A D-aminotransferase mutant and its application in the preparation of L-phosphinothricin ammonium

Abstract

The invention discloses a D-transaminase mutant and its application in the preparation of L-glufosinate, wherein the D-transaminase mutant is to mutate the 265th tryptophan of the amino acid sequence shown in SEQ ID NO.1 to serine. The D-transaminase mutant of the present invention has high enantioselectivity (ee>99.9%), and only catalyzes the conversion of D-glufosinate into 4-(hydroxy (methyl) phosphoryl)-2-oxobutyric acid, and L-glufosinate is retained, reducing the difficulty of the reaction. Using a one-pot method, commercially available D, L-glufosinate is used as a substrate, and D-glufosinate without herbicidal activity is synthesized into L-glufosinate, with higher atomic utilization efficiency, meeting the requirements of green chemistry. Simple operation, carried out under mild reaction conditions, suitable for large-scale production, with the potential for industrial application, the yield of L-glufosinate product is up to 100%, and the enantiomeric excess (ee) is greater than 99.9%.

Description

(I) Technical field

[0001] The invention relates to the field of bioindustry, and in particular to a D-aminotransferase mutant and a method for preparing L-glufosinate by a biological multi-enzyme cascade one-pot method using racemic D,L-glufosinate as a substrate. (II) Background technology

[0002] Glufosinate, also known as glufosinate, is called Phsophinothricin (PPT for short) in English. Its chemical name is 2-amino-4-[hydroxy(methyl)phosphonyl]-butyric acid. It is a highly efficient, broad-spectrum, low-toxic non-selective herbicide. It is an ideal herbicide for genetically modified resistant crops and has a very broad application prospect. Glufosinate has two enantiomers, but only the L-configuration is phytotoxic and is easily decomposed in the soil. It has low toxicity to humans and animals and low destructive power to the environment. At present, the glufosinate sold on the market is generally a racemic mixture. If the glufosinate product can be used in the form of an optically pure isomer of the L-configuration, the amount of glufosinate used can be reduced by 50%, which is of great significance for improving atom economy, reducing costs, and alleviating environmental pressure.

[0003] There are three main methods for preparing optically pure L-glufosinate: chiral resolution, chemical synthesis and biocatalysis.

[0004] The chiral separation method is to separate the D-type and L-type isomers by chiral separation of racemic D,L-glufosinate or its derivatives, thereby obtaining optically pure L-glufosinate. This process has the following main disadvantages: it requires the use of expensive chiral separation reagents, the theoretical yield can only reach 50%, the single separation rate is low, the process is relatively complicated, and the cost is high.

[0005] The chemical synthesis method is to synthesize optically pure L-glufosinate from chiral raw materials. The chemical asymmetric synthesis method has many process steps and low yield. The expensive chiral raw materials lead to high production costs, which is not conducive to the large-scale preparation of L-glufosinate.

[0006] The biocatalytic method for producing L-glufosinate has the advantages of strict stereoselectivity, mild reaction conditions, and high yield, and is the dominant method for producing L-glufosinate. It mainly includes the following two categories:

[0007] (1) Using L-phosphinothricin derivatives as substrates, the product is obtained by direct enzymatic hydrolysis. The main advantages are high conversion rate and high product ee value, but it requires expensive and difficult to obtain chiral raw materials as precursors.

[0008] (2) Using the precursor of racemic glufosinate as substrate, it is obtained by selective resolution of enzymes. The main advantages are that the raw materials are relatively easy to obtain and the catalyst activity is high, but the theoretical yield can only reach 50%, resulting in a waste of raw materials.

[0009] In addition to these two traditional biocatalytic methods, the deracemization synthesis method using D,L-glufosinate as raw material highlights a huge cost advantage. Since the commercially available glufosinate is D,L-glufosinate, its industrial production technology is very mature. The deracemization synthesis method directly uses D,L-glufosinate as raw material, which is simple and easy to obtain, has low cost, and can better meet the existing L-glufosinate industrial production system.

[0010] Transaminase (EC 2.6.1.X) is a typical 5'-pyridoxal phosphate (PLP)-dependent enzyme that mediates the transamination reaction between an amino donor and an amino acceptor. It has high enantioselectivity, a broad substrate spectrum, and high catalytic activity, and is widely used in the synthesis of chiral amino acids and chiral amines. The transaminase-catalyzed transamination reaction between α-ketoacids and α-amino acids is a reversible reaction with a low reaction equilibrium constant (Keq=1). It is often inhibited by products or by-products, resulting in a theoretical 100% conversion rate that cannot be achieved. Transaminase is divided into PLP folding type I and PLP folding type IV according to the folding type of PLP. Among them, D-transaminase belongs to PLP folding type IV, has unique enantioselectivity, and can catalyze the reversible conversion between D-amino acids and ketoacids. (III) Summary of the invention

[0011] The present application provides a D-transaminase mutant and its application in the one-pot splitting of racemic D,L-phosphinothricin to prepare L-phosphinothricin, using racemic D,L-phosphinothricin as substrate, using biological multi-enzyme cascade one-pot method to prepare L-phosphinothricin, converting D-phosphinothricin without herbicidal activity into L-phosphinothricin with herbicidal activity. The enzyme catalysis system includes a D-transaminase mutant for catalyzing D-phosphinothricin in D,L-phosphinothricin to 2-carbonyl-4-[hydroxy(methyl)phosphinothricin]butyric acid, and a glutamate dehydrogenase for catalytic reduction of 2-carbonyl-4-[hydroxy(methyl)phosphinothricin to L-phosphinothricin. The method has the advantages of high catalytic substrate concentration, few by-products, high conversion rate and yield, and greater potential for industrial application.

[0012] The technical solution adopted by the present invention is:

[0013] The present invention provides a D-aminotransferase mutant, wherein the tryptophan at position 265 of the amino acid sequence shown in SEQ ID NO.1 is mutated to serine (W265S); the amino acid sequence of the D-aminotransferase mutant is shown in SEQ ID NO.3.

[0014] The present invention also provides a gene encoding the D-aminotransferase mutant, the nucleotide sequence of which is shown in SEQ ID NO.4.

