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Method for efficiently producing transglutaminase and special engineering bacterium thereof

A technology of amino acids and recombinant microorganisms, applied in the biological field, can solve the problems of increasing industrial production costs, time costs, and low expression levels

Active Publication Date: 2021-10-22
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, more and more expression systems are used to express MTG, but there are still problems such as low expression level and secondary treatment with protease in the later stage
There are also some studies that use intein to successfully express MTG in E. coli and Bacillus subtilis, but it still needs to undergo secondary treatment of pH or temperature changes to realize the shearing of the pro region, which greatly increases the cost of industrial production. and time cost

Method used

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  • Method for efficiently producing transglutaminase and special engineering bacterium thereof
  • Method for efficiently producing transglutaminase and special engineering bacterium thereof
  • Method for efficiently producing transglutaminase and special engineering bacterium thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Embodiment 1, construct recombinant plasmid and recombinant bacterium

[0074] 1. Construction of recombinant plasmids

[0075] 1. Construction of recombinant plasmid pXMJ19-pro-ssp-dnaB 155M -MTG.

[0076] Recombinant plasmid pXMJ19-pro-ssp-dnaB 155M -MTG is a circular plasmid, as shown in sequence 1 of the sequence listing.

[0077] In sequence 1 of the sequence listing, nucleotides 6304-6322 constitute the T7 promoter, nucleotides 6323-6347 constitute the lac operator, nucleotides 6350-6385 constitute the RBS, and nucleotides 6386-8002 constitute the nucleotide Acid is the complete open reading frame (encoding fusion protein). In sequence 1 of the sequence listing, the 6386-6388th position is the start codon, the 6389-6523rd nucleotide codes the pro region, the 6524-6985th nucleotide codes the ssp-dnaB intein, and the 6986-6988th nucleotide The first nucleotide encodes the amino acid residue M, the 6989-7981 nucleotide encodes the mature MTG, and the 7982-7999 n...

Embodiment 2

[0094] Embodiment 2, shake flask fermentation of recombinant bacteria

[0095] 1. Strain CG-pXMJ19-pro-ssp-dnaB 155M -Induced fermentation culture of MTG

[0096] 1. The strain CG-pXMJ19-pro-ssp-dnaB 155M Inoculate a single colony of -MTG into a test tube filled with 5 mL of liquid BHI medium containing 17 μg / mL chloramphenicol, and culture with shaking at 30°C and 200 r / min for 12 hours to obtain the seed solution.

[0097] 2. Inoculate 1mL seed liquid into a 250mL Erlenmeyer flask filled with 50mL fermentation medium containing 17μg / mL chloramphenicol, culture at 30°C, 200r / min shaking until OD 600nmvalue=1.

[0098] 3. After completing step 2, add IPTG to the culture system so that the concentration in the system is 0.5mM, and then shake and culture at 25°C and 200r / min for 48h. At this time, the system OD 600nm The value is about 20, and the system at this time is named as a fermentation product.

[0099] 4. Get the fermentation product (the amount of bacteria is: OD ...

Embodiment 3

[0130] Embodiment 3, the fermenter cultivation of recombinant bacterium

[0131] 1. Preparation of seed solution

[0132] Strain CG-pXMJ19-pro-ssp-dnaB 155M Inoculate a single colony of -MTG into a test tube filled with 5 mL of liquid BHI medium containing 17 μg / mL chloramphenicol, and culture with shaking at 30°C and 200 r / min for 12 hours to obtain the seed solution. Multiple seed solutions were prepared using multiple tubes.

[0133] 2. Inoculate 100mL of seed liquid into a 2.5L fermenter equipped with 1L fermentation medium containing 17μg / mL chloramphenicol, and ferment for 48h. During the fermentation process, the temperature is controlled at 25°C, and the pH value is controlled at about 7.0 by feeding ammonia water. During fermentation, when the system OD 600nm When the value reaches 8-10, add IPTG and make its concentration in the system 0.5mM. During the fermentation process, monitor the glucose concentration of the system, and maintain the glucose concentration ...

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Abstract

The invention discloses a method for efficiently producing transglutaminase and a special engineering bacterium thereof. The invention provides a protein as shown in a sequence 2 in a sequence table. The invention also provides a recombinant microorganism obtained by introducing a DNA molecule for expressing the protein into corynebacterium glutamicum. The recombinant microorganism can be used for preparing transglutaminase. According to the invention, MTG derived from streptomyces moocenogenus is expressed under a T7 promoter, and intein ssp-dnaB is inserted between a pro region and a mature region of MTG, so that efficient expression of MTG in corynebacterium glutamicum is successfully realized, and after thalli obtained by fermentation are subjected to ultrasonication, MTG with biological activity can be obtained without protease treatment or other condition change. The method can be used for preparing transglutaminase, is further applied to the fields of tissue engineering, textiles, leather processing and the like, and has application and popularization values.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for efficiently producing transglutaminase and special engineering bacteria thereof. Background technique [0002] Transglutaminase (EC 2.3.2.13, TGase) belongs to the family of transferases that catalyze the conversion of γ-carboxamide groups (donors) from glutamine residues to various primary amines (acyl acceptors). Acyl transfer. At present, TGase has been used in the field of food processing for more than 10 years and has been generally recognized as safe (GRAS) by the US Food and Drug Administration (FDA). TGase can improve the properties of proteins through intramolecular and intermolecular cross-linking of proteins. Therefore, the enzyme shows wider application value in the fields of tissue engineering, textile and leather processing. [0003] TGase exists widely in animals and plants, but its yield is low, and the cost of separation and purification i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/77C12N1/21C12R1/15
CPCC12N9/1044C12N15/77C12Y203/02013
Inventor 董志扬张楠张山何永志张岩峰
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI