanine A at the 152nd position is mutated to glycine G, and its amino acid sequence is shown in SEQ ID NO.3; using the mutant L-amino acid dehydrogenase mutant A152G shown in SEQ ID NO.3 as a template, the L-amino acid dehydrogenase mutant AADH22-A152G / V354G after valine V at the 354th position is mutated to glycine G, and its amino acid sequence is shown in SEQ ID NO.4; using the mutant L-amino acid dehydrogenase mutant AADH22-A152G / V354G shown in SEQ ID NO.4 as a template, the L-amino acid dehydrogenase mutant AADH22-A152G / V354G / N156A after asparagine N at the 156th position is mutated to alanine A, and its amino acid sequence is shown in SEQ ID NO.5.

[0010] According to the second aspect of the present invention, there is provided a coding gene for the L-amino acid dehydrogenase mutant as described above.

[0011] According to the third aspect of the present invention, there is provided a recombinant vector and a genetically engineered bacterium containing the coding gene for the L-amino acid dehydrogenase mutant as described above.

[0012] According to the fourth aspect of the present invention, there is provided an application of the L-amino acid dehydrogenase mutant as described above in catalyzing the preparation of L-glufosinate from 2-oxo-4-(hydroxymethylphosphinyl)butyric acid (PPO).

[0013] According to a preferred embodiment of the present invention, the application includes the following steps: 1) preparing a genetically engineered bacterium expressing the L-amino acid dehydrogenase mutant, and the amino acid sequence of the L-amino acid dehydrogenase mutant is shown in SEQ ID NOs.2-5; 2) culturing the genetically engineered bacterium to prepare an enzyme solution or freeze-dried bacterial powder; 3) adding the enzyme solution or freeze-dried bacterial powder to a reaction system containing the substrate 2-oxo-4-(hydroxymethylphosphinyl)butyric acid, an amino donor, and a reduced coenzyme, and performing a reductive amination reaction to obtain L-glufosinate.

[0014] Preferably, in step 3), the temperature of the reductive amination reaction is 28-32 °C, and the pH value of the reaction solution is 9.

[0015] Preferably, in step 3), the reduced coenzyme is NADH.

[0016] Preferably, in step 3), the reaction system further includes a coenzyme regeneration system, and the coenzyme regeneration system is: a glucose dehydrogenase coenzyme regeneration system with glucose dehydrogenase as the coenzyme regeneration enzyme, glucose as the coenzyme regeneration substrate, and containing NADH and NAD + +.

[0017] Preferably, in step 3), the amino donor is ammonium triphosphate.

[0018] According to the fifth aspect of the present invention, there is provided an application of the L-amino acid dehydrogenase mutant as described above in catalyzing the preparation of L-glufosinate from D,L-glufosinate.

[0019] The wild-type L-glutamate dehydrogenase used in the present invention is derived from Xenophilus azovorans WP_038202116.1. This wild-type L-glutamate dehydrogenase (AADH22) consists of 426 amino acid residues and has very low enzyme activity when catalyzing the substrate PPO, which is not suitable for industrial production. The inventors performed site-directed mutagenesis screening on different amino acid sites of the above wild-type enzyme against the substrate PPO and found that some mutants at the A152, V354, and N156 sites had significantly improved enzyme activity towards the new substrate PPO. Then, combinatorial mutagenesis was performed on these sites and the L-glutamate dehydrogenase mutant described in the present invention was screened out from them.

[0020] Specifically, the present invention is implemented by the following technical solutions: 1) Using the amino acid sequence of AADH22 as a template, the crystal structure is obtained by homology modeling. After substrate docking, the mutation sites are selected; 2) Design mutation primers; 3) Perform whole plasmid PCR using the plasmid carrying the gene encoding L-amino acid dehydrogenase AADH22 as a template to introduce mutations; 4) After digesting the PCR product with DpnⅠ, transform it into Escherichia coli BL21(DE3); 5) Pick single colonies and sequence to verify whether they are positive mutant strains; 6) Induce culture, collect the bacteria, and measure the change in enzyme activity after mutation.

