Glutamine transaminase with improved heat stability and application thereof

A technology of glutamine and transaminase, applied in the field of transglutaminase, can solve problems such as poor thermal stability, and achieve the effects of improving production efficiency and reducing production costs

Active Publication Date: 2013-03-27
TAIXING YIMING BIOLOGICAL PRODS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In order to improve the poor thermal stability of MTG, this study added a tag to the C-terminal amino acid of MTG, thereby en

Method used

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  • Glutamine transaminase with improved heat stability and application thereof
  • Glutamine transaminase with improved heat stability and application thereof
  • Glutamine transaminase with improved heat stability and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0019] Example 1: Simulation of MTG crystal structure derived from Streptomyces hygroscopicus

[0020] Using the reported TGase crystal structure of S. mobaaensis as a template, the crystal structure of S. hygoscopicus TGase was simulated on the swiss-model website (http: / / swissmodel.expasy.org / ).

Embodiment 2

[0021] Example 2: Transglutaminase with Improved Thermostability

[0022] Based on the transglutaminase encoding gene published by Genbank: EU477523, an amino acid tag is added to its C-terminus, wherein the amino acid sequence of the tag is shown in SEQ ID NO.1 or SEQ ID NO.2, among which SEQ ID NO.1 is preferred .

Embodiment 3

[0023] Example 3: Obtaining mutant strains with improved thermostability (linker9, linker13)

[0024] 1. Use chemical total synthesis or PCR to clone the gene encoding transglutaminase Genbank: EU477523, add different types of tags that help improve thermal stability at the C-terminus of MTG, and clone the modified transglutaminase gene into carrier.

[0025] 2. Transform the plasmid with correct sequencing into E.coli BL 21, select the transformant and inoculate it into LB liquid medium, culture at 37°C for 12 hours, and transfer it to TB medium with an inoculation volume of 3%. Bacteria grow to OD 600 When it was 2, IPTG was added to induce, and the culture temperature was lowered to 20°C, and cultured for 48h.

[0026] 3. Collect the fermentation supernatant, detect the enzyme activity of the fermentation supernatant, and purify the sample with His-nickel column.

[0027] 4. The specific enzyme activity and Km value of the purified MTG were measured, and the results are ...

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Abstract

The invention discloses a glutamine transaminase with improved heat stability and application thereof. High-efficiency expression of microbial transglutaminase (MTG) in escherichia coli serves as a modification platform, a label facilitating improvement of heat stability is added at the C end of MTG maturase, preference is given to IGCIILT to obtain a mutant strain with good enzymatic property, and the heat stability is improved by 2.5 times and 3 times. Modified enzyme is suitable for industrial application, the production cost can be reduced, and the production efficiency can be improved.

Description

technical field [0001] The invention relates to a transglutaminase, in particular to a transglutaminase with improved thermostability. Background technique [0002] Microbial transglutaminase (Transglutaminase EC 2.3.2.13 full name R-glutaminyl-peptide:amine-γ-glutayle-transferase referred to as MTG) can catalyze the formation of ε-(γ-glutamyl) lysine between or within molecules of proteins Amino acid covalent bonds, causing intramolecular and intermolecular crosslinking of proteins, connections between proteins and amino acids, and hydrolysis of glutamine groups in protein molecules. The unique catalytic function of TGase makes it have broad application prospects in food, textile, biopharmaceutical and other fields. However, due to some defects of MTG itself, such as thermal stability and other factors, the application range of MTG is limited. Therefore, based on the obtained expression platform of MTG in Escherichia coli, molecular modification of MTG was carried out by ...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/10C12N1/21C12N5/10C12N15/70C12R1/19
Inventor 陈坚刘松堵国成陈康康王广圣
Owner TAIXING YIMING BIOLOGICAL PRODS
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