[0015] The present invention also relates to a recombinant vector containing a gene encoding the D-aminotransferase mutant and a recombinant genetic engineering bacterium constructed by the recombinant vector. The recombinant vector can use any expression plasmid in the art as an expression vector, such as pET-24a(+), pET-28b(+), pET-Duet1, pCDF-Duet1, pACYC-Duet1, pRSF-Duet1, etc., and the use of the preferred pET-28a(+) plasmid has a higher expression efficiency. The recombinant genetic engineering bacterium uses Escherichia coli BL21(DE3) as a host. The construction of the expression plasmid can be carried out by any method in the art such as one-step seamless cloning, double enzyme digestion, single enzyme digestion, etc., and the constructed plasmid can be introduced into Escherichia coli BL21(DE3) for expression by transformation.

[0016] The present invention also provides an application of the D-aminotransferase mutant in the one-pot splitting of racemic D,L-glufosinate to prepare L-glufosinate. The application method comprises the following steps: using wet bacteria obtained by fermentation culture of a recombinant genetic engineering bacterium containing a D-aminotransferase mutant encoding gene as a catalyst, racemic D,L-glufosinate as a substrate, an amino donor and D-glucose as cosubstrates, amino acid dehydrogenase and glucose dehydrogenase as coenzymes, nicotinamide adenine dinucleotide (NADH) as a redox cofactor, adding an amino acceptor and pyridoxal phosphate, using a buffer solution with a pH of 6.5-8.0 as a reaction medium to form a reaction system, reacting at 25-50° C. for 8-24 hours, separating and purifying the reaction solution to obtain L-glufosinate; the amino acceptor is pyruvate, α-ketoglutarate, α-ketosuccinate or 3-ketobutyrate, preferably pyruvate; the amino donor is ammonium sulfate or ammonium formate; and the amino acid dehydrogenase comprises glutamate dehydrogenase.

[0017] Preferably, in the reaction system, the catalyst is added to a final concentration of 5-50 g / L (preferably 10-15 g / L) based on the weight of wet cells, and 5-15 g / L based on the dry weight of wet cells; the substrate is added to a final concentration of 40-400 mM, preferably 40 mM; the D-glucose is added to a final concentration of 30-300 mM, preferably 30 mM; the amino acceptor is added to a final concentration of 20-800 mM, preferably 24 mM; the nicotinamide adenine dinucleotide is added to a final concentration of 0.05-1.0 mM, preferably 0.1 mM; the pyridoxal phosphate is added to a final concentration of 0.05-1.0 mM, preferably 0.1 mM; the amino donor is added to a final concentration of 20-300 mM, preferably 26 mM. The molar ratio of the amino acceptor to D-phosphinothricin in D,L-phosphinothricin is 1.1-1.5:1, preferably 1.2:1. The molar ratio of the pyridoxal phosphate to the D-glufosinate in the substrate D,L-glufosinate is 1:200-4000, preferably 1:2000; the molar ratio of the amino donor to the D-glufosinate in the D,L-glufosinate is 1.2-2:1, preferably 1.3:1; the molar ratio of the nicotinamide adenine dinucleotide to the D-glufosinate in the D,L-glufosinate is 1:400-4000, preferably 1:2000.

[0018] Preferably, in the reaction system, amino acid dehydrogenase and glucose dehydrogenase are added in the form of wet cells obtained by fermentation culture of recombinant genetic engineering bacteria containing co-expression of amino acid dehydrogenase encoding genes and glucose dehydrogenase encoding genes, and the final concentration of the wet cells is 5-50 g / L (preferably 10-15 g / L) based on the weight of the wet cells, and 5-15 g / L based on the dry weight of the wet cells, preferably 10 g / L. The amino acid dehydrogenase is glutamate dehydrogenase GluDH, the amino acid sequence is shown in SEQ ID NO.5, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.6; the amino acid sequence of glucose dehydrogenase GDH is shown in SEQ ID NO.7, and the nucleotide sequence is shown in SEQ ID NO.8.

[0019] Preferably, the reaction conditions are: reaction temperature 30-40°C, reaction time 8-12 hours. Most preferably, reaction at 35°C for 12 hours.

[0020] Preferably, the reaction pH is preferably 7.0-8.0, more preferably 8.0. Preferably, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer with pH=6.5-8.0, Tris-hydrochloric acid buffer with pH=8.0-9.5, more preferably disodium hydrogen phosphate-sodium dihydrogen phosphate buffer with pH=8.0.

[0021] Preferably, the catalyst is prepared as follows: a recombinant genetically engineered bacterium containing a D-aminotransferase mutant encoding gene (preferably a recombinant Escherichia coli BL21 (DE3) / pET-28a (+) -D-TAW265S) is inoculated into a LB liquid culture medium containing 50 μg / mL kanamycin resistance, and cultured at 37 ° C, 200 rpm for 8-10 hours, and then inoculated into a fresh LB liquid culture medium containing 50 μg / mL kanamycin resistance at a 1% (v / v) inoculation amount, and cultured at 37 ° C, 180 rpm until the bacterial OD600 reaches 0.6-0.8, IPTG with a final concentration of 0.1 mM is added, and after induction culture at 28 ° C for 12 hours, the culture is centrifuged at 4 ° C, 8000 rpm for 10 minutes, the supernatant is discarded, and the wet bacteria are collected.

[0022] Preferably, the coenzyme is prepared as follows: a recombinant genetic engineering bacterium co-expressing an amino acid dehydrogenase encoding gene and a glucose dehydrogenase encoding gene (preferably a recombinant Escherichia coli BL21 (DE3) / pET-Duet1-GluDH-GDH) is inoculated into a LB liquid medium containing 50 μg / mL ampicillin resistance, and cultured at 37 ° C, 200 rpm for 8-10 hours, and then inoculated into a fresh LB liquid medium containing 50 μg / mL ampicillin resistance at a 1% (v / v) inoculation amount, and cultured at 37 ° C, 180 rpm until the bacterial OD600 reaches 0.6-0.8, IPTG with a final concentration of 0.1 mM is added, and after induction culture at 24 ° C for 14 hours, the culture is centrifuged at 4 ° C, 8000 rpm for 10 minutes, the supernatant is discarded, and the wet bacteria are collected.

