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Thermal-stable microbial transglutaminase, and coding gene thereof

A technology of transglutaminase and microorganisms, applied in plant gene improvement, transferase, genetic engineering, etc.

Inactive Publication Date: 2018-06-01
TAIXING DONGSHENG FOOD TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, MTG has not been well applied in some food processing links that require high temperature conditions.

Method used

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  • Thermal-stable microbial transglutaminase, and coding gene thereof
  • Thermal-stable microbial transglutaminase, and coding gene thereof
  • Thermal-stable microbial transglutaminase, and coding gene thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1: Cloning and sequencing analysis of MTG gene

[0019] Search several Streptomyces transglutaminase sequences from NCBI website, conduct bioinformatics analysis, use CODEHOP (Consensus-degenