Alcohol dehydrogenase and application thereof in asymmetric synthesis of L-glufosinate-ammonium

Abstract

The invention relates to the technical field of enzyme engineering, and provides alcohol dehydrogenase, application of the alcohol dehydrogenase in asymmetric synthesis of L-glufosinate-ammonium and alcohol dehydrogenase, in order to solve the problem of high production cost caused by poor enzyme activity of alcohol dehydrogenase in production of L-glufosinate-ammonium by a biological catalysis method in the prior art, the alcohol dehydrogenase is alcohol dehydrogenase KPA or alcohol dehydrogenase A18.8, and the alcohol dehydrogenase can be applied to asymmetric synthesis of L-glufosinate-ammonium. The nucleotide sequence of the alcohol dehydrogenase KPA is as shown in SEQ ID NO.1, the amino acid sequence coded by the alcohol dehydrogenase KPA is as shown in SEQ ID NO.2, the nucleotide sequence of the alcohol dehydrogenase A18.8 is as shown in SEQ ID NO.3, and the amino acid sequence coded by the alcohol dehydrogenase A18.8 is as shown in SEQ ID NO.4. The two alcohol dehydrogenases have excellent catalytic activity, and can be used for asymmetric synthesis of L-glufosinate-ammonium reaction to keep high yield of L-glufosinate-ammonium and reduce the use amount of coenzyme.

Description

Technical Field

[0001] The invention relates to the technical field of enzyme engineering, in particular to alcohol dehydrogenase and application thereof in asymmetric synthesis of L-glufosinate ammonium. Background Art

[0002] Glufosinate (PPT for short) is an efficient, broad-spectrum, contact herbicide belonging to the class of organophosphine herbicides. Its mechanism of action is to interfere with the metabolic pathway of glutamine synthetase in plants. It has the advantages of low toxicity, harmlessness, and easy degradation. Currently, the glufosinate sold on the market is usually a racemic mixture, but only the L-configuration of the mixture has herbicidal activity. Therefore, providing a pure L-configuration of glufosinate products can significantly reduce the amount of glufosinate used, reduce the cost of use, and alleviate environmental pressure.

[0003] The biocatalytic method for producing glufosinate has the advantages of strict stereoselectivity, mild reaction conditions, and high yield. Since the industrial production technology of D,L-glufosinate is very mature, the route for producing L-glufosinate by biocatalysis is to directly use D,L-glufosinate as a raw material and directly deracemize it using a biocatalyst to prepare L-glufosinate. The specific process is to first use D-amino acid oxidase to catalyze D-glufosinate in D,L-glufosinate into 2-carbonyl-4-[hydroxy(methyl)phosphonyl]butyric acid (PPO), and then catalytically reduce PPO to L-glufosinate by glutamate dehydrogenase. When glutamate dehydrogenase is used to catalyze the reduction of PPO, additional coenzyme NADH or NADPH is required, but because the amount of NADH and NADPH required to form a cycle in the reaction of this system is large and expensive, a biological enzyme is usually added to provide a coenzyme cycle. The biological enzyme used in the conventional coenzyme cycle is glucose dehydrogenase, which uses glucose as a substrate and provides NAD(P)H for the cycle while producing gluconic acid as a byproduct. Because gluconic acid and refined glufosinate are both highly water-soluble and have similar properties, gluconic acid is difficult to remove during post-treatment or requires complicated steps to remove. Using alcohol dehydrogenase to provide a coenzyme cycle, low-boiling alcohols such as isopropanol, methanol, and ethanol can be used as substrates, and its byproducts can be removed by heating and rotary evaporation, which is simple to operate and convenient for post-treatment. For example, the invention patent with publication number CN116162665A, "A method and device for preparing L-glufosinate from D,L-glufosinate reaction liquid", uses an alcohol dehydrogenase coenzyme regeneration system with alcohol dehydrogenase as a coenzyme regeneration enzyme and isopropanol as a coenzyme regeneration substrate, but the alcohol dehydrogenase used in this system has a low regeneration efficiency for the coenzyme, resulting in a large amount of coenzyme, which increases the preparation cost of L-glufosinate. Summary of the invention

