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Glutamyltranspeptidase for synthesizing gamma-polyglutamic acid and coding gene thereof

A technology of glutamyl transpeptidase and polyglutamic acid, which is applied in the fields of genetic engineering and enzyme engineering, can solve the problems such as the lack of glutamyl transpeptidase, and achieve the effect of good application value

Inactive Publication Date: 2013-08-07
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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IPC IPC(8): C12N9/10C12N15/54C12N15/10C12N15/70C12P13/02
Inventor 叶茂
Owner GUANGDONG IND TECHN COLLEGE
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