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A d-amino acid oxidase mutant and its application

An amino acid, oxidase technology, applied in the directions of oxidoreductase, application, enzyme, etc., can solve the problems of low single resolution rate, waste of raw materials, complicated process, etc., achieve high tolerance, good application value, and improve yield. rate effect

Active Publication Date: 2019-12-17
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This process mainly has the following disadvantages: it needs to use expensive chiral resolution reagents, the theoretical yield can only reach 50%, the single resolution rate is low, and the process is relatively complicated
The main advantage is 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 waste of raw materials

Method used

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  • A d-amino acid oxidase mutant and its application
  • A d-amino acid oxidase mutant and its application
  • A d-amino acid oxidase mutant and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Construction and screening of D-amino acid oxidase mutant library

[0057] The D-amino acid oxidase gene (the amino acid sequence shown in SEQ ID No.1, the nucleotide sequence shown in SEQ ID No.2) was constructed as an expression vector pET-24a(+), transformed into Escherichia coli, and the starting strain E.coli was obtained BL21(DE3) / pET-24a.

[0058] The D-amino acid oxidase mutant library was prepared through five rounds of mutation, and the primers were designed as shown in Table 1.

[0059] The specific method is as follows:

[0060] In the first round, using the genome of the wild fungus Rhodotorula gracilis as a template, using epPCR-F and epPCR-R as upstream and downstream primers, through error-prone PCR, transformation, smearing, screening of dominant strains, and sequencing to find Beneficial mutation sites 213, 54, 58, 52, 335 were identified.

[0061] In the second round, using the genome of the wild fungus Rhodotorula gracilis as a template,...

Embodiment 2

[0082] Embodiment 2 Construction of high-throughput screening method

[0083] Using D,L-glufosinate-ammonium as substrate, enzymes with catalytic activity to D-glufosinate-ammonium were screened by 2,4-dinitrophenylhydrazine colorimetric method.

[0084] Quantitatively weigh D, L-glufosinate-ammonium into a 50mM phosphate buffer solution with pH=8, and place it in the reaction vessel so that the final concentration of D,L-glufosinate-ammonium is 50mM, and the concentration of the crude enzyme solution is 50g / L. The concentration of catalase was 2g / L. The reaction temperature was controlled at 30° C. by a water bath, and samples were taken regularly.

[0085] Detect the content of 2-carbonyl-4-[hydroxy(methyl)phosphono]butyric acid by 2,4-dinitrophenylhydrazine chromogenic method: take 60 μL of the reaction solution, add 2 mM 2,4-dinitro 40 μl of phenylhydrazine, incubated in a constant temperature bath at 37°C for 20 minutes, added 100 μl of 1M sodium hydroxide, and mixed fo...

Embodiment 3

[0093] The cultivation of embodiment 3 thalline and the purification of mutant

[0094] 1. Bacteria culture

[0095] After the engineering bacteria containing the D-amino acid oxidase gene were activated by streaking on a plate, a single colony was picked and inoculated into 5 mL LB liquid medium containing 50 μg / mL kanamycin, and cultured with shaking at 37°C for 12 hours. Transfer to 50mL LB liquid medium also containing 50μg / mL kanamycin according to 2% inoculum amount, and culture with shaking at 37°C until OD 600 When it reaches about 0.8, add IPTG with a final concentration of 0.5mM, and culture with shaking at 28°C for 16h. After the cultivation, the culture solution was centrifuged at 8000rpm for 10min, the supernatant was discarded, the bacteria were collected, and stored in a -80°C ultra-low temperature refrigerator until use.

[0096] 2. Preparation of crude enzyme solution

[0097] After the culture, the collected cells were washed twice with pH 8 phosphate buff...

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Abstract

The invention discloses a D-amino acid oxidase mutant and application thereof. The mutant is obtained by carrying out single mutagenesis or multiple mutagenesis on a 52nd site, a 54th site, a 58th site, a 213rd site and a 335th site of amino acid with an amino acid sequence shown in SEQ ID NO.1, wherein glycine at the 52nd site is mutated into leucine, asparagine at the 54th site is mutated into valine, phenylalanine at the 58th site is mutated into glutamine, methionine at the 213rd site is mutated into serine, and serine at the 335th site is mutated into glycine. According to the D-amino acid oxidase mutant and the application thereof, a D-amino acid oxidase gene with an amino acid sequence as shown in SEQ ID NO.2 is mutated by utilizing a site-saturation mutagenesis technology, so thatthe enzyme activity and the product conversion rate are far higher than those of a wild type, and therefore the product yield in a 4-(hydroxymethylphosphoryl)-2-carbonyl-butanoic acid production process is increased.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a D-amino acid oxidase mutant and its application. Background technique [0002] Glufosinate-ammonium, also known as glufosinate, the English name is Phosphinothricin (abbreviated as PPT), the chemical name is 2-amino-4-[hydroxy (methyl) phosphono] butyric acid, it is the second largest transgenic crop resistant to weeding in the world Agent, developed and produced by Hearst (now owned by Bayer after several mergers). Glufosinate-ammonium is a phosphonic acid herbicide, a glutamine synthetase inhibitor, and a non-selective (killing) contact herbicide. [0003] As we all know, the total herbicide market is huge. At present, the world's three major herbicides are paraquat, glyphosate, and glufosinate-ammonium. In terms of market use, glyphosate is the champion, but due to its long-term use, a large number of weeds have developed resistance, and glyphosate tends to be ineffective; pa...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/06C12N15/53C12N15/70C12N1/21C12P7/42
CPCC12N9/0024C12N15/70C12P7/42C12Y104/03003
Inventor 薛亚平程峰王柳玉徐建妙郑裕国
Owner ZHEJIANG UNIV OF TECH
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