[0021] According to the present invention, an L - amino acid dehydrogenase mutant is provided. The sequence of the L - amino acid dehydrogenase mutant includes the sequence after mutating the 152nd amino acid residue A, and / or the 354th amino acid residue V, and / or the 156th amino acid residue N shown in SEQ ID NO.1 into basic, hydrophilic or small - steric - hindrance amino acid residues. The present invention detected the activity of each mutant in the catalytic reductive amination reaction. To analyze the synergistic effect among them, the mutants with significantly improved activity were combined, and the optimal triple - mutant AADH22 - A152G / V354G / N156A was determined, that is, the amino acid residues at the 152nd, 354th, and 156th positions mutated simultaneously. Through these protein - engineering strategies, multiple mutants with improved L - amino acid dehydrogenase activity were obtained, and the acquisition of these mutants is very beneficial for industrial production.

[0022] Based on the wild - type amino acid dehydrogenase shown in SEQ ID NO.1 as a template, the present invention determined the amino acid sites crucial for activity through analysis, and obtained the following multiple leucine dehydrogenase mutants, including: replacing A at the 152nd position with G to form the mutant A152G; replacing V at the 354th position with G to form the mutant V354G; replacing N at the 156th position with A to form the mutant N156A; jointly mutating the 152nd and 354th positions to form the mutant A152G / V354G; jointly mutating the amino acids at the 152nd, 354th, and 156th positions to form the mutant A152G / V354G N156A.

[0023] Furthermore, the present invention applied the above - mentioned L - amino acid dehydrogenase mutant to the asymmetric reductive amination synthesis of L - glufosinate. It was found that after 12 hours of reaction, the conversion rate of the wild - type L - amino acid dehydrogenase AADH22 was still lower than 40%. By protein engineering, AADH22 was modified to obtain multiple mutants with improved catalytic performance of L - amino acid dehydrogenase. The relative enzyme activities of AADH22 - A152G, AADH22 - V354G, AADH22 - A152G / V354G, and AADH22 - A152G / V354G / N156A were 300%, 453%, 1973%, and 2660% respectively, and the highest was 25.6 times higher than that of the wild - type. When using the triple - mutant AADH22 - A152G / V354G / N156A with the highest activity of L - amino acid dehydrogenase to catalyze 0.5 M PPO, the triple - mutant could completely convert PPO to generate L - glufosinate within 4 h, and the ee value reached 99.9%.

[0024] In summary, according to the L - amino acid dehydrogenase provided by the present invention, it can maintain a high catalytic activity during the process of catalytically reducing amination of PPO to produce L - glufosinate, and has a high enantioselectivity; according to multiple L - amino acid dehydrogenase mutants provided by the present invention, the problem of low enzyme activity of L - amino acid dehydrogenase in the process of preparing L - glufosinate by the reductive amination method is solved. Compared with the wild - type L - glutamate dehydrogenase, when using the L - amino acid dehydrogenase mutants of the present invention to prepare L - glufosinate, the catalytic efficiency has been significantly improved at the same substrate concentration, the reaction time is significantly shortened, the efficiency of the enzyme is improved, the reaction cost is reduced, which is conducive to industrial production.

[0025] It should be understood that the capital letters in the present invention represent amino acids well - known to those skilled in the art, and according to this application, they represent the corresponding amino acid residues here.

[0026] The experimental methods in the present invention are all conventional methods unless otherwise specified. For gene cloning operations, specific reference can be made to "Molecular Cloning: A Laboratory Manual" edited by J. Sambrook et al.

[0027] Description of some sequences in the sequence listing:

[0028] SEQ ID NO.1 is the amino acid sequence of L - amino acid dehydrogenase (AADH22) annotated as such in WP_012202150.1 derived from Delftia acidovorans;

[0029] SEQ ID NO.2 is the amino acid sequence of the mutant AADH22 - V354G of AADH22;

[0030] SEQ ID NO.3 is the amino acid sequence of the mutant AADH22 - A152G of AADH22;

[0031] SEQ ID NO.4 is the amino acid sequence of the mutant AADH22 - A152G / V354G of AADH22;

[0032] SEQ ID NO.5 is the amino acid sequence of the mutant AADH22 - A152G / V354G / N156A of AADH22.