[0004] The present invention firstly overcomes the problem of high production cost caused by poor alcohol dehydrogenase activity in the production of L-phosphinothricin ammonium by biocatalytic method under the prior art, and provides two alcohol dehydrogenases with excellent catalytic activity. The alcohol dehydrogenases can be used in the asymmetric synthesis of L-phosphinothricin ammonium to maintain a high yield of L-phosphinothricin ammonium and reduce the amount of coenzyme used.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions: Alcohol dehydrogenase, wherein the alcohol dehydrogenase is alcohol dehydrogenase KPA or alcohol dehydrogenase A18.8, The nucleotide sequence of alcohol dehydrogenase KPA is shown in SEQ ID NO.1, and the amino acid sequence encoded by it is shown in SEQ ID NO.2. The nucleotide sequence of alcohol dehydrogenase A18.8 is shown in SEQ ID NO.3, and the amino acid sequence encoded by it is shown in SEQ ID NO.4.

[0006] Alcohol dehydrogenase KPA and alcohol dehydrogenase A18.8 can catalyze the regeneration cycle of NADPH and NADH and have excellent catalytic activity.

[0007] A method for asymmetric synthesis of L-glufosinate-ammonium comprises the following steps: (1) 2-carbonyl-4-[hydroxy(methyl)phosphonyl]butyric acid is used as a substrate, mixed with a buffer, an ammonium salt and an organic co-solvent, and then glutamate dehydrogenase, the alcohol dehydrogenase and a coenzyme are added to react to obtain a reaction solution; (2) Separate and purify the reaction solution to obtain L-phosphinothricin.

[0008] The reaction process of the present invention is as follows: PPO is converted into L-phosphinothricin ammonium under the action of glutamate dehydrogenase and coenzyme, and alcohol dehydrogenase participates in the regeneration cycle of coenzyme. In this method, the yield of L-phosphinothricin ammonium is high, the catalytic activity of alcohol dehydrogenase is high, and the regeneration speed of coenzyme is fast, so the amount used is small.

[0009] Preferably, the glutamate dehydrogenase in (1) is glutamate dehydrogenase GluGDH1, whose nucleotide sequence is shown in SEQ ID NO.5, amino acid sequence is shown in SEQ ID NO.6, and coenzyme is NADP + .

[0010] GluGDH1 was cloned from Pseudomonas monteilii WP_060477601.1 and is an NADPH-dependent glutamate dehydrogenase.

[0011] Preferably, the glutamate dehydrogenase in (1) is glutamate dehydrogenase GluGDH2, whose nucleotide sequence is shown in SEQ ID NO.7, amino acid sequence is shown in SEQ ID NO.8, and coenzyme is NAD + .

[0012] GluGDH2 was cloned from Lysinibacillus composti WP_124765558.1 and is a NADH-dependent glutamate dehydrogenase.

[0013] Preferably, in (1), glutamate dehydrogenase and alcohol dehydrogenase are added in the form of wet bacteria, broken enzyme solution or membrane-permeated bacteria solution, the added amount of alcohol dehydrogenase is 10 to 500 g / L based on the wet weight of bacteria, and the added amount of glutamate dehydrogenase is 10 to 500 g / L based on the wet weight of bacteria.

[0014] Preferably, the alcohol dehydrogenase in (1) is obtained by the following steps: constructing a gene encoding alcohol dehydrogenase into a recombinant expression vector, transforming the recombinant expression vector into a host bacterium to obtain an alcohol dehydrogenase-expressing engineered bacterium, and culturing the alcohol dehydrogenase-expressing engineered bacterium to obtain alcohol dehydrogenase; and the glutamate dehydrogenase is obtained by the following steps: constructing a gene encoding glutamate dehydrogenase into a recombinant expression vector, transforming the recombinant expression vector into a host bacterium to obtain an glutamate dehydrogenase-expressing engineered bacterium, and culturing the glutamate dehydrogenase-expressing engineered bacterium to obtain glutamate dehydrogenase.

