A kind of method utilizing L-glutamic acid oxidase to catalyze the production of α-ketoglutarate

Abstract

The invention discloses a method for producing alpha-oxoglutarate under catalysis of L-glutamate oxidase. The method comprises the following steps: performing optimal design and synthesis on codon of an L-glutamate oxidase (L-GOX) gene; constructing a fusion expression vector pET-28a-LGOX-ELP of the L-GOX and Elastin-Like Proteins (ELP), transferring the vector into escherichia coli Rosetta (DE3), selecting a converter for cultivation, adding an inducer for expression, and obtaining recombined L-glutamate oxidase fusion protein crude enzyme fluid; performing purification by an ITC method, thus obtaining recombined L-glutamate oxidase fusion proteins, performing enzyme digestion on the recombined L-glutamate oxidase fusion proteins by protease, and obtaining mature L-glutamate oxidase; producing oxoglutarate under the catalysis of the mature L-glutamate oxidase. The method is simple, quick and efficient, can be used for large-scale separation and purification of L-glutamate oxidase, and is higher in actual application value in the enzyme production of oxoglutarate.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for catalyzing and producing alpha-ketoglutarate by utilizing L-glutamic acid oxidase. Background technique [0002] α-ketoglutarate (α-KG) is an important biological compound. As one of the important intermediate metabolites of the tricarboxylic acid cycle, α-ketoglutarate participates in important physiological processes such as amino acid, sugar, protein and fat metabolism, and plays an important role in the regulation of microbial carbon and nitrogen metabolism. Due to the importance of α-ketoglutaric acid in cell metabolism, it is widely used in food, medicine, fine chemical and cosmetic industries, and the market demand is huge. In particular, L-arginine-α-ketoglutarate (1:1) and L-arginine-α-ketoglutarate (2:1) is an amino acid with increasing sales in the international market Salt health products, as functional nutrition enhancers and liver-protecting d...

AI Technical Summary

Problems solved by technology

Although affinity chromatography has the characteristics of high selectivity, high activity recovery and high purity, and has become one of the most effective techniques for purifying biological macromolecules such as proteins, its shortcomings are also obvious, mainly including the consumption of These defects not only make the production process of this purified protein more costly, but also limit its large-scale industrial application.

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