[0023] The co-expression recombinant genetic engineering bacteria containing the amino acid dehydrogenase encoding gene and the glucose dehydrogenase encoding gene of the present invention are constructed according to the following method: after full gene synthesis, the gene fragment of the amino acid dehydrogenase (preferably glutamate dehydrogenase GluDH, the nucleotide sequence is shown in SEQ ID NO.6) and the gene fragment of the glucose dehydrogenase GDH (the nucleotide sequence is shown in SEQ ID NO.8) are respectively inserted between the two restriction sites NcoⅠ and NotⅠ of the cloning site 1 of the expression plasmid pET-Duet1 and between the two restriction sites NdeⅠ and XhoⅠ of the cloning site 2 to obtain pET-Duet1-GluDH-GDH; after sequencing verification, pET-Duet1-GluDH-GDH is transferred into the expression host Escherichia coli E. coli BL21 (DE3) to construct the recombinant Escherichia coli E. coli BL21 (DE3) / pET-Duet1-GluDH-GDH.

[0024] The D-aminotransferase mutant of the present invention has strict enantioselectivity, and transfers the amino group of D-phosphinothricin without herbicidal activity in racemic D,L-phosphinothricin to the amino receptor pyruvate, and deaminates to generate the intermediate product 4-(hydroxy(methyl)phosphoryl)-2-oxobutyric acid (PPO), while L-phosphinothricin with herbicidal activity does not participate in the reaction and is completely retained. 14 -10 18 ), the intermediate product 4-(hydroxy(methyl)phosphoryl)-2-oxobutanoic acid is completely converted into L-phosphinothricin ammonium.

[0025] The amino acid dehydrogenase of the present invention can be any amino acid dehydrogenase with 4-(hydroxy(methyl)phosphoryl)-2-oxobutanoic acid activity known in the art. The amino acid dehydrogenase can convert 4-(hydroxy(methyl)phosphoryl)-2-oxobutanoic acid into L-phosphinothricin in the presence of an amino donor, such as ammonium sulfate and ammonium formate.

[0026] The glucose dehydrogenase of the present invention can be any known in the art having a catalytic cofactor NAD + Oxidation occurs to generate NADH dehydrogenase, realizing the redox cofactor cycle, which requires the presence of a cosubstrate.

[0027] The D-aminotransferase mutant, glutamate dehydrogenase and glucose dehydrogenase of the present invention can be added in the form of purified enzymes, ultrasonically broken crude enzyme solutions, wet bacteria, immobilized bacteria, immobilized enzymes, complete fermentation broth or any combination thereof.

[0028] The principle of the one-pot method for synthesizing L-phosphinothricin ammonium is as follows: a D-aminotransferase mutant catalyzes D-phosphinothricin ammonium to synthesize 4-(hydroxy(methyl)phosphoryl)-2-oxobutyric acid in the presence of an amino receptor (pyruvic acid, α-keto acid) and pyridoxal phosphate, and an amino acid dehydrogenase catalyzes 4-(hydroxy(methyl)phosphoryl)-2-oxobutyric acid to synthesize L-phosphinothricin ammonium in the presence of an amino donor and nicotinamide adenine dinucleotide.

[0029] The yield and conversion rate of the present invention can be determined by any method known in the art. For example, the content of D-phosphinothricin and L-phosphinothricin can be determined by pre-column derivatization high performance liquid chromatography (HPLC).

[0030] Compared with the prior art, the beneficial effects of this application are mainly reflected in:

[0031] (1) The D-aminotransferase mutant of the present invention has high enantioselectivity (ee>99.9%), and only catalyzes the conversion of D-phosphinothricin to 4-(hydroxy(methyl)phosphoryl)-2-oxobutanoic acid, while L-phosphinothricin is retained, thereby reducing the difficulty of the reaction required to convert 4-(hydroxy(methyl)phosphoryl)-2-oxobutanoic acid (PPO) into L-phosphinothricin.

[0032] (2) The introduction of the amino acid dehydrogenase of the present invention promotes the reaction equilibrium of the D-aminotransferase mutant to move toward the product 4-(hydroxy(methyl)phosphoryl)-2-oxobutyric acid, and the conversion efficiency far exceeds the catalysis of the D-aminotransferase mutant alone, thereby improving the yield and reducing the amount of amino receptor used.

[0033] (3) The present invention adopts a one-pot method, using commercially available D,L-phosphinothricin as a substrate to synthesize L-phosphinothricin from D-phosphinothricin without herbicidal activity, without the need for chemical synthesis of the intermediate keto acid 4-(hydroxy(methyl)phosphoryl)-2-oxobutanoic acid, and has higher atomic utilization efficiency, meeting the requirements of green chemistry.

[0034] (4) The one-pot method of the present invention for splitting D,L-glufosinate to prepare L-glufosinate is simple to operate and is carried out under mild reaction conditions. It is suitable for large-scale production and has the potential for industrial application. The yield of the L-glufosinate product is as high as 100%, and the enantiomeric excess (ee) is greater than 99.9%. (IV) Description of the drawings

[0035] Figure 1 The invention discloses a reaction formula for synthesizing L-glufosinate by deracemizing D,L-glufosinate ammonium using a multi-enzyme one-pot method.

[0036] Figure 2 This is the construction diagram of pET-28a(+)-D-TA plasmid.

[0037] Figure 3 This is the construction diagram of the pET-Duet1-GluDH-GDH plasmid.

[0038] Figure 4 The D,L-PPT spectrum was detected by pre-column derivatization HPLC.

[0039] Figure 5 This is Example 4, which shows the effect of the amount of pyruvate added on the conversion rate of recombinant Escherichia coli BL21 (DE3) / pET-28a (+) -D-TAW265S single enzyme deracemization of D, L-phosphinothricin to prepare D-PPT.

[0040] Figure 6 This is the effect of reaction temperature and pH on the one-pot deracemization of D,L-phosphinothricin to synthesize L-phosphinothricin in Example 5; A is the effect of temperature, and B is the effect of pH.

[0041] Figure 7 The effect of the amount of amino acceptor and amino donor added on the one-pot deracemization of D,L-phosphinothricin ammonium by three-enzyme cascade to synthesize L-phosphinothricin ammonium is shown in Example 6. A is the effect of the amount of pyruvic acid added, and B is the effect of the amount of ammonium sulfate added.

[0042] Figure 8 The effect of the amount of pyridoxal phosphate and nicotinamide adenine dinucleotide added on the three-enzyme cascade one-pot deracemization of D,L-phosphinothricin to synthesize L-phosphinothricin ammonium is shown in Example 7. A is the effect of the amount of pyridoxal phosphate added, and B is the effect of the amount of nicotinamide adenine dinucleotide added.