[0015] Preferably, the alcohol dehydrogenase and glutamate dehydrogenase in (1) are obtained by the following steps: constructing the coding gene of the alcohol dehydrogenase and the coding gene of the glutamate dehydrogenase into the same recombinant expression vector, transforming the recombinant expression vector into host bacteria to obtain an engineered bacterium that co-expresses the alcohol dehydrogenase and glutamate dehydrogenase, and culturing the engineered bacterium that co-expresses the alcohol dehydrogenase and glutamate dehydrogenase to obtain the alcohol dehydrogenase and glutamate dehydrogenase.

[0016] The alcohol dehydrogenase and glutamate dehydrogenase provided by the present invention can be used in the form of a co-expression strain to catalyze the synthesis of L-phosphinothricin ammonium, or can be used in combination in the form of a separate recombinant strain.

[0017] Preferably, the vector is a plasmid, a phage or a viral vector.

[0018] More preferably, the vector is pACYCDuet-1.

[0019] Preferably, the host bacterium is Escherichia coli.

[0020] More preferably, the host bacterium is Escherichia coli BL21 (DE3).

[0021] Preferably, the ammonium salt in (1) is one or more of ammonium sulfate, ammonium chloride, and ammonium formate, the organic co-solvent is one or more of isopropanol, methanol, and ethanol, and the buffer is a phosphate buffer with a pH of 5.5 to 8.0.

[0022] Preferably, the initial concentration of the substrate in (1) is 10-1500 mmol / L, the molar ratio of the organic co-solvent to the substrate is (1-1.2):1, the molar ratio of the ammonium salt to the substrate is (0.6-1.5):1, and the amount of coenzyme added is 0.05-0.5 g / L.

[0023] Preferably, the reaction temperature in (1) is 20-50°C.

[0024] Therefore, the present invention has the following beneficial effects: alcohol dehydrogenase has better catalytic activity, can catalyze the synthesis of L-glufosinate with a concentration of 100 to 300 g / L, with a conversion rate of 99%, a yield of 98%, and an optical purity of more than 99%, accelerates the regeneration cycle of NADH and NADPH, reduces the usage of NADH and NADPH in the synthesis reaction, and thus reduces the synthesis cost, and has a good application development prospect. DETAILED DESCRIPTION

[0025] The present invention is further described below in conjunction with specific implementation methods.

[0026] Example 1 Asymmetric synthesis of L-phosphinothricin ammonium using alcohol dehydrogenase KPA, the steps are as follows: S1. Construction of co-expression strain of alcohol dehydrogenase KPA and glutamate dehydrogenase GluGDH1: S1.1. Construct a co-expression vector pACYCDuet-1-KPA-GluGDH1 for alcohol dehydrogenase KPA (nucleotide sequence is shown in SEQ ID NO.1, amino acid sequence is shown in SEQ ID NO.2) and glutamate dehydrogenase GluGDH1 (nucleotide sequence is shown in SEQ ID No.5, amino acid sequence is shown in SEQ ID No.6), and retain the alcohol dehydrogenase N-terminal His-tag gene; S1.2, transforming pACYCDuet-1-KPA-GluGDH1 into E. coli BL21 (DE3) to obtain the alcohol dehydrogenase and glutamate dehydrogenase co-expression strain E. coli BL21 (DE3)-pACYCDuet-KPA-GluGDH1; S2, Inducible expression of alcohol dehydrogenase KPA and glutamate dehydrogenase GluGDH1 co-expression strain: The E. coli BL21(DE3)-pACYCDuet-KPA-GluGDH1 obtained in S1 was inoculated into LB medium containing 50 μg / mL chloramphenicol, and cultured at 37°C, 200 rpm overnight. Then, 1% inoculum (v / v) was inoculated into LB medium containing 50 μg / mL chloramphenicol, and cultured at 37°C, 200 rpm until the bacterial concentration OD600 reached about 0.6. IPTG was added at a final concentration of 0.1 mM, and after induction culture at 26°C for 6 h, the bacterial cells were collected by centrifugation at 4°C, 8000 rpm for 10 min, and stored at -80°C for later use. S3. Fermentation tank culture of the strain co-expressing alcohol dehydrogenase KPA and glutamate dehydrogenase GluGDH1: The E. coli BL21 (DE3) -pACYCDuet-KPA-GluGDH1 obtained in S1 was inoculated into LB medium containing 50 μg / mL kanamycin, and cultured at 37 ° C, 200 rpm overnight, and then inoculated into a medium containing 50 μg / mL kanamycin at a 2% inoculum (v / v) and cultured at 37 ° C, 200 rpm, and inoculated into a fermenter containing 15 L fermentation medium containing 50 μg / mL kanamycin at a 10% inoculum (v / v) in the mid-logarithmic phase, and cultured at 37 ° C for 14 hours (mid-to-late logarithmic phase), lactose was added for induction for 20 hours, and the wet cells were collected by centrifugation in a tubular centrifuge for later use; S4, alcohol dehydrogenase KPA and glutamate dehydrogenase GluGDH1 co-expression strain catalyzes high concentration of 2-carbonyl-4-[hydroxy(methyl)phosphonyl]butyric acid (PPO): The wet cells obtained in S3 were crushed by a homogenizer to obtain an enzyme solution with a crushing concentration of 500 g / L. The reaction system was 1 L, and the enzyme solution was added with a final concentration of 50 g / L, 2-carbonyl-4-[hydroxy(methyl)phosphonyl]butyric acid (PPO) 500 mM, ammonium sulfate 400 mM, isopropanol 4% (V / V), NADP + 0.2 g / L, 50 mM pH 7.0 phosphate buffer, 35°C, 800 rpm.