[0033] The invention clones an L-amino acid dehydrogenase gene from Xenophilus azovorans; WP_038202116.1, and realizes heterologous expression of the gene in Escherichia coli. The enzyme can be coupled with D-amino acid oxidase to catalyze the conversion of D,L-phosphinothricin ammonium (D,L-PPT) into L-phosphinothricin ammonium (L-PPT) by a "one-pot method". However, the activity of the enzyme to its intermediate prochiral keto acid product 2-carbonyl-4-(hydroxymethylphosphonyl)butyric acid (PPO) is not high enough, which becomes the rate-limiting step in the "one-pot method" process, thereby limiting its industrial application. Therefore, the present invention uses the reported crystal structure of L-amino acid dehydrogenase and molecular simulation to determine the spatial structure of the enzyme and possible amino acid sites related to activity, and improves the catalytic activity of L-amino acid dehydrogenase to 2-carbonyl-4-(hydroxymethylphosphonyl)butyric acid through site-directed mutagenesis technology, eliminates the rate-limiting link, and further increases the yield of L-phosphinothricin ammonium, which has strong industrial application value.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] The present invention improves the catalytic activity of L-amino acid dehydrogenase to 2-carbonyl-4-(hydroxymethylphosphonyl)butyric acid (PPO) by a molecular modification method of mutating a specific alanine in a substrate binding pocket of L-amino acid dehydrogenase to glycine, and / or mutating valine to glycine, with the highest improvement multiple reaching 26.6 times, thereby solving the problem of low enzyme activity of L-amino acid dehydrogenase in the process of preparing L-glufosinate by reductive amination method. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The structural formulas of D-phosphinothricin (D-PPT) and L-phosphinothricin (L-PPT) are shown respectively;

[0037] Figure 2 The reaction formula for preparing L-phosphinothricin ammonium by coupling L-amino acid dehydrogenase and glucose dehydrogenase;

[0038] Figure 3 This is the HPLC spectrum of racemic glufosinate standard sample; wherein, the retention time is: L-glufosinate is 20.3min, and D-glufosinate is 25.2min;

[0039] Figure 4Time course diagrams of the preparation of L-glufosinate by asymmetric amination reduction of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid by coupling glucose dehydrogenase with wild-type (AADH22) and mutant (AADH22-V354G, AADH22-A152G, AADH22-A152G / V354G, AADH22-A152G / V354G / N156A) of glufosinate dehydrogenase respectively. Detailed implementation mode

[0040] The present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited to the scope of the described embodiments. For the experimental methods without specific conditions in the following embodiments, they are carried out according to conventional methods and conditions, or according to the product instructions.

[0041] Materials and methods

[0042] Reagents used in upstream genetic engineering: The genomic DNA extraction kit, plasmid extraction kit, DNA purification and recovery kit, and one-step cloning kit used in the examples were purchased from Nanjing Novizan Biotech Co., Ltd.; E. coli BL21(DE3), plasmid pET-28a(+), etc. were purchased from Shanghai Xuguan Biotech Development Co., Ltd.; DNA marker, low molecular weight standard protein, and protein gel were purchased from Beijing GenStar Co., Ltd.; ClonExpress II One Step Cloning Kit seamless cloning kit was purchased from Nanjing Novizan Biotech Co., Ltd.; Dpn I endonuclease was purchased from Thermo Fisher Scientific (China) Co., Ltd.; Primer synthesis and sequence sequencing work were completed by Hangzhou Qingke Zixi Biotechnology Co., Ltd., and the full gene synthesis was completed by Hangzhou Qingke Zixi Biotechnology Co., Ltd. The KOD-One site-directed mutagenesis kit was purchased from Toyobo Co., Ltd. Japan. The usage methods of the above reagents refer to the product instructions.

