Supplementary strategy for improving catalytic synthesis of L-theanine from gamma-glutamyl transpeptidase

Abstract

The invention discloses to further optimization of a process for generating L-theanine from L-glutamine and ethylamine catalyzed by gamma-glutamyl transpeptidase secreted from recombinant pMA5-ggt / Bacillus. subtilis by using a method that double substrates are added in different batches, and belongs to the field of fermentation engineering and enzyme engineering. The process comprises the following steps: culturing the strain of bacillus subtilis which is established in previous operation and is used for secreting gamma-glutamyl transpeptidase in a fermentation tank; concentrating supernate crude enzyme liquid for conversion of theanine; on the basis, adding the substrates in different batches so as to further regulate and control the conversion process, that is, in a reaction system with 60U / mL of the enzyme liquid, adding substrates glutamine and ethylamine into the system every other two hours, wherein the addition amount of glutamine is fixed to be 20 mM, the ratio of a donor to a receptor is controlled to be 1:12, the pH value is adjusted to be 10, 164.2 mM of theanine can be obtained after 18 hours, and the conversion rate of glutamine can be up to 91.2%. The method has the advantages that the operation is simple, the cost is low, the yield of theanine is high, the substrate conversion rate is high, and the like, and is beneficial for industrial large-scale production.

Description

technical field [0001] The invention relates to improving the yield of L-theanine produced by transforming γ-glutamyl transpeptidase secreted and expressed by Bacillus subtilis by adopting a fed-batch strategy, and belongs to the fields of enzyme engineering and fermentation engineering. technical background [0002] L-theanine (N-ethyl-L-glutamic acid), a natural amino acid originally extracted from tea leaves, has various health effects on the body. L-theanine has been certified as a safe food additive by the US Food and Drug Administration (FAD). Due to the many benefits of theanine appeal, its demand is increasing day by day. [0003] γ-glutamyl transpeptidase is widely used in the enzymatic production of theanine. This enzyme catalyzes the hydrolysis of the γ-glutamine bond of γ-glutamyl compounds, and at the same time, the γ-glutamyl molecules produced by hydrolysis transferred to the receptor molecule. When L-glutamine is selected as the donor, ethylamine is used a...

AI Technical Summary

Problems solved by technology

[0004] The inventor previously disclosed a strain of recombinant Bacillus subtilis / pMA5-ggt that can efficiently secrete and express soluble γ-glutamyl transpeptidase (patent number: 201410747714.9), but the yield of batch catalytic synthesis of L-theanine is still relatively low , can not meet the needs of industrialization, so the present invention adopts the method of adding double substrates in batches on the basis of using γ-glutamyl transpeptidase to produce L-theanine in batches in the early stage, which can not only solve the problem of supplying The low solubility of L-glutamine and its inhibitory effect on enzymes can solve the problem of too low donor caused by too high concentration of ethylamine acceptor when other scholars only add donor L-glutamine. Receptor ratio issues and poor pH control adversely affect the reaction