[0027] Example 2 Asymmetric synthesis of L-phosphinothricin ammonium using alcohol dehydrogenase A18.8, the steps are as follows: S1. Construction of a strain co-expressing alcohol dehydrogenase A18.8 and glutamate dehydrogenase GluGDH2: S1.1. Constructing a vector pACYCDuet-1-A18.8-GluGDH2 for co-expressing alcohol dehydrogenase A18.8 (nucleotide sequence is shown in SEQ ID NO.3, amino acid sequence is shown in SEQ ID NO.4) and glutamate dehydrogenase GluGDH2 (nucleotide sequence is shown in SEQ ID No.7, amino acid sequence is shown in SEQ ID No.8), retaining the N-terminal His-tag gene of alcohol dehydrogenase; S1.2, transforming pACYCDuet-1-A18.8-GluGDH2 into Escherichia coli E. coli BL21 (DE3) to obtain the alcohol dehydrogenase and glutamate dehydrogenase co-expression strain E. coli BL21 (DE3)-pACYCDuet-1-A18.8-GluGDH2; S2, induction expression of alcohol dehydrogenase A18.8 and glutamate dehydrogenase GluGDH2 co-expression strain: the steps are the same as those in Example 1; S3, fermentation tank culture of alcohol dehydrogenase A18.8 and glutamate dehydrogenase GluGDH2 co-expression strain: the steps are the same as those in Example 1; S4, alcohol dehydrogenase A18.8 and glutamate dehydrogenase GluGDH2 co-expression strain catalyzes high concentration of 2-carbonyl-4-[hydroxy(methyl)phosphonyl]butyric acid (PPO): NADP + Replaced by NAD + The remaining steps and reaction conditions are the same as in Example 1.