[0043] Fig. 9 This is the reaction process of the three-enzyme cascade one-pot method for deracemization of D,L-phosphinothricin to synthesize L-phosphinothricin in Example 8. (V) Specific implementation plan

[0044] In order to better explain the invention of the present application, the present application will be described in detail below in conjunction with a specific implementation scheme. The implementation scheme is only used to explain the present invention and does not limit the scope of protection of the present invention.

[0045] LB liquid culture medium: peptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L, solvent is distilled water.

[0046] LB solid medium: 20 g / L of agar was added to LB liquid medium.

[0047] Kanamycin, ampicillin, streptomycin, chloramphenicol, and isopropylthio-β-D-galactoside (IPTG) were purchased from Shanghai Bioengineering; racemic D,L-phosphinothricin was provided by Shandong Luba Chemical Co., Ltd. (Jinan, China); except for the reagents specified, all others were domestic analytical grade. PLP stands for pyridoxal phosphate.

[0048] Materials and reagents used in genetic engineering: Plasmid Miniprep Kit and PCR product Cleanup Kit were purchased from AxyPrep, one-step seamless cloning kit was purchased from Novezan (Nanjing, China), restriction endonuclease Dpn I was purchased from Takara; primers and whole gene synthesis, gene sequencing were completed in Hangzhou Qingke Biotechnology Co., Ltd.

[0049] Example 1: Construction of recombinant Escherichia coli

[0050] 1. Recombinant E. coli BL21(DE3) / pET-28a(+)-D-TA

[0051] Schematic diagram of plasmid construction Figure 2As shown, the sequence (amino acid sequence as shown in SEQ ID NO.1, nucleotide sequence as shown in SEQ ID NO.2) annotated as D-aminotransferase D-TA (GenBank No.: AAA22252.1) from Bacillus sp was fully synthesized and inserted between the NcoⅠ and NotⅠ sites of the expression plasmid pET-28a(+) to obtain pET-28a(+)-D-TA. After sequencing verification, pET-28a(+)-D-TA was transferred into the expression host Escherichia coli E.coliBL21(DE3) to construct E.coliBL21(DE3) / pET-28a(+)-D-TA, which was used as a wild-type strain for the expression of the recombinant enzyme. After sequencing verification, the plasmid pET-28a(+)-D-TA was extracted.

[0052] SEQ ID NO.1

[0053] MGYTLWNDQIVKDEEVKIDKEDRGYQFGDGVYEVVKVYNGEMFTVNEHIDRLYASAEKIRITIPYTKDKFHQLLHELVEKNELNTGHIYFQVTRGTSPRAHQFPENTVKPVIIGYTKENPRPLENLEKGVKATFVEDIRWLR CDIKSLNLLGAVLAKQEAHEKGCYEAILHRNNTVTEGSSSNVFGIKDGILYTHPANNMILKGITRDVVIACANEINMPVKEIPFTTHEALKMDELFVTSTTSEITPVIEIDGKLIRDGKVGEWTRKLQKQFETKIPKPLHI.

[0054] 2. Construction of recombinant E. coli BL21(DE3) / pET-Duet1-GluDH-GDH

[0055] Schematic diagram of plasmid construction Figure 3As shown, the sequence annotated as glutamate dehydrogenase GluDH (GenBank No.: WP_012292398.1) from Lysinibacillus (amino acid sequence shown in SEQ ID NO.5, nucleotide sequence shown in SEQ ID NO.6) and the sequence annotated as glucose dehydrogenase GDH (GenBank No.: AIZ68241.1) from Exiguobacterium sibiricum (amino acid sequence shown in SEQ ID NO.7, nucleotide sequence shown in SEQ ID NO.8) were fully synthesized and inserted into the expression plasmid pET-Duet1 between the two restriction sites NcoⅠ and NotⅠ in the cloning site 1 and between the two restriction sites NdeⅠ and XhoⅠ in the cloning site 2, respectively, to obtain pET-Duet1-GluDH-GDH. After sequencing verification, pET-Duet1-GluDH-GDH was transferred into the expression host Escherichia coli E. coli BL21 (DE3) to construct recombinant E. coli BL21 (DE3) / pET-Duet1-GluDH-GDH for the expression of the recombinant enzyme. After sequencing verification, the plasmid pET-Duet1-GluDH-GDH was extracted.

[0056] SEQ ID NO.5:

[0057] MSENLNLFTSTQDVIQDALNKLGYDEAMYELLKEPLRMLQVRIPVKMDDGTTKVFTGYRAQHNDAVGPTKGGVRFHPQVSEEEVKALSMWMTLKCGIVDLPYG GGKGGVICDPRQMSMGEIERLSRGYVRAVSQIVGPTKDIPGPDVFTNAQIMAWMMDEYSRMDEFNSPGFITGKPLVLGGSQGRDRATAQGVTIVEEAAKKRGI DIKGARVVIQGFGNAGSFLAKFMHDLGAKVIGISDAYGALHDPEGLDIDYLLDRRDSFGTVTTLFENTISNKELLELDCDILVPAAIENQITADNAHNIKADIVVEAANGPTTAEATKILTERGILLVPDVLASAGGVTVSYFEWVQNNQGYYWTEEEVEERLYKKMVEAFDNVYTTATTRNINMRLAAYMVGVRRTAEASRFRGWV.

[0058] SEQ ID NO.7:

[0059] MGYNSLKGKVAIVTGGSMGIGEAIIRRYAEEGMRVVINYRSHPEEAKKIAEDIKQAGGEALTVQGDVSKEEDMINLVKQTVDHFGQLDVFVNNAGVEMPSPSHEMSLEDWQKVIDVNLTGAFLGAREALKY FVEHNVKGNIINMSSVHEIIPWPTFVHYAASKGGVKLMTQTLAMEYAPKGIRINAIGPGAINTPINAEKFEDPKQRADVESMIPMGNIGKPEEISAVAAWLASDEASYVTGITLFADGGMTLYPSFQAGRG.

[0060] Example 2: Screening of D-aminotransferase mutations

[0061] 1. Construct a D-aminotransferase mutant library by error-prone PCR.

[0062] Using the plasmid pET-28a(+)-D-TA extracted from E. coli BL21(DE3) / pET-28a(+)-D-TA (wild-type D-aminotransferase) in Example 1 as a template, PCR amplification was performed using the primers in Table 1 to randomly introduce mutations.

