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Constructing method and application of gamma-glutamyl transpeptidase and chaperonin coexpression recombinant plasmid

A technology of glutamyl transpeptidase and glutamyl transpeptidase activity, which is applied in the field of genetic engineering, can solve the problems that the large and small subunits cannot be folded correctly, cannot interact effectively, and cannot form mature enzymes, etc., and achieve easy Effects of amplification, easy operation, and promotion of transformation

Inactive Publication Date: 2015-04-29
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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  • Constructing method and application of gamma-glutamyl transpeptidase and chaperonin coexpression recombinant plasmid
  • Constructing method and application of gamma-glutamyl transpeptidase and chaperonin coexpression recombinant plasmid
  • Constructing method and application of gamma-glutamyl transpeptidase and chaperonin coexpression recombinant plasmid

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Embodiment Construction

[0026] 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.

[0027] 1. Construction of co-expression system of glutamyl transpeptidase and five Escherichia coli chaperone systems:

[0028] Transfer the Escherichia coli clone cryopreservation solution containing four chaperone protein expression plasmids (see Table 1) into the anti- In the test tube of the corresponding LB liquid medium; 200rpm, 37 ℃ shaking culture for 12h after the small extraction of the plasmid; the five chaperone protein expression plasmids after the small extraction were respectively combined with the glutamyl transpeptidase expression plasmid pGEX-4T-1 / γ-ggt was mixed at a ratio of 1:1. After mixing, the total amount of nucleic acid contained in the fi...

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Abstract

The invention provides a constructing method and application of a recombinant glutamyl transpeptidase coexpression plasmid as well as a method for efficiently accumulating theanine. Through coexpression of chaperonin and GST-labelled glutamyl transpeptidase, a genetically-engineered escherichia coli bacterium BL21 / C4 with very high enzyme activity of the glutamyl transpeptidase is successfully constructed, and after the coexpression, the optimal enzyme activity reaches 15,500 U / L. By the constructing method, the enzyme expression and the enzyme activity can be significantly improved, transformation of a substrate is promoted, and the production capacity of the theanine is further increased; the process cycle is shortened (the enzyme activity is high); the production cost is reduced (the enzyme consumption cost is lowered in enzymatic reaction); the method is easy to operate, easy to popularize and high in stability and is suitable for large-scale industrial production; a new thought is provided for producing other valuable products by a biological enzyme method.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a production method for constructing an Escherichia coli genetically engineered bacterium with extremely high glutamyl transpeptidase activity and synthesizing theanine through enzymatic conversion. 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, ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P13/04C12R1/19
CPCC12N9/1044C12P13/04C12Y203/02013
Inventor 殷志敏王期蒋毅兰蕾
Owner NANJING NORMAL UNIVERSITY
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