[0028] Example 3 Asymmetric synthesis of L-phosphinothricin ammonium using alcohol dehydrogenase KPA, the steps are as follows: S1. Construction of alcohol dehydrogenase KPA expression strain and glutamate dehydrogenase GluGDH1 expression strain: S1.1, construct the vector pACYCDuet-1-KPA for expressing alcohol dehydrogenase KPA (nucleotide sequence is shown in SEQ ID NO.1, amino acid sequence is shown in SEQ ID NO.2), retain the N-terminal His-tag gene of alcohol dehydrogenase, and add pACYCDuet-1-KPA to E. coli BL21 (DE3) to obtain the alcohol dehydrogenase expression strain E. coli BL21 (DE3)-pACYCDuet-KPA; S1.2, construct the vector pACYCDuet-1-GluGDH1 for expressing glutamate dehydrogenase GluGDH1 (nucleotide sequence is shown in SEQ ID NO.5, amino acid sequence is shown in SEQ ID NO.6), and add pACYCDuet-1-GluGDH1 to E. coli BL21 (DE3) to obtain the glutamate dehydrogenase GluGDH1 expression strain E. coli BL21 (DE3)-pACYCDuet-GluGDH1; S2, induced expression of alcohol dehydrogenase KPA expression strain and glutamate dehydrogenase GluGDH1 expression strain: the induction conditions are the same as those in Example 1; S3, alcohol dehydrogenase KPA expression strain and glutamate dehydrogenase GluGDH1 expression strain fermentation tank culture: culture conditions are the same as in Example 1, to obtain alcohol dehydrogenase KPA wet bacteria and glutamate dehydrogenase GluGDH1 wet bacteria; S4, alcohol dehydrogenase KPA expression strain and glutamate dehydrogenase GluGDH1 expression strain jointly catalyze high concentration of 2-carbonyl-4-[hydroxy(methyl)phosphonyl]butyric acid (PPO): The alcohol dehydrogenase KPA wet cells obtained in S3 were crushed by a homogenizer to obtain an alcohol dehydrogenase KPA enzyme solution with a crushing concentration of 500 g / L, and the glutamate dehydrogenase GluGDH1 wet cells obtained in S3 were crushed by a homogenizer to obtain a glutamate dehydrogenase GluGDH1 enzyme solution with a crushing concentration of 500 g / L. The reaction system was 1 L, the alcohol dehydrogenase KPA enzyme solution was added to a final concentration of 50 g / L, the glutamate dehydrogenase GluGDH1 enzyme solution was added to a final concentration of 50 g / L, 2-carbonyl-4-[hydroxy(methyl)phosphonyl]butyric acid (PPO) 500 mM, ammonium sulfate 400 mM, isopropanol (V / V), NADP + 0.2 g / L, 50 mM pH 7.0 phosphate buffer, 35°C, 800 rpm.

[0029] Comparative Example 1 The asymmetric synthesis of L-glufosinate using alcohol dehydrogenase is carried out in the same manner as in Example 1, except that the alcohol dehydrogenase LbADH (gene accession number: CAD66648) derived from Lactobacillus brevis is used to replace the alcohol dehydrogenase KPA.

[0030] In the above-mentioned embodiments and comparative examples, the reaction system was heated at high temperature after reacting for 6 hours, and then the solid impurities were removed by suction filtration. The obtained filtrate was subjected to rotary evaporation to remove water until the reaction liquid was in a colloid state, and four times the volume of methanol was added for crystallization and desalination. Four times the volume of ethanol was added to the crystallization filtrate, and the pH was adjusted to 9 with ammonia water and then allowed to stand at 4°C for crystallization. The crude L-glufosinate was obtained by suction filtration, and the crude L-glufosinate was dissolved and then detected by liquid chromatography, and the PPO conversion rate, L-glufosinate ee value and PPT yield were calculated. The results are shown in the following table. project PPO conversion rate / % L-Glufosinate ee value / % PPT yield / % Example 1 99 99 98 Example 2 99 99 98 Example 3 98 99 95 Comparative Example 1 15 99 12

[0031] It can be seen from the PPO conversion rate, L-phosphinothionine ee value and PPT yield of the above embodiments and comparative examples that, compared with the alcohol dehydrogenase LbADH conventionally used in existing industrial production, alcohol dehydrogenase KPA and alcohol dehydrogenase A18.8 have better reaction activity and can obtain a higher PPT yield at a lower coenzyme usage.

Claims

1. Alcohol dehydrogenase, characterized in that The alcohol dehydrogenase is alcohol dehydrogenase KPA or alcohol dehydrogenase A18.8, The nucleotide sequence of alcohol dehydrogenase KPA is shown in SEQ ID NO.1, and the amino acid sequence encoded by it is shown in SEQ ID NO.

2. The nucleotide sequence of alcohol dehydrogenase A18.8 is shown in SEQ ID NO.3, and the amino acid sequence encoded by it is shown in SEQ ID NO.

4.