[0063] PCR reaction system (50 μL): template 0.5-20 ng, 1× Taq Buffer (without Mg 2+ ), 0.2mM dNTP, 0.3mM MnCl2, 2mM MgCl2, 0.2M each of primers T7 promoter and T7 terminator, 5U of Taq DNA polymerase.

[0064] PCR conditions: (1) pre-denaturation at 95°C for 5 min; (2) denaturation at 94°C for 50 s; (3) annealing at 55°C for 60 s; (4) extension at 72°C for 120 s, steps (2) to (4) for a total of 30 cycles; (5) final extension at 72°C for 10 min, and storage at 4°C.

[0065] The PCR product was digested with endonuclease Dpn I at 37°C for 2h, analyzed by agarose gel electrophoresis, and then excised and recovered. The recovered PCR product was ligated to the pET-28a(+) vector by T4 ligase. The ligated product was transformed into E. coli BL21(DE3), spread on LB plates containing 50μg / mL kanamycin, and cultured at 37°C overnight.

[0066] Pick a single colony and culture it in a 96-well plate. Add 1 mL of LB liquid medium containing 50 μg / mL kanamycin and 1 mM IPTG to each well and culture at 37°C for 18 h. Centrifuge the bacteria in the 96-well plate for 30 min (3000 rpm, 4°C) using a 96-well plate centrifuge. After discarding the supernatant, resuspend the bacteria in each well with 1.5 mL of sodium dihydrogen phosphate-disodium hydrogen phosphate buffer (200 m M, pH 8.0).

[0067] Table 1 Primer design table

[0068]

[0069] 2. Screening of mutant strains using ELISA

[0070] Take out 500 μL of bacterial suspension and put it into a new 96-well plate. Add substrate reaction solution (D,L-phosphinothricin 100mM, coenzyme PLP 0.1mM) to each well and react at 35°C for 10min. Mix the reaction solution with the screening reagent in a volume ratio of 1:1, react at 30°C for 30s, detect the fluorescence value at an emission wavelength of 350nm and an excitation wavelength of 460nm, and screen mutant strains with higher fluorescence values ​​than the wild-type strain E. coli BL21(DE3) / pET-28a(+)-D-TA.

[0071] Screening reagents: 0.013 g of o-phthalaldehyde and 0.032 g of N-acetyl-L-cysteine, dissolved in boric acid buffer at pH = 9.8 and made up to 50 mL.

[0072] 3. HPLC analysis method for rescreening mutants

[0073] The reaction solution of the mutant strain screened in step 2 was diluted 40 times with ultrapure water, 200 μL of the diluted reaction solution was taken, 400 μL of the derivatization reagent was added, and the mixture was derivatized at 30°C and 600 rpm for 5 min, and 400 μL of ultrapure water was added to make it 1 mL, and the mixture was mixed and passed through a 0.22 μm microfiltration membrane as a liquid phase sample to be tested. L-phosphinothricin was detected by HPLC analysis, and 5 positive mutant strains and 7 negative mutant strains were screened. Among them, the activity of the positive mutant strain increased by 1.5 to 7 times, and the activity of the negative mutant strain decreased by 40% to 80%. By comparing the 5 positive mutant strains, a high-yield L-phosphinothricin mutant strain was obtained and sent for sequencing, and the high-yield mutant strain E.coliBL21 (DE3) / pET-28a (+) -D-TAW265S was verified, which was recorded as mutant strain W265S. The amino acid sequence of the D-aminotransferase mutant of mutant strain W265S is shown in SEQ ID No.3, and the nucleotide sequence is shown in SEQ ID No.4.

[0074] Pre-column derivatization HPLC detection method: Ultimate 3000 HPLC system (ThermoFisher, Dionex, USA) equipped with a fluorescence detector (UltiMate FLD-3100) was used. C18 (4.6×250mm, 5μm, China). The retention time of L-PPT is 12.9 minutes, and the retention time of D-PPT is 16.1 minutes. Mobile phase: methanol: 0.05M ammonium acetate (pH5.7), volume ratio is 10:90; flow rate is 1.0mL / min, detection wavelength Ex=340nm, Em=450nm, injection volume is 10μL, column temperature is 35℃. The peak diagram of HPLC detection of D,L-phosphinothricin is as follows Figure 4 shown.

[0075] Preparation of derivatization reagent: 0.1 g o-phthalaldehyde (OPA), 0.12 g N-acetyl-L-cysteine ​​(NAC), dissolved in 10 mL anhydrous ethanol, fixed to 50 mL with boric acid buffer (0.1 mol / L pH 9.8), and stored at 4°C for later use.

[0076] 4. Detection of D-aminotransferase mutant enzyme activity

[0077] The recombinant Escherichia coli E. coli BL21 (DE3) / pET-28a (+) -D-TAW265S screened in step 3 was inoculated into 8 mL of LB liquid culture medium containing 50 μg / mL kanamycin resistance, and cultured at 37 ° C, 200 rpm for 8-10 h, and then inoculated into fresh 100 mL of LB liquid culture medium containing 50 μg / mL kanamycin resistance at a 1% (v / v) inoculum. Culture at 37 ° C, 180 rpm until the bacterial OD600 reaches 0.6-0.8, add IPTG with a final concentration of 0.1 mM, and induce culture at 28 ° C for 12 h. After centrifugation at 4 ° C, 8000 rpm for 10 min, the supernatant was discarded, and the precipitate was collected to obtain the recombinant Escherichia coli E. coli BL21 (DE3) / pET-28a (+) -D-TAW265S wet bacteria, which was recorded as mutant W265S wet bacteria.

[0078] 1 g of mutant W265S wet bacteria was suspended in 10 mL of 20 mM phosphate buffer (pH 8.5), and then ultrasonically disrupted for 10 min at 40 W. The ultrasonic disruption conditions were as follows: power 40 W, disruption for 1 s, and pause for 3 s. The disrupted mixture was centrifuged at 4 °C and 8000 r / min for 10 min, the precipitate was discarded, and the supernatant was collected and purified using a nickel column (40×12.6 mm, Bio-Rad, USA). The specific steps were as follows:

[0079] (1) Rinse the Ni column with Binding Buffer at a flow rate of 1 mL / min until the UV baseline is balanced. The amount used is approximately 6 times the volume of the Ni column.

