Method by using co-expression recombinant bacteria strain to convert and produce alpha-ketoglutarate

Abstract

The invention discloses a method by using a co-expression recombinant bacteria strain to convert and product alpha-ketoglutarate, and belongs to the technical field of biology. The method is characterized in that novel L-glutamic oxidase is screened from streptomycete and is induced to express and purify in escherichia coli, and the enzymatic property is studied; when the pH (potential of hydrogen) value is 5.5 to 7.0, the enzyme has higher activity, the most suitable reaction temperature is 30 to 45 DEG C, Vmax is 100 to 150U / mg, and Km is 8 to 10mM; the original gene sequence of the L-glutamic oxidase is subject to codon optimization, and the plasmid co-expression with a catalase gene from the escherichia coli is performed, so as to construct the co-expression recombinant bacteria strain; the recombinant bacteria strain is used as a whole-cell catalyst to convert the L-glutamic acid (salt), the output of alpha-ketoglutarate reaches 76.08g / L after reacting for 9h, and the molar conversion rate is 96.8%. The method has the advantages that the problems of complicated production steps, low yield, pollution to environment and the like in the alpha-ketoglutarate production process aresolved, the high-yield and one-step type production of the alpha-ketoglutarate is realized, and the industrial application value is higher.

Description

technical field [0001] The invention relates to a method for transforming and producing α-ketoglutarate by co-expressing recombinant bacterial strains, and belongs to the field of biotechnology. Background technique [0002] L-glutamate oxidase (LGOX) is a flavoproteinase with flavin adenine dinucleotide (FAD) as the prosthetic group, which can Specific oxidation of glutamate to hydrogen peroxide, ammonia and α-ketoglutarate [1] . α-ketoglutarate (α-KG), as an important dibasic acid in the tricarboxylic acid cycle and amino acid metabolism, plays an important role in the formation of amino acids and nitrogen transport, and is widely used in medicine, fine chemicals, food and animals Feed and other fields [2] . [0003] At present, the production methods of α-KG include chemical synthesis, fermentation, and biocatalysis. The traditional α-KG production adopts chemical synthesis, but the harmful reagents such as strong acid and alkali, cyanide, etc. used in the chemical s...

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Problems solved by technology

[0012] In order to solve the above-mentioned technical problems, the object of the present invention is to provide a method for transforming and producing α-ketoglutaric acid by co-expressing recombinant strains, which can efficiently convert L-glutamic acid (salt) into α-ketoglutarate Diacid, thereby solving the problems of high production cost, long cycle and low efficiency in the industrial production of α-ketoglutaric acid, which is conducive to the large-scale production of α-ketoglutaric acid.

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