[0043] The high-performance liquid analysis method for D,L-PPT is as follows:

[0044] Liquid chromatography column: OA5000L column (4.6 mm x 150 mm); Mobile phase: An aqueous solution containing 2 mM copper sulfate: 0.3% acetonitrile; Conditions: Flow rate 0.7 mL / min, injection volume 3 μL, UV wavelength 254 nm, column temperature 30 °C. According to the present invention, the high-performance liquid chromatogram of the racemic glufosinate standard sample is as Figure 3 shown.

[0045] Example 1: Construction and screening of a mutant library of L-amino acid dehydrogenase

[0046] Step 1: Activation of recombinant bacteria and plasmid extraction

[0047] The engineered Escherichia coli strain (AADH22) carrying the L-amino acid dehydrogenase gene from Delftia acidovorans (WP_012202150.1) was activated and cultured using LB medium.

[0048] Preparation of LB liquid medium: Weigh 10 g of peptone, 5 g of yeast extract, and 10 g of sodium chloride, dissolve them in 1 L of deionized water, dispense into Erlenmeyer flasks and sterilize, sterilize at 121 °C for 20 min, and use for culturing Escherichia coli after cooling.

[0049] Preparation of LB solid medium: Weigh 1.5 g of agar into 100 mL of the above liquid LB medium, sterilize under the same conditions and cool for later use.

[0050] Streak the glycerol tube containing L-amino acid dehydrogenase AADH22 onto a petri dish containing LB solid medium and incubate statically at 37 °C for 12 h. Pick a single colony from the petri dish and inoculate it into 5 mL of LB medium containing 50 μg / mL kanamycin, culture at 37 °C and 200 rpm for 12 h. After obtaining the culture solution, extract the plasmid according to the operation manual of the plasmid extraction kit. The obtained plasmid can be used for subsequent operations or stored at -20 °C for long term.

[0051] Step 2: Site-directed mutagenesis

[0052] Design of mutagenic primers: The primers used for PCR site-directed mutagenesis were designed on the website QuikChange Primer Design. Upload the target gene fragment to the website, translate it into protein, select the specific site, then select the target amino acid, and click Design Primers to obtain the base sequence of the primers. The primer sequences are shown in Table 1 below.

[0053] Table 1 Primers used for mutagenesis

[0054]

[0055] The mutagenic PCR system is shown in Table 2 below.

[0056] Table 2 Mutagenic PCR reaction system

[0057]

[0058] PCR amplification conditions are: pre-denaturation at 95 °C for 3 min; denaturation at 95 °C for 15 s; annealing at T m -5 °C for 20 s; extension at 72 °C for 5 min (cycles ②③④ are repeated 30 times); extension at 72 °C for 10 min; store at 4 °C.

[0059] After PCR, perform positive verification by DNA agarose gel electrophoresis. Demethylate the PCR product, add 0.5 μL of DpnⅠ to the PCR product, mix well by pipetting, and digest in a 37°C metal bath for 2 h 30 min before transformation.

[0060] Transformation of the recombinant plasmid: After digestion, place the PCR product and the prepared competent cells on ice for an ice bath for 5 min. In a laminar flow hood, take 10 μL of the digested product and add it to the competent cells, and continue the ice bath for at least 30 min. During this period, turn on the 42°C water bath to prepare for the ice bath. After the ice bath is completed, perform a heat shock at 42°C in the water bath for exactly 90 s. After taking it out, continue the ice bath for at least 2 min, then add 900 μL of antibiotic-free LB medium, place it in a 37°C shaker and culture for 45 min, then spread it on a plate supplemented with kanamycin and culture it overnight in a 37°C incubator. Then send it to the company for sequencing confirmation and save it.

[0061] Step 3: Induced expression

[0062] Pipette 5 μL of glycerol bacteria from the preservation tube and inoculate it into a test tube containing 5 mL of liquid LB medium supplemented with kanamycin, place it at 37°C, and shake culture in a shaker at 200 rpm for 10 - 12 h. Then pipette 1 mL of the bacterial solution into 50 mL of liquid LB medium, continue to place it at 37°C, and shake culture in a shaker at 200 rpm for 2 - 3 h until the OD 600 reaches between 0.6 - 0.8. Add 50 μL of pre-prepared IPTG stored in the refrigerator to the cultured bacterial solution, place it in a shaker at 20°C and 200 rpm for induced expression. After culturing for 16 h - 18 h, transfer the bacterial solution to a centrifuge tube for balancing, use a low-temperature high-speed centrifuge to centrifuge at 8,000 rpm for 10 min to collect the bacteria, pour off the supernatant, add physiological saline to wash twice, and then place it in the refrigerator for standby.

