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Amino acid dehydrogenase mutant and application of mutant in synthesizing L-glufosinate

An amino acid and dehydrogenase technology, which is applied to amino acid dehydrogenase mutants and its application in the synthesis of L-glufosinate-ammonium, can solve the problems of low substrate concentration and low asymmetric amination reduction activity, and achieve The effect of shortened reaction time and good industrial application prospects

Active Publication Date: 2019-04-12
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The object of the present invention is to provide an amino acid dehydrogenase for the problems of low activity of the existing amino acid dehydrogenase to the asymmetric amination reduction of 2-carbonyl-4-(hydroxymethylphosphinyl)-butyric acid and low substrate concentration. The hydrogenase mutant and the amino acid dehydrogenase mutant gene recombinant bacteria and its crude enzyme solution are used as biocatalysts for L-glufosinate-ammonium chiral biosynthesis, the activity of the catalyst is increased by 33 times, and the substrate concentration is increased 5 times

Method used

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  • Amino acid dehydrogenase mutant and application of mutant in synthesizing L-glufosinate
  • Amino acid dehydrogenase mutant and application of mutant in synthesizing L-glufosinate
  • Amino acid dehydrogenase mutant and application of mutant in synthesizing L-glufosinate

Examples

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Embodiment 1

[0033] Example 1: Construction and screening of amino acid dehydrogenase mutant library

[0034] The amino acid dehydrogenase gene (nucleotide sequence shown in SEQ ID No.1, amino acid sequence shown in SEQ ID No.2) cloned from Pseudomonas monteiliiWP_060477601.1 was constructed expression vector pETDEut- dygdh was transformed into Escherichia coli to obtain the starting strain E.coli BL21(DE3) / pETDEut-dygdh.

[0035] The preparation of the amino acid dehydrogenase mutant library was achieved through 4 rounds of site-directed saturation mutagenesis. The primers were designed as shown in Table 1. In the first round, the vector pETDEut-dygdh was used as a template, and F95F and F95R in Table 1 were used as primers, and saturation mutation PCR was carried out. , the 95th phenylalanine in the amino acid sequence of amino acid dehydrogenase shown in SEQID No.2 was mutated into the remaining 19 amino acids, and transformed, plated, and the amino acid dehydrogenase mutant pDyGDH-F95L...

Embodiment 2

[0039] Example 2: Induced expression of amino acid dehydrogenase parent, mutant and glucose dehydrogenase

[0040] Glucose dehydrogenase gene esgdh (nucleotide sequence is shown in SEQ ID No.3, amino acid sequence is shown in SEQ ID No.4) cloned from Exiguobacterium sibiricum, and connected to pET-28b(+) by double restriction enzymes On the vector, the recombinant plasmid was introduced into Escherichia coli E.coli BL21(DE3) to obtain the recombinant glucose dehydrogenase strain E.coliBL21(DE3) / pET28b-esgdh.

[0041] The starting strain E.coli BL21(DE3) / pETDEut-dygdh and the amino acid dehydrogenase mutant strain in Example 1 were respectively inoculated into LB liquid medium containing ampicillin at a final concentration of 50 μg / mL, cultured at 37°C for 8 hours, Inoculate the inoculum with a volume concentration of 2% into fresh LB liquid medium containing ampicillin at a final concentration of 50 μg / mL, culture at 37°C and 180 rpm for 1.5 hours, and then add a final concent...

Embodiment 3

[0044] Example 3: Mutant library screening

[0045] Mix the wet cells of the mutant strain induced and expressed in Example 2 and the wet cells of glucose dehydrogenase at a mass ratio of 3:1, add the amount of 50 g / L of the total amount of cells into pH 7.4, and resuspend in 100 mM phosphate buffer , ultrasonic crushing on ice-water mixture for 15 minutes, ultrasonic crushing conditions: power 400W, crushing for 1 second, pausing for 5 seconds, to obtain the crude enzyme solution of the mutant strain. Under the same conditions, replace the wet cells of the mutant strain with the starting strain E.coli BL21(DE3) / pET2Deut-dygdh to prepare the crude enzyme solution of the starting strain.

[0046] The crude enzyme solution of the mutant strain or the crude enzyme solution of the original strain was used as a catalyst, 2-carbonyl-4-(hydroxymethylphosphinyl)-butyric acid was used as a substrate, glucose was used as an auxiliary substrate, and no exogenous NADPH was added. Or NADP...

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Abstract

The invention discloses an amino acid dehydrogenase mutant and application of the mutant in synthesizing L-glufosinate. The amino acid dehydrogenase mutant is obtained by carrying out single mutationor multiple mutation on the 95th, 108th, 172nd and 303rd amino acid as shown in SEQ ID No.2. The specific enzyme activity of the amino acid dehydrogenase mutant DyGDH-F95I-A108T-R172P-R303H prepared by the method is improved by 33 times than that of female parent aldo-keto reductase, and the maximum feeding amount of a substrate 2-carbonyl-4-(hydroxyl methyl phosphinyl)-butyric acid reaches 500 mM. The amino acid dehydrogenase mutant has better industrial application prospect. The amino acid dehydrogenase mutant is used for producing L-glufosinate, and the reaction time is remarkably shortened. The general process requires 20 hours, while the reaction time of the present invention takes only 120 minutes, which shows that the amino acid dehydrogenase mutant has a good Industrial applicationprospect.

Description

technical field [0001] The present invention designs the construction of amino acid dehydrogenase mutants and the construction of DyGDH mutants, develops amino acid dehydrogenase recombinant bacteria and enzymes in 2-amino-4-(hydroxymethylphosphinyl)-L-ammonium butyrate (commonly known as L -glufosinate-ammonium) chiral biosynthesis. Background technique [0002] Glufosinate-ammonium is the second most resistant herbicide in genetically modified crops in the world. It was developed and produced by Hearst (now owned by Bayer). It is a phosphoric acid herbicide and a glutamine synthetase inhibitor. It is non-selective ( Natural) contact herbicides. [0003] The activity of glufosinate-ammonium is between that of glyphosate and paraquat, and has the advantages of high activity, low toxicity, easy degradation, and environmental friendliness; in addition, it can also be used to screen transgenic crops resistant to glufosinate-ammonium, so it is widely used , are generally favor...

Claims

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Application Information

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IPC IPC(8): C12N9/06C12N15/54C12N1/21C12P13/04C12R1/19
CPCC12N9/0014C12P13/04C12Y104/01015C12N9/0016
Inventor 薛亚平程峰李恒郑裕国徐建妙
Owner ZHEJIANG UNIV OF TECH
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