2. A method for asymmetric synthesis of L-phosphinothricin ammonium, characterized in that: The steps include: (1) 2-Carbonyl-4-[hydroxy(methyl)phosphonyl]butyric acid is used as a substrate, mixed with a buffer, an ammonium salt and an organic co-solvent, and then glutamate dehydrogenase, the alcohol dehydrogenase as claimed in claim 1 and a coenzyme are added to react to obtain a reaction solution; (2) Separate and purify the reaction solution to obtain L-phosphinothricin.

3. A method for asymmetric synthesis of L-phosphinothricin ammonium according to claim 2, characterized in that: The glutamate dehydrogenase in (1) is glutamate dehydrogenase GluGDH1, the nucleotide sequence of which is shown in SEQ ID NO.5, the amino acid sequence of which is shown in SEQ ID NO.6, and the coenzyme is NADP + .

4. A method for asymmetric synthesis of L-phosphinothricin ammonium according to claim 2, characterized in that: The glutamate dehydrogenase in (1) is glutamate dehydrogenase GluGDH2, the nucleotide sequence of which is shown in SEQ ID NO.7, the amino acid sequence of which is shown in SEQ ID NO.8, and the coenzyme is NAD + .

5. A method for asymmetric synthesis of L-phosphinothricin according to claim 2, 3 or 4, characterized in that: In the above (1), glutamate dehydrogenase and alcohol dehydrogenase are added in the form of wet bacteria, broken enzyme solution or membrane-permeated bacteria solution, the added amount of alcohol dehydrogenase is 10-500 g / L based on the wet weight of bacteria, and the added amount of glutamate dehydrogenase is 10-500 g / L based on the wet weight of bacteria.

6. The method for asymmetric synthesis of L-phosphinothricin ammonium according to claim 5, characterized in that: The alcohol dehydrogenase in (1) is obtained by the following steps: constructing the coding gene of alcohol dehydrogenase into a recombinant expression vector, transforming the recombinant expression vector into a host bacterium to obtain an alcohol dehydrogenase-expressing engineered bacterium, and culturing the alcohol dehydrogenase-expressing engineered bacterium to obtain alcohol dehydrogenase; the glutamate dehydrogenase is obtained by the following steps: constructing the coding gene of glutamate dehydrogenase into a recombinant expression vector, transforming the recombinant expression vector into a host bacterium to obtain an glutamate dehydrogenase-expressing engineered bacterium, and culturing the glutamate dehydrogenase-expressing engineered bacterium to obtain glutamate dehydrogenase.

7. The method for asymmetric synthesis of L-phosphinothricin ammonium according to claim 5, characterized in that: The alcohol dehydrogenase and glutamate dehydrogenase in (1) are obtained by the following steps: constructing the coding gene of the alcohol dehydrogenase and the coding gene of the glutamate dehydrogenase into the same recombinant expression vector, transforming the recombinant expression vector into host bacteria to obtain an engineered bacterium that co-expresses the alcohol dehydrogenase and glutamate dehydrogenase, and culturing the engineered bacterium that co-expresses the alcohol dehydrogenase and glutamate dehydrogenase to obtain the alcohol dehydrogenase and glutamate dehydrogenase.

8. The method for asymmetric synthesis of L-phosphinothricin ammonium according to claim 2, characterized in that: The ammonium salt in (1) is one or more of ammonium sulfate, ammonium chloride, and ammonium formate, the organic co-solvent is one or more of isopropanol, methanol, and ethanol, and the buffer is a phosphate buffer with a pH of 5.5 to 8.

0.

9. The method for asymmetric synthesis of L-phosphinothricin ammonium according to claim 8, characterized in that: In the above (1), the initial concentration of the substrate is 10-1500 mmol / L, the molar ratio of the organic co-solvent to the substrate is (1-1.2):1, the molar ratio of the ammonium salt to the substrate is (0.6-1.5):1, and the amount of the coenzyme added is 0.05-0.5 g / L.

10. The method for asymmetric synthesis of L-phosphinothricin ammonium according to claim 2, characterized in that: The reaction temperature in (1) is 20-50°C.

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