[0063] Add a certain volume of PB buffer (20 mM, pH 7.0) to the collected bacterial cells, vortex to mix well, and place it in ice water. Use an ultrasonic cell disruptor to break the cells to obtain a crude enzyme solution. Set the disruption program as: on for 5 s, off for 3 s, and disrupt for 15 min. After disruption, balance and centrifuge to obtain the supernatant, which is the crude enzyme solution. Add 4 mL of 75% glycerol, mix well, dispense, and store at -80°C for standby. All the above operations are carried out on ice.

[0064] Step 4: Mutant enzyme activity assay

[0065] The enzyme activity detection system includes: an appropriate amount of enzyme, 100 mM of the substrate PPO, 20 mM of the coenzyme (NADH), 500 mM of ammonium chloride buffer at pH 9.0, and the total system is 500 μL. React at 30 °C in a shaker at 250 rpm for 10 min. After completion, heat the reaction solution in a 95 °C water bath for 10 min to terminate the reaction. Measure the initial rate of product formation within the time when the product conversion is ≤ 5%.

[0066] The enzyme activity unit (U) is defined as: the amount of enzyme required to catalyze the conversion of 1 μmol of substrate into L-PPT per minute is defined as one activity unit.

[0067] According to the above enzyme activity determination method, the enzyme activities of AADH22 wild-type and the mutant strains AADH22-A152G, AADH22-V354G, AADH22-A152G / V354G, AADH22-A152G / V354G / N156A modified using the above modification strategy were determined. Taking the enzyme activity of AADH22 wild-type as 100%, the relative activities of the mutants were calculated. The results showed that the relative enzyme activities of AADH22-A152G, AADH22-V354G, AADH22-A152G / V354G, AADH22-A152G / V354G / N156A were 300.5%, 453%, 1973%, and 2660% respectively, with the highest being 25.6 times higher than the wild-type. Using the triple mutant AADH22-A152G / V354G / N156A with the highest activity to react with 500 mM of the substrate PPO, the conversion rate reached over 99% within 4 h, ee > 99.9%, and the space-time yield reached 24.5 g·L -1 ·h -1 .

[0068] Example 2: Preparation of L-glufosinate by double enzyme coupling of wild-type L-amino acid dehydrogenase (AADH22-WT) and glucose dehydrogenase

[0069] The activation and induction expression of the wild-type engineering bacteria of L-amino acid dehydrogenase, the mutant engineering bacteria of L-amino acid dehydrogenase, and the engineering bacteria of glucose dehydrogenase are as described in Example 1.

[0070] Among them, the engineering bacteria of glucose dehydrogenase are the strains preserved in this laboratory. This glucose dehydrogenase is derived from Glucose1-dehydrogenase[Bacillus subtilis QB928], GenBank: AFQ56330.1. The reaction mechanism for preparing L-glufosinate by double enzyme coupling of L-amino acid dehydrogenase and glucose dehydrogenase is as Figure 2 shown.

[0071] The reaction system is: 500 mM of the substrate PPO, 120 g / L of glucose, NAD+ 0.1 mM, adjust the pH to 9.0 with ammonia water, 10 g / L of freeze-dried bacterial powder of L-amino acid dehydrogenase (L AADH22), and 5 g / L of bacterial powder of glucose dehydrogenase (GDH).

[0072] Samples were taken at the 0 h, 0.2 h, 0.4 h, 1 h, 1.5 h, 2 h, 3 h, 5 h, 7 h, 9 h, and 12 h of the reaction.

[0073] Sample pretreatment: Take 100 μL of the sample to be detected from the shake flask, place it in a water bath at 95 °C and heat for 10 min, then centrifuge at 14,000 rpm for 10 min. Take the supernatant, dilute it tenfold with pure water and centrifuge again. Absorb a certain amount of the supernatant and place it in a liquid phase vial for liquid phase detection.

