Cloning of novel glutamate decarboxylase gene and application thereof

Abstract

The invention relates to the cloning of a novel glutamate decarboxylase gene and the application thereof. A glutamate decarboxylase is obtained from bacillus, and then the glutamate decarboxylase is induced to express in escherichia coli. The study on the properties of the glutamate decarboxylase shows that, the glutamate decarboxylase is higher in activity at pH 4.0 to pH 6.0, and the optimum temperature of the glutamate decarboxylase is 45-65 DEG C. The Vmax of the glutamate decarboxylase is 150-200 U / mg, and the Km of the glutamate decarboxylase is 7-9 mmol / L. The recombinant escherichia coli containing the glutamate decarboxylase is subjected to whole-cell transformation, and then 500 g / L L-glutamic acid is added in batches. At 37 DEG C, the recombinant escherichia coli is converted for 12 hours in a sodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution at the pH 7.0, and 347.8 g / L gamma-aminobutyric acid is finally generated. The molar conversion rate is 99.4%. Compared with an existing glutamate decarboxylase, the above novel glutamate decarboxylase is higher in catalytic efficiency and relatively wide in pH range, thus being more suitable for industrial application.

Description

technical field [0001] The invention relates to a novel glutamic acid decarboxylase and its application in the production of gamma-aminobutyric acid, belonging to the field of biocatalysis. Background technique [0002] Glutamate decarboxylase is a 5'-pyridoxal phosphate (PLP)-dependent enzyme that catalyzes the decarboxylation of L-glutamate to γ-aminobutyric acid (GABA) and carbon dioxide. The product γ-aminobutyric acid is an important inhibitory neurotransmitter, which has various physiological functions such as anti-anxiety, regulating blood pressure, and treating epilepsy, and has important application value in food, medicine and other industries. At present, the main methods of producing GABA include chemical synthesis, plant enrichment and microbial fermentation. The microbial fermentation method refers to the use of GAD contained in the organism to biocatalyze the decarboxylation of L-glutamic acid or L-glutamate to generate GABA. Due to its mild reaction conditio...

AI Technical Summary

Problems solved by technology

Express the glutamic acid decarboxylase gene of Escherichia coli or Lactobacillus brevis in Escherichia coli, and use the whole cell transformation method to produce GABA, and the yield can reach 280-300 g / L respectively [6] and 278.3 g / L [7] , although the catalytic efficiency is high, it is still necessary to reduce the pH of the conversion environment to 4.5 or below, which will cause a large loss of equipment

[0004] The glutamic acid decarboxylase found so far generally has low activity and the optimum pH of the enzyme is less than 5.0. When the pH is greater than 6.0, the enzyme activity will drop sharply. This characteristic greatly limits the application of glutamic acid decarboxylase in industry.

Images