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Recombinant vector for expressing L-glutamic oxidase and catalase, engineering bacteria, applications thereof and method for producing Alpha-ketoglutarate

A technology of glutamate oxidase and catalase, which is applied in the fields of molecular biotechnology and enzyme engineering, can solve the problems of unrealized industrial production, long fermentation period, serious pollution, etc., and achieves the effect of good practical use value.

Active Publication Date: 2019-04-26
SICHUAN JISHENG BIOPHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003]The traditional methods of organic synthesis include acyl nitrile hydrolysis and ethyl oxalate hydrolysis, which take several steps to reach, which are cumbersome and seriously polluting; Great breakthroughs have been made. The accumulation of α-ketoglutaric acid in Torulopsis glabrata with glucose can reach 43.7g / L in 64 hours; Yarrowia lipolytica can reach 186g / L in 117 hours when cultured with glycerol. Although the yield is already very high, other miscellaneous There are many acids, it is difficult to separate and purify, and the fermentation cycle is too long, so industrial production has not yet been realized

Method used

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  • Recombinant vector for expressing L-glutamic oxidase and catalase, engineering bacteria, applications thereof and method for producing Alpha-ketoglutarate
  • Recombinant vector for expressing L-glutamic oxidase and catalase, engineering bacteria, applications thereof and method for producing Alpha-ketoglutarate
  • Recombinant vector for expressing L-glutamic oxidase and catalase, engineering bacteria, applications thereof and method for producing Alpha-ketoglutarate

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Experimental program
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Embodiment 1

[0034] This embodiment provides a recombinant vector and engineering bacteria expressing L-glutamic acid oxidase and catalase, the recombinant vector includes L-glutamic acid oxidase gene LGOX expressing L-glutamic acid oxidase, The catalase gene CAT of catalase and the regulation sequence SD of regulating L-glutamate oxidase gene LGOX and catalase gene CAT expression; The base sequence of L-glutamate oxidase gene LGOX is as SEQ As shown in ID No.1, the base sequence of the catalase gene CAT is shown in SEQ ID No.2, and the base sequence of the regulatory sequence SD is shown in SEQ ID No.3.

[0035] The obtaining process of the recombinant vector and engineering bacteria expressing L-glutamic acid oxidase and catalase is as follows:

[0036] 1.1 According to the L-glutamic acid oxidase gene of Streptomyces afghaniensis 772, the full-length sequence was optimized and the signal peptide of the sequence was removed to obtain the L-glutamic acid oxidase gene, and the amplificatio...

Embodiment 2

[0043] This embodiment provides a production method of α-ketoglutaric acid, the steps of the production method are as follows:

[0044] 1.1 Refer to the method of Example 1 to construct pET32a-LGOX-SD-CAT recombinant vector and engineering bacteria pET32a-LGOX-SD-CAT / BL21(DE3);

[0045] 1.2 Inoculate the engineering bacteria pET32a-LGOX-SD-CAT / BL21(DE3) into the primary culture medium LB, and carry out the primary culture stage of the primary culture. The condition of the primary culture is 37°C, 220rpm for 12h ; to OD600 is 3.8;

[0046] 1.3 Transfer the bacterial liquid from the primary culture to the secondary culture medium TB to continue the secondary culture stage of the primary culture. The condition of the secondary culture is 37°C and 200rpm for 4 hours;

[0047] 1.4 Inoculate the bacteria of the secondary culture into the fermentation medium added (fermentation medium contains yeast extract 28g / L, peptone 15g / L, glycerol 10g / L, dipotassium hydrogen phosphate trihydr...

Embodiment 3

[0052] This embodiment provides a production method of α-ketoglutaric acid, the steps of the production method are as follows:

[0053] 1.1 Refer to the method of Example 1 to construct pET32a-LGOX-SD-CAT recombinant vector and engineering bacteria pET32a-LGOX-SD-CAT / BL21(DE3);

[0054] 1.2 Inoculate the engineered bacteria pET32a-LGOX-SD-CAT / BL21(DE3) into the primary culture medium LB, and carry out the primary culture stage of the primary culture. The condition of the primary culture is 37°C, 250rpm for 8h ; to OD600 is 4.1;

[0055] 1.3 Transfer the bacterial liquid from the primary culture to the secondary culture medium TB to continue the secondary culture stage of the primary culture. The conditions for the secondary culture are 37°C and 235rpm for 5 hours;

[0056] 1.4 Inoculate the bacteria of the secondary culture into the fermentation medium added (fermentation medium contains yeast extract 28g / L, peptone 15g / L, glycerol 10g / L, dipotassium hydrogen phosphate trihyd...

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Abstract

The invention provides a recombinant vector for expressing L-glutamic oxidase and catalase, engineering bacteria, applications thereof and a method for producing Alpha-ketoglutarate and belongs to thefield of molecular biological technique and enzyme engineering. The recombinant vector for expressing L-glutamic oxidase and catalase provided by the invention is capable of promoting capacity of converted substrates of L-glutamic oxidase and catalase to generate Alpha-ketoglutarate by inserting a regulating sequence SD for regulating expressions of L-glutamic oxidase gene LGOX and catalase geneCAT into a vector and has a higher practical use value.

Description

technical field [0001] The invention relates to the fields of molecular biotechnology and enzyme engineering, in particular to a recombinant vector expressing L-glutamic acid oxidase and catalase, an engineering bacterium and its application, and a production method of α-ketoglutarate. Background technique [0002] α-Ketoglutaric acid is a key node in the tricarboxylic acid cycle and amino acid metabolism in organisms. It can generate glutamic acid through glutamic acid dehydrogenase or transaminase. It is widely used in food, medical, cosmetics, fine chemicals and other fields , and even in the field of sports, it is used as an auxiliary tonic to reduce the damage caused by ammonia accumulation. The market demand is greater than the supply, and the price is relatively high. Currently, there are three main methods for synthesizing α-ketoglutarate: organic chemical synthesis, fermentation accumulation and enzymatic conversion. [0003] The traditional methods of organic synt...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12P7/50
CPCC12N9/0022C12N9/0065C12N15/70C12P7/50C12Y104/03011C12Y111/01006
Inventor 王苓李晚军伍万兵曾帅刘鑫
Owner SICHUAN JISHENG BIOPHARM CO LTD
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