Cloning and Application of a Glutamic Acid Decarboxylase Gene

Abstract

The present invention relates to the cloning and application of a novel glutamic acid decarboxylase gene. The glutamic acid decarboxylase obtained from Bacillus is induced and expressed in Escherichia coli and its properties are studied. It is found that the enzyme has a pH of 4.0 It has higher activity at ~pH6.0, the optimum temperature is between 45℃~60℃, Vmax is 150~200 U / mg, and Km is 7~9 mmol / L. The recombinant Escherichia coli containing the enzyme was transformed into whole cells, and 500 g / L L-glutamic acid was added by fed-batch feeding, at 37°C, in disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, pH 7.0 After 12 h of conversion, 347.8 g / L of γ-aminobutyric acid was finally produced, and the molar conversion rate was 99.4%. Compared with the existing glutamic acid decarboxylase, the novel glutamic acid decarboxylase has higher catalytic efficiency and wider pH range, and is more suitable for industrial application.

Description

technical field [0001] The invention relates to a 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 can catalyze 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 conditions,...

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 280-300g / L respectively [6] and 278.3g / 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