[0080] (2) Load the supernatant onto the Ni column equilibrated in step (1) at a flow rate of 1 mL / min and a loading volume of 1 to 2 column volumes to allow the target protein to fully bind to the Ni column.

[0081] (3) After loading in step (2), wash the impurities with washing buffer at a flow rate of 1 mL / min until the UV baseline is balanced. The amount used is approximately 4 times the volume of the nickel column.

[0082] (4) Wash the Ni column in step (3) with elution buffer to elute the target protein at a flow rate of 1 mL / min. Start collecting when the absorbance reaches 0.25 and increases, and stop collecting when the absorbance drops to 0.25.

[0083] (5) After collecting the eluate in step (4), the Ni column is continuously rinsed with Elution Buffer at a flow rate of 1 mL / min until the baseline is flushed. The Elution Buffer is replaced with Binding Buffer and the Ni column is continuously rinsed. The Binding Buffer is used to balance the Ni column at a flow rate of 1 mL / min until the baseline is flushed. The amount used is approximately 6 times the volume of the Ni column. The Ni column is preserved with 8 times the volume of the column of 20% ethanol and stored in a 4°C refrigerator.

[0084] (7) Removal of salt ions: The eluate collected in step (4) was placed in a dialysis bag (MD44, MW: 8000-14000), placed in a PBS buffer (20 mM, pH 8.5), and dialyzed for desalination at 4°C for 8 h. The mixture was dialyzed again in the same manner, and the retentate was the pure enzyme solution. Pure enzyme solutions E. coli BL21(DE3) / pET-28a(+)-D-TAW265S and pure enzyme solutions E. coli BL21(DE3) / pET-28a(+)-D-TA were obtained, respectively.

[0085] Equilibration buffer (20 mM phosphate buffer, 300 mM NaCl, 20 mM imidazole, pH 8.5): Weigh 3.12 g of sodium dihydrogen phosphate dihydrate (final concentration 20 mM) and 17.53 g of sodium chloride (final concentration 300 mM) into a 1 L beaker, add ultrapure water to dissolve, and finally adjust the volume to 1 L, and adjust the buffer pH to 8.5 with phosphoric acid or sodium hydroxide.

[0086] Washing buffer (20mM phosphate buffer, 300mM NaCl, 50mM imidazole, pH 8.5): Weigh 3.12g of sodium dihydrogen phosphate dihydrate (final concentration 20mM), 17.53g of sodium chloride (final concentration 300mM) and 3.4g of imidazole (final concentration 50mM) into a 1L beaker, add ultrapure water to dissolve, finally make up to 1L, and adjust the buffer pH to 8.5 with phosphoric acid or sodium hydroxide.

[0087] Elution buffer (50 mM phosphate buffer, 300 mM NaCl, 500 mM imidazole, pH 8.5): Weigh 3.12 g of sodium dihydrogen phosphate dihydrate (final concentration 20 mM), 17.53 g of sodium chloride (final concentration 300 mM) and 34 g of imidazole (final concentration 500 mM) into a 1 L beaker, add ultrapure water to dissolve, finally make up to 1 L, and adjust the buffer pH to 8.5 with phosphoric acid or sodium hydroxide.

[0088] Determination of specific enzyme activity of D-aminotransferase and its mutants:

[0089] The enzyme activity unit (U) is defined as: the amount of enzyme required to generate 1 μmol of L-phosphinothion per minute at 35°C and pH 8.0. The specific enzyme activity is defined as the number of activity units per milligram of enzyme protein, U / mg.

[0090] Standard conditions for enzyme activity detection: 100 mM D,L-glufosinate, 0.1 mM PLP, appropriate amount of enzyme solution, 35°C, pH 8.0, 600 rpm for 10 minutes, sample treatment and HPLC analysis.

[0091] The protein concentration was determined using the BCA protein assay kit (Nanjing KeyGen Biotechnology Development Co., Ltd., Nanjing).

[0092] The pure enzyme solution of D-aminotransferase mutant was obtained, with a protein concentration of 4.25 μg / μL and a specific enzyme activity of 88.92 U / mg. The pure enzyme solution of wild-type D-aminotransferase was prepared in the same way, with a protein concentration of 4.51 μg / μL and a specific enzyme activity of 10.78 U / mg.

[0093] Example 3: Cultivation and expression of recombinant Escherichia coli

[0094] The recombinant E. coli BL21 (DE3) / pET-28a (+) -D-TA and E. coli BL21 (DE3) / pET-28a (+) -D-TA W265S constructed in Example 1 and Example 2 were inoculated into 8 mL of LB liquid culture medium containing 50 μg / mL kanamycin resistance, respectively, and cultured at 37 ° C, 200 rpm for 8-10 h, and then inoculated into a fresh 100 mL of LB liquid culture medium containing 50 μg / mL kanamycin resistance at a 1% (v / v) inoculum amount, and cultured at 37 ° C, 180 rpm until the bacterial OD600 reached 0.6-0.8, IPTG with a final concentration of 0.1 mM was added, and after induction culture at 28 ° C for 12 h, the culture was centrifuged at 4 ° C, 8000 rpm for 10 min, the supernatant was discarded, and the precipitate was collected to obtain recombinant E. coli E. coli BL21(DE3) / pET-28a(+)-D-TA and E.coli BL21(DE3) / pET-28a(+)-D-TAW265S wet cells were recorded as E.coliBL21(DE3) / pET-28a(+)-D-TA wet cells (WT) and E.coli BL21(DE3) / pET-28a(+)-D-TAW265S wet cells (W265S).

[0095] The recombinant E. coli BL21 (DE3) / pET-Duet1-GluDH-GDH constructed in Example 1 was inoculated into 8 mL of LB liquid culture medium containing 50 μg / mL ampicillin resistance, and cultured at 37 ° C, 200 rpm for 8-10 h, and then inoculated into a fresh 100 mL of LB liquid culture medium containing 50 μg / mL ampicillin resistance at a 1% (v / v) inoculation amount, and cultured at 37 ° C, 180 rpm until the bacterial OD600 reached 0.6-0.8, IPTG with a final concentration of 0.1 mM was added, and after induction culture at 24 ° C for 14 h, the culture was centrifuged at 4 ° C, 8000 rpm for 10 min, the supernatant was discarded, and the precipitate was collected to obtain the recombinant E. coli BL21 (DE3) / pET-Duet1-GluDH-GDH co-expression wet bacteria, which was recorded as E. coli BL21(DE3) / pET-Duet1-GluDH-GDH co-expression wet bacteria.