[0074] After the reaction ended, the data was as Figure 4 shown. The results showed that 300 mM of PPO remained at 12 h of the reaction for the wild-type AADH22-WT, the conversion rate was lower than 40%, and ee > 99.9%.

[0075] Example 3: Preparation of L-glufosinate by double enzyme coupling of L-amino acid dehydrogenase mutant (AADH22-V354G) and glucose dehydrogenase

[0076] The induced expression of the engineering bacteria of L-amino acid dehydrogenase mutant and glucose dehydrogenase and the acquisition of bacterial powder were as described in Example 1.

[0077] The reaction system was: 500 mM of substrate PPO, 120 g / L of glucose, NAD + 0.1 mM, adjust the pH to 9.0 with ammonia water, 10 g / L of freeze-dried bacterial powder of L-amino acid dehydrogenase (LAADH22), and 5 g / L of bacterial powder of glucose dehydrogenase (GDH).

[0078] Samples were taken at the 0 h, 0.3 h, 1 h, 1.5 h, 2 h, 3 h, 5 h, 7.5 h, and 12 h of the reaction, and were processed for liquid phase detection after treatment.

[0079] After the reaction ended, the data was as Figure 4 shown. The conversion rate of AADH22-V354G was 89% after 12 hours, 50 mM of PPO remained, and ee > 99.9%.

[0080] Example 4: Preparation of L-glufosinate by double enzyme coupling of L-amino acid dehydrogenase mutant (AADH22-A152G) and glucose dehydrogenase

[0081] The induced expression of the engineering bacteria of L-amino acid dehydrogenase mutant and glucose dehydrogenase and the acquisition of bacterial powder were as described in Example 1.

[0082] The reaction system is as follows: substrate PPO 500 mM, glucose 120 g / L, NAD + 0.1 mM, pH is adjusted to 9.0 with ammonia water, freeze-dried bacterial powder of L-amino acid dehydrogenase (L AADH22) 10 g / L, bacterial powder of glucose dehydrogenase (GDH) 5 g / L.

[0083] Samples are taken at the 0 h, 0.3 h, 1 h, 1.5 h, 2 h, 3 h, 5 h, 7.5 h, and 12 h during the reaction, and after treatment, they are used for liquid phase detection.

[0084] After the reaction is completed, the data is as Figure 4 shown. AADH22-A152G reaches a conversion rate of 99% at 12 hours, ee > 99.9%

[0085] Example 5: Preparation of L-glufosinate by dual-enzyme coupling of L-amino acid dehydrogenase mutant (AADH22-A152G / V354G) and glucose dehydrogenase

[0086] The induced expression of the engineered bacteria of L-amino acid dehydrogenase mutant and the engineered bacteria of glucose dehydrogenase and the acquisition of bacterial powder are as described in Example 1.

[0087] The reaction system is as follows: substrate PPO 500 mM, glucose 120 g / L, NAD + 0.1 mM, pH is adjusted to 9.0 with ammonia water, freeze-dried bacterial powder of L-amino acid dehydrogenase (LAADH22) 10 g / L, bacterial powder of glucose dehydrogenase (GDH) 5 g / L.

[0088] Samples are taken at the 0 h, 0.3 h, 1 h, 1.5 h, 2 h, 3 h, 5 h, 7.5 h, and 12 h during the reaction, and after treatment, they are used for liquid phase detection.

[0089] After the reaction is completed, the data is as Figure 4 shown. AADH22-A152G / V354G reaches a conversion rate of 99% at 5 hours during the reaction, ee > 99.9%.

[0090] Example 6: Preparation of L-glufosinate by dual-enzyme coupling of L-amino acid dehydrogenase mutant (AADH22-A152G / V354G / N156A) and glucose dehydrogenase

[0091] The induced expression of the engineered bacteria of L-amino acid dehydrogenase mutant and the engineered bacteria of glucose dehydrogenase and the acquisition of bacterial powder are as described in Example 1.

