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Glutamyltranspeptidase for synthesizing gamma-polyglutamic acid and coding gene thereof

A glutamyl transpeptidase-based and glutamyl transpeptidase technology, which is applied in the field of glutamyl transpeptidase for synthesizing γ-polyglutamic acid and its encoding gene, and can solve the problem of glutamyl transpeptidase Enzymes and other problems, to achieve the effect of good application value

Inactive Publication Date: 2015-01-28
GUANGDONG IND TECHN COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far, there is no research report on glutamyl transpeptidase that can synthesize γ-polyglutamic acid

Method used

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  • Glutamyltranspeptidase for synthesizing gamma-polyglutamic acid and coding gene thereof
  • Glutamyltranspeptidase for synthesizing gamma-polyglutamic acid and coding gene thereof
  • Glutamyltranspeptidase for synthesizing gamma-polyglutamic acid and coding gene thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Obtaining of glutamyl transpeptidase gene and verification of the function of synthesizing γ-polyglutamic acid

[0036] 1. Acquisition of glutamyl transpeptidase gene

[0037] A mangrove metagenomic library was constructed from coastal mangrove silt samples in Zhanjiang City, Guangdong Province. A medium containing 100 μg / ml ampicillin, 0.1% γ-glutamyl-p-nitroaniline, 1% glucose and 0.5% amino acid complex was used as the selection medium to screen glutamyl transpeptidase-positive clones from the library. Copy the cells in the library onto a solid selective medium plate with a replica inoculation needle, culture at 37°C for 3 days, and screen for positive clones with yellow hydrolysis circles around the colonies.

[0038] In order to screen out clones that synthesize γ-polyglutamic acid, pick the clones that produce yellow hydrolysis circles on the selection medium, and inoculate them into 10ml containing 100μg / ml ampicillin and 0.5mM isopropylthio-β-D -Gala...

Embodiment 2

[0049] Example 2 Study on conditions for the synthesis of γ-polyglutamic acid by recombinant glutamyl transpeptidase GGT_015

[0050] 1. Effect of pH value on the ability to synthesize γ-polyglutamic acid

[0051] Take 10 μl of purified enzyme solution, add 1ml of 20% glutamine, and react in water bath at 30°C for 2h, 4h, 6h, and 8h respectively in buffer solutions with pH 6.0, 6.5, 7.0, 7.5, and 8.0, and add 1ml 15% acetic acid solution was used to terminate the reaction, and the light absorption value was measured at 214nm wavelength. The greater the absorption value, the greater the amount of synthesized γ-polyglutamic acid. At the same time, the inactivated enzyme solution was used as a control. The result is as image 3 The results showed that the optimal pH value of GGT_015 for synthesizing γ-polyglutamic acid was 7.5; below or above this pH value, the synthesis ability of GGT_015 would be relatively reduced.

[0052] 2. Effect of temperature on the ability to synthes...

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Abstract

The invention provides glutamyltranspeptidase for synthesizing gamma-polyglutamic acid and a coding gene thereof. The protein molecule of the glutamyltranspeptidase has the amino acid sequence as shown in SEQ ID NO: 2; and the gene of the glutamyltranspeptidase has the nucleotide sequence as shown in SEQ ID NO: 1. The invention also discloses a method for synthesizing the gamma-polyglutamic acid by utilizing recombinant glutamyltranspeptidase. By adopting the method, the gamma-polyglutamic acid is synthesized by utilizing the recombinant glutamyltranspeptidase and taking glutamine monomers as raw materials. The glutamyltranspeptidase can be used for synthesizing the gamma-polyglutamic acid by taking the glutamine monomers as the raw materials, thus having importance significance in enriching glutamyltranspeptidase family members and developing a new gamma-polyglutamic acid synthesis route.

Description

technical field [0001] The invention belongs to the field of genetic engineering and enzyme engineering, in particular to a glutamyl transpeptidase for synthesizing γ-polyglutamic acid and its coding gene. Background technique [0002] γ-polyglutamic acid (Poly-γ-glutamic acid, γ-PGA) is an anionic polymer composed of D- or L-glutamic acid through α-amino and γ-carboxyl to form a γ-amide bond . γ-PGA is a new water-soluble and biodegradable green biomaterial, which has the characteristics of edible, non-toxic, film-forming, cohesive, and moisturizing properties. Thickeners and humectants are used in medicine, food and cosmetics, and can also be used as water-retaining agents, antifreeze agents and fresh-keeping agents for fruits and vegetables, as well as flocculants and heavy metal chelating agents for water treatment. With the increasing awareness of environmental protection, γ-polyglutamic acid has attracted much attention as a biodegradable polymer material. [0003] ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/10C12N15/70C12P13/02
Inventor 叶茂
Owner GUANGDONG IND TECHN COLLEGE
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