Preparation method of gamma-glutamyltranspeptidase
A technology of glutamyl transpeptidase and glutamine, which is applied in the field of preparation of γ-glutamyl transpeptidase, can solve the problems that the large and small subunits cannot be folded correctly, cannot interact effectively, and cannot form mature enzymes, etc. , to achieve the effect of being suitable for large-scale industrial production, easy to scale up, and reducing the cost of enzyme consumption
Inactive Publication Date: 2016-01-06
NANJING NORMAL UNIVERSITY
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Problems solved by technology
The reason is mainly due to the complex post-translational processing of glutamyl transpeptidase, which causes the large and small subunits after expression and transport to not fold correctly and interact effectively, so they cannot form natural mature enzymes
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[0021] The present invention can be better understood from the following examples. However, those skilled in the art can easily understand that the content described in the embodiments is only for illustrating the present invention, and should not and will not limit the present invention described in the claims.
[0022] 1. Using a shaker to induce expression of glutamyl transpeptidase:
[0023] Transfer the Escherichia coli clone cryopreservation solution containing the expression plasmid into a test tube containing 4ml of LB liquid medium corresponding to the resistance of the expression plasmid at a ratio of 1:100; 220rpm, shake culture at 37°C for 12h and then transfer Put into 80ml M9 liquid medium, when OD600 reaches 0.7, lower the temperature of the shaker to 20°C and add IPTG or lactose for shaking culture, when OD600 reaches 2.5, add the compound of L-glutamine and NAD, centrifuge after 3 hours The bacteria were harvested, and the enzyme activity was measured after t...
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The invention provides a preparation method of gamma-glutamyltranspeptidase. The method includes the steps of converting glutamyltranspeptidase expression plasmids into escherichia coli so as to establish genetically engineered bacteria, conducting fermentation on the genetically engineered bacteria, and conducting induction expression through IPTG or lactose, wherein compounds containing L-glutamine and NAD are added 3 hours before induction expression ends to promote expression. The genetically engineered bacteria, containing glutamyltranspeptidase, of escherichia coli are bred in a high density mode, the genetically engineered bacteria are combined with protein to efficiently express an activating agent, and the optimal enzyme activity reaches 350000 U / L after expression. By means of the method, the expression amount and the enzyme activity of enzymes can be obviously increased, the efficiency for converting substrate glutamine into L-theanine is promoted, and therefore the production of theanine is improved. Meanwhile, the process cycle is shortened, the production cost is reduced, and the method is easy to operate and amplify, high in stability and suitable for large-scale industrialized production.
Description
technical field [0001] The present invention relates to the technical field of genetic engineering. Specifically, the combination of high-density fermentation combined with L-glutamine and NAD greatly improves the expression and soluble expression of glutamyl transpeptidase transferase, making GGTase activity Provide 3-5 times, obtain a gamma-glutamyl transpeptidase (gamma-GGTase) high-efficiency preparation technology, significantly reduce the production cost of enzymatic synthesis of L-theanine. 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 ...
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IPC IPC(8): C12N9/10C12R1/19
CPCC12N9/1044C12Y203/02013
Inventor 殷志敏朱岩岩黄正才
Owner NANJING NORMAL UNIVERSITY