[0096] Example 4: Recombinant E. coli BL21 (DE3) / pET-28a (+)-D-TAW265S single enzyme deracemizes D,L-phosphinothricin.

[0097] Reaction system 10mL: pyruvic acid (20mM, 40mM, 60mM, 100mM, 200mM, 400mM and 800mM), D,L-phosphinothricin 40mM, pyridoxal phosphate 0.1mM, 10g / L E.coliBL21(DE3) / pET-28a(+)-D-TAW265S wet bacteria (W265S), solvent is 50mM phosphate buffer (pH=8.0). React at 35°C for 12h. Under the same conditions, 10g / L E.coliBL21(DE3) / pET-28a(+)-D-TA wet bacteria (WT) prepared by the method of Example 3 was used as the control group.

[0098] After the reaction, 200 μL of the reaction solution was taken, 5 μL of 6M hydrochloric acid was added to terminate the reaction, 5 μL of 6M NaOH aqueous solution was added to neutralize, and the reaction solution was centrifuged (12000 rpm, 1 min), and after being diluted with ultrapure water by an appropriate multiple, the D-phosphinothricin conversion rate was detected by the HPLC method described in Example 2 to investigate the effect of the amount of amino receptor pyruvate on the catalytic reaction of mutant W265S. The reaction results are shown in FIG. Figure 5 As shown, with the increase of the amount of pyruvic acid, the conversion rate of D-PPT gradually increased. When 800 mM pyruvic acid (molar ratio = 40 / 1 pyruvic acid / D-phosphinothricin) was used, the conversion rate was 46.89%, and complete conversion could not be achieved.

[0099] Example 5: Effects of reaction temperature and pH on the one-pot deracemization of D,L-phosphinothricin by three-enzyme cascade.

[0100] 1. Reaction temperature

[0101] Reaction system 10mL: D, L-glufosinate 40mM, pyridoxal phosphate 0.1mM, nicotinamide adenine dinucleotide 0.1mM, 30mM pyruvate, 30mM ammonium sulfate, 30mM D-glucose, 10g / L E.coli BL21 (DE3) / pET-28a (+) -D-TAW265S prepared by the method of Example 3 and 10g / L E.coli BL21 (DE3) / pET-Duet1-GluDH-GDH co-expression wet bacteria prepared by the method of Example 3, solvent is 50mM phosphate buffer (pH = 8.0). React at different temperatures (25℃, 30℃, 35℃, 40℃, 45℃ and 50℃) for 12h, and use the HPLC method described in Example 2 to determine the yield of L-glufosinate. The results are shown in Table 1. Figure 6 In A, when the reaction temperature is low (25°C and 30°C), the yield is low. The final yield is the highest at 35°C, reaching 92.15%. When the temperature increases, the yield decreases. Therefore, 35°C is determined to be the most suitable catalytic temperature.

[0102] 2. pH

[0103] The temperature in step 1 was set to 35°C, and the solvents were 50 mM phosphate buffer (pH = 7.0, 7.5, 8.0) and 50 mM Tris-HCl buffer (pH = 8.5, 9.0), respectively. Other operations were the same, and the results are shown in Figure 6 In B, when the reaction pH is 8.0 and phosphate buffer is used, the final yield is the highest.

[0104] Therefore, the reaction conditions for the three-enzyme cascade catalysis were selected as: pH 8.0 and 35°C.

[0105] Example 6: Effects of amino group acceptors and ammonia donors on the one-pot deracemization of D,L-phosphinothricin to synthesize L-phosphinothricin via a three-enzyme cascade.

[0106] 1. Amount of pyruvic acid added

[0107] Reaction system 10mL: D, L-glufosinate 40mM, pyridoxal phosphate 1mM, nicotinamide adenine dinucleotide 0.5mM, different final concentrations of pyruvic acid (20mM, 22mM, 24mM, 26mM, 28mM and 30mM), 30mM ammonium sulfate, 30mM D-glucose, 10g / L E.coliBL21(DE3) / pET-28a(+)-D-TAW265S prepared by the method of Example 3 and 10g / L E.coliBL21(DE3) / pET-Duet1-GluDH-GDH co-expression wet bacteria prepared by the method of Example 3, solvent is 50mM phosphate buffer (pH=8.0). React at 35℃ for 12h, and measure the yield of L-glufosinate by the method of Example 2. The results are shown in Figure 7 In Figure A, when the amount of pyruvate added reached 24 mM, the final yield was 99.9%. Therefore, when the amount of pyruvate used reached a molar ratio of ≥1.2:1 (pyruvate / D-phosphinothricin), it was sufficient to support the entire reaction.

[0108] 2. Ammonium sulfate addition amount

[0109] In step 1, the final concentration of pyruvate was set to 30 mM, and the final concentration of ammonium sulfate was changed to 20 mM, 22 mM, 24 mM, 26 mM, 28 mM and 30 mM. Other operations were the same. The results are shown in Figure 7 In B, when the addition amount reaches 26 mM, the final yield is 99.9%. Therefore, when the amount of ammonium sulfate used reaches a molar ratio ≥ 1.3:1 (ammonium sulfate / D-phosphinothricin), it is sufficient to support the entire reaction.

[0110] Example 7: Effect of coenzyme addition on the one-pot deracemization of D,L-phosphinothricin to synthesize L-phosphinothricin by three-enzyme cascade.

[0111] 1. Amount of pyridoxal phosphate added

[0112] Reaction system 10mL: D, L-glufosinate 40mM, different amounts of pyridoxal phosphate (0.05mM, 0.1mM, 0.2mM, 0.3mM, 0.5mM and 1mM), 0.5mM nicotinamide adenine dinucleotide, 24mM pyruvate, 26mM ammonium sulfate, 30mM D-glucose, 10g / L E. coli BL21 (DE3) / pET-28a (+) -D-TAW265S wet bacteria prepared by the method of Example 3 and 10g / L E. coli BL21 (DE3) / pET-Duet1-GluDH-GDH co-expression wet bacteria prepared by the method of Example 3, the solvent is 50mM phosphate buffer (pH = 8.0). The reaction was carried out at 35°C for 12h, and the L-glufosinate yield was detected by the HPLC method described in Example 2. The results are shown in FIG. Figure 8 In Figure A, when the addition amount of pyridoxal phosphate reaches 0.1 mM, the final yield can reach 100%. Therefore, when the addition amount of pyridoxal phosphate reaches 0.1 mM, it is sufficient to support the entire reaction.

