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Mutant of L-amino acid oxidase

An amino acid and oxidase technology, applied in the field of genetic engineering, can solve the problem of low yield and achieve the effect of single target product, easy control and improved catalytic efficiency

Active Publication Date: 2019-02-12
JIANGNAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] However, the current method of utilizing L-amino acid transformation has improved the output and conversion rate of α-ketoisovaleric acid to a certain extent, but the output is still very low. Therefore, in order to improve the output and conversion rate of α-ketoisovaleric acid, enhanced Its industrial application value urgently needs to improve the catalytic efficiency of L-amino acid oxidase to L-valine

Method used

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  • Mutant of L-amino acid oxidase

Examples

Experimental program
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Effect test

Embodiment 1

[0028] Example 1 Preparation of L-amino acid oxidase mutant

[0029] Using the genome of Corynebacterium glutamicum as a template, primers were designed for PCR, and the gene encoding L-amino acid oxidase derived from Corynebacterium glutamicum was cloned. The primers used were: as the forward primer, with Obtain the target gene for the amplification of the reverse primer (the bold parts are the restriction sites of XhoⅠ and hindⅢ respectively); PCR program: pre-denaturation at 95°C for 3 minutes, denaturation at 95°C for 3 minutes, and then enter the following cycle: denaturation at 98°C for 10 seconds, 55°C Anneal for 30s, extend at 72°C for 1min and 40s; 34 cycles; extend at 72°C for 10min, hold at 4°C.

[0030] Digest the target gene and expression vector pET28a with restriction endonuclease XhoI and hindIII at 37°C for 2 hours, then use T4 ligase to digest and gel-recover the target gene and plasmid pET28a at 16°C for 10 hours

[0031] Using the pET28a plasmid (abbr...

Embodiment 2

[0041] Example 2 Induced expression of L-amino acid oxidase mutant Q183A

[0042] The mutant Q183A-pET28a was transformed into Escherichia coli BL21(DE3) cells, and the transformants were selected for sequencing verification. The verified positive transformants were cultured overnight at 37°C in LB medium, then transferred to TB medium with 2% inoculum, and cultured to OD 600 When =0.6-0.8, add IPTG with a final concentration of 0.04mM, and induce at 25°C for 12h.

[0043] Collect the fermentation broth, centrifuge at 8000rpm for 8min at 4°C to collect the cells.

Embodiment 3

[0044] Embodiment 3: the catalytic efficiency of recombinant bacteria to L-valine

[0045] The catalytic reaction system is 20mL, including recombinant E. coli whole cells with a final concentration of 30g / L, substrate 70g / L-valine, and 20mM Tris buffer (pH=8.0), and reacted at 200rpm and 25°C for 24h After sampling, the samples were analyzed by HPLC. Catalytic efficiency is calculated from the amount of product formed.

[0046] The recombinant bacteria expressing the L-amino acid oxidase mutant Q183A catalyzed L-valine to obtain the mass spectrum of the reaction product as shown in figure 1 As shown, according to the comparison of the mass spectrograms of the ketovaline standard substance and the reaction solution, it is proved that the reaction product is ketovaline. Catalytic results such as figure 2 As shown, the yield of recombinant bacteria expressing L-amino acid oxidase mutant Q183A can reach 54.6g / L after catalysis for 24 hours, and the conversion rate reaches 84....

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Abstract

The invention discloses a mutant of L-amino acid oxidase and a preparation method and application thereof, and belongs to the technical field of gene engineering. Through performing site-specific mutagenesis on the L-amino acid oxidase derived from corynebacterium glutamicum, a mutant Q183A is obtained. Heterologous expression is performed on escherichia coli through the mutant Q183A performs, catalytic efficiency of obtained recombinant bacteria to L-valine is improved by 18.7%, a yield of alpha-ketoisocaproic acid reaches 54.6 g / L, and a molar conversion rate of a substrate is 84.6%. Compared with a chemical method, the provided whole-cell transformation method has the advantages of moderate reaction condition, single target product, easy separation, environment-friendliness and the like, and is simple in process, easy to control, and easy for popularization and application.

Description

technical field [0001] The invention relates to a mutant of L-amino acid oxidase and its preparation method and application, belonging to the technical field of genetic engineering. Background technique [0002] L-amino acid oxidase is an oxidoreductase that can catalyze the formation of α-keto acids from L-amino acids. Researchers have done a lot of research on the spatial structure, substrate specificity, and catalytic ability of L-amino acid oxidase on unnatural substrates. In recent years, the application of this enzyme in the biotransformation of α-keto acid Gradually attract the attention of researchers. [0003] α-keto acid is an important intermediate, which is mainly used in food, medicine, cosmetics and other fields, which makes the enzymatic conversion of α-keto acid widely used in industrial production. L-amino acid oxidase is a flavoprotease, and the oxidative deamination reaction of L-amino acid can be divided into two steps: first, amino acid C α The hydrog...

Claims

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Application Information

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IPC IPC(8): C12N9/06C12N15/53C12N15/10C12N15/70C12N1/21C12P7/40C12R1/19
CPCC12N9/0022C12N15/1031C12N15/70C12P7/40C12Y104/03002C12Q2531/113
Inventor 吴静裴杉杉刘佳陈修来罗秋玲张权
Owner JIANGNAN UNIV
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