[0092] The reaction system is as follows: substrate PPO 500 mM, glucose 120 g / L, NAD +0.1 mM, adjust the pH to 9.0 with ammonia water, 10 g / L of freeze-dried bacterial powder of L-amino acid dehydrogenase (L AADH22), and 5 g / L of bacterial powder of glucose dehydrogenase (GDH).

[0093] Samples were taken at the 0 h, 0.3 h, 1 h, 1.5 h, 2 h, 3 h, 5 h, 7.5 h, and 12 h of the reaction, and were processed for liquid phase detection after treatment.

[0094] After the reaction ended, the data was as Figure 4 shown. The conversion rate of AADH22-A152G / V354G / N156A could reach over 99% within 4 hours, and the space-time yield reached 24.5 g·L -1 ·h -1 , and ee > 99.9%.

[0095] The above are only the preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various changes can be made to the above embodiments of the present invention. All simple, equivalent changes and modifications made according to the claims and the content of the specification of the present invention application fall within the scope of the claims of the present invention patent. Those not described in detail in the present invention are all conventional technical contents.

Claims

1. An L-amino acid dehydrogenase mutant with improved activity, characterized in that, Comprising: An L - amino acid dehydrogenase mutant AADH22 - V354G with the valine V at position 354 mutated to glycine G, using the wild - type L - amino acid dehydrogenase AADH22 shown in SEQ ID NO.1 as a template, and its amino acid sequence is as shown in SEQ ID NO.2; An L - amino acid dehydrogenase mutant AADH22 - A152G with the alanine A at position 152 mutated to glycine G, using the wild - type L - amino acid dehydrogenase AADH22 shown in SEQ ID NO.1 as a template, and its amino acid sequence is as shown in SEQ ID NO.3; An L - amino acid dehydrogenase mutant AADH22 - A152G / V354G after the valine V at position 354 is mutated to glycine G, using the mutant L - amino acid dehydrogenase mutant AADH22 - A152G shown in SEQ ID NO.3 as a template, and its amino acid sequence is as shown in SEQ ID NO.4; An L - amino acid dehydrogenase mutant AADH22 - A152G / V354G / N156A after the asparagine N at position 156 is mutated to alanine A, using the mutant L - amino acid dehydrogenase mutant AADH22 - A152G / V354G shown in SEQ ID NO.4 as a template, and its amino acid sequence is as shown in SEQ ID NO.

5.

2. A coding gene for the L - amino acid dehydrogenase mutant as claimed in claim 1.

3. A recombinant vector and a genetically engineered bacterium comprising the coding gene for the L - amino acid dehydrogenase mutant as claimed in claim 1.

4. Use of the L - amino acid dehydrogenase mutant as claimed in claim 1 in the preparation of L - glufosinate from 2 - oxo - 4 - (hydroxymethylphosphinyl) butyric acid.

5. The application according to claim 4, wherein Comprising the following steps: 1) Prepare a genetically engineered bacterium expressing the glutamate dehydrogenase mutant, and the amino acid sequence of the glutamate dehydrogenase mutant is as shown in SEQ ID NO.2 - 5; 2) Culture the genetically engineered bacterium to prepare an enzyme solution or freeze - dried bacterial powder; 3) Add the enzyme solution or freeze - dried bacterial powder to a reaction system containing the substrate 2 - oxo - 4 - (hydroxymethylphosphinyl) butyric acid, an amino donor, and a reduced coenzyme for a reductive amination reaction to obtain L - glufosinate.

6. The application according to claim 5, wherein In step 3), the temperature of the reductive amination reaction is 28 - 32 °C, and the pH value of the reaction solution is 9.

7. The application according to claim 5, wherein In step 3), the reduced coenzyme is NADH.

8. The application according to claim 5, wherein In step 3), the reaction system further includes a coenzyme regeneration system, which is: a glucose dehydrogenase coenzyme regeneration system using glucose dehydrogenase as the coenzyme regeneration enzyme, glucose as the coenzyme regeneration substrate, and containing NADH and NAD + +.

9. The application according to claim 5, characterized in that, In step 3), the amino donor is triammonium phosphate.

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