[0113] 2. Amount of Nicotinamide Adenine Dinucleotide Added

[0114] In step 1, pyridoxal phosphate was set to 1 mM, and the amount of nicotinamide adenine dinucleotide added was changed to 0.05 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.5 mM and 1 mM. Other operations were the same. The results are shown in Figure 8 In Figure B, when the amount of nicotinamide adenine dinucleotide added reaches 0.1 mM, the final yield can reach 100%. Therefore, when the amount of nicotinamide adenine dinucleotide added reaches 0.1 mM, it is sufficient to support the entire reaction.

[0115] Example 8: Reaction process of three-enzyme cascade one-pot deracemization of D,L-phosphinothricin to synthesize L-phosphinothricin

[0116] The reaction system was 30 mL: 400 mM D, L-phosphinothricin, 0.1 mM pyridoxal phosphate, 0.1 mM nicotinamide adenine dinucleotide, 240 mM pyruvate, 260 mM ammonium sulfate, 260 mM D-glucose, 15 g / L E. coli BL21 (DE3) / pET-28a (+) -D-TAW265S wet bacteria (W265S) prepared in Example 3 and 15 g / L E. coli BL21 (DE3) / pET-Duet1-GluDH-GDH co-expression wet bacteria prepared by the method of Example 3, and the solvent was 50 mM phosphate buffer (pH = 8.0). Under the same conditions, 15 g / L of E. coli BL21 (DE3) / pET-28a (+) -D-TA wet bacteria (WT) prepared by the method of Example 3 and 15 g / L of E. coli BL21 (DE3) / pET-Duet1-GluDH-GDH co-expression wet bacteria prepared by the method of Example 3 were used as control groups. They were reacted at 35°C for 12 hours, and the HPLC method described in Example 2 was used to detect the yield of L-phosphinothricin ammonium. The results are shown in Table 1. Fig. 9 As shown, under the optimized reaction system, the substrate concentration was increased to 400 mM, and the final yield reached 100% after 10 hours of reaction, and the racemic D,L-glufosinate was completely converted into L-glufosinate.

[0117] As shown in the above cases, the biological multi-enzyme one-pot method of deracemizing D,L-phosphinothricin to synthesize L-phosphinothricin has high efficiency and potential for industrial application.

Claims

1. A D-aminotransferase mutant, characterized in that: The D-aminotransferase mutant is obtained by mutating the tryptophan at position 265 of the amino acid sequence shown in SEQ ID NO.1 to serine.

2. A gene encoding the D-aminotransferase mutant according to claim 1.

3. A recombinant genetically engineered bacterium containing a gene encoding the D-aminotransferase mutant according to claim 2.

4. Use of the D-aminotransferase mutant according to claim 1 in the one-pot resolution of racemic D,L-glufosinate to prepare L-glufosinate.

5. The use according to claim 4, characterized in that The application method comprises the following steps: using wet bacteria obtained by fermentation culture of a recombinant genetic engineering bacterium containing a D-transaminase mutant encoding gene as a catalyst, using racemic D,L-phosphinothricin as a substrate, an amino donor and D-glucose as co-substrates, amino acid dehydrogenase and glucose dehydrogenase as coenzymes, using NADH as a redox cofactor, adding an amino acceptor and pyridoxal phosphate, using a pH 7.0-8.0 buffer as a reaction medium to form a reaction system, reacting at 30-40° C. for 8-12 hours, separating and purifying the reaction solution to obtain L-phosphinothricin; the amino acceptor is pyruvic acid; and the amino donor is ammonium sulfate.

6. The use according to claim 5, characterized in that In the reaction system, the catalyst is added to a final concentration of 10-15 g / L based on the weight of the wet cells; the substrate is added to a final concentration of 40-400 mM; the D-glucose is added to a final concentration of 30-300 mM; the amino acceptor is added to a final concentration of 20-30 mM; the NADH is added to a final concentration of 0.05-1.0 mM; the pyridoxal phosphate is added to a final concentration of 0.05-1.0 mM; the amino donor is added to a final concentration of 20-30 mM; the amino acid dehydrogenase and the glucose dehydrogenase are added in the form of wet cells obtained by fermentation culture of a co-expressed recombinant genetic engineering bacterium containing an amino acid dehydrogenase encoding gene and a glucose dehydrogenase encoding gene, and the wet cells are added to a final concentration of 10-15 g / L based on the weight of the wet cells; the nucleotide sequence of the amino acid dehydrogenase encoding gene is shown in SEQ ID NO.6, and the nucleotide sequence of the glucose dehydrogenase encoding gene is shown in SEQ ID NO.

8.

7. The use according to claim 5, characterized in that The catalyst is prepared as follows: a recombinant genetically engineered bacterium containing a D-aminotransferase mutant encoding gene is inoculated into a LB liquid culture medium containing 50 μg / mL kanamycin resistance, and cultured at 37°C and 200 rpm for 8-10 h, and then inoculated into a fresh LB liquid culture medium containing 50 μg / mL kanamycin resistance at a volume concentration of 1%, and cultured at 37°C and 180 rpm until the bacterial OD600 reaches 0.6-0.8, IPTG with a final concentration of 0.1 mM is added, and after induction culture at 28°C for 12 h, the culture is centrifuged at 4°C and 8000 rpm for 10 min, the supernatant is discarded, and the wet bacterial cells are collected.

8. The use according to claim 6, characterized in that The coenzyme is prepared as follows: inoculate the co-expressed recombinant genetic engineering bacteria containing the amino acid dehydrogenase encoding gene and the glucose dehydrogenase encoding gene into a LB liquid culture medium containing 50 μg / mL ampicillin resistance, culture at 37°C and 200 rpm for 8-10 h, then inoculate into a fresh LB liquid culture medium containing 50 μg / mL ampicillin resistance at a volume concentration of 1%, culture at 37°C and 180 rpm until the bacterial OD600 reaches 0.6-0.8, add IPTG with a final concentration of 0.1 mM, induce and culture at 24°C for 14 h, centrifuge at 4°C and 8000 rpm for 10 min, discard the supernatant, and collect the wet bacteria.

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