Method for efficiently synthesizing L-theanine by using gamma-glutamyl transpeptidase produced by Escherichia coli

Abstract

The invention relates to a method for efficiently synthesizing L-theanine by using gamma-glutamyl transpeptidase produced by Escherichia coli. The method comprises the following steps of: a) filtering a raw fermentation liquor of Escherichia coli by using an ultrafiltration membrane with the aperture of 0.001-0.01um; b) adding an ethylamine solution into an enzyme and bacterium trapped fluid to regulate the pH to be 10-11, and standing for 2 to 3 hours to obtain an enzyme concentrated solution; and c) adding the enzyme concentrated solution obtained in the step b) into an aqueous solution containing L-glutamine to obtain an enzyme reaction system, adding an ethylamine solution into the enzyme reaction system to control the pH of the enzyme reaction system to be 9-10.5, and controlling the enzymatic conversion temperature to be 30-35DEG C. The method is easy to implement and low in time and energy consumption, avoids the introduction of impurities and the deactivation of gamma-glutamyl transpeptidase, avoids the corrosion of a reactor due to the introduction of hydrochloric acid, improves the yield and purity of the L-theanine and reduces cost.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for efficiently synthesizing L-theanine by using Escherichia coli to produce γ-glutamyl transpeptidase. Background technique [0002] L-theanine (N-ethyl-γ-glutamine, theanine) is a natural amino acid, a characteristic amino acid in tea, and an effective taste substance in tea. L-theanine is mainly used for: (1) As an emerging food additive, it can be used as a quality improver for tea drinks, an additive for improving food flavor, an additive for functional food, and an additive for "emotional food"; It is widely used in Europe, America, Japan, China Taiwan and other countries and regions. For example, Japan has developed chocolate, jelly, pudding, chewing gum, health tea and various refreshing drinks with theanine added. (2) In the field of pharmaceutical intermediates, theanine can be used in anti-tumor, blood pressure-lowering, sedative, anti-fatigue and other drugs....

AI Technical Summary

Problems solved by technology

[0005] The gamma-glutamyl transpeptidase in the above fermentation broth is produced in Escherichia coli cells, and 60% of the gamma-glutamyl transpeptidase is secreted outside the cells , 40% of the γ-glutamyl transpeptidase is still in the cells. In order to make full use of the enzymes in the cells, some people centrifuge to collect the bacteria at the end of fermentation, and then break the wall to collect the enzyme solution. Currently, the main methods reported in the world are boiling, Freeze-thawing, enzymatic hydrolysis, ultrasonic and other wall-breaking methods break the microbial cells and release enzyme liquid; these wall-breaking methods are complicated to operate, time-consuming, energy-consuming, and will also introduce impurities, and worse, will reduce the activity of the enzyme , to reduce the conversion rate of L-theanine; another method is to add excess ethylamine during the reaction to facilitate the enzyme reaction, and then add hydrochloric acid to adjust the pH, but this way of adjusting the pH will consume a lot of hydrochloric acid , a large amount of hydrochloric acid will cause great corrosion to the metal reactor, and at the same time introduce impurity chloride ions, which will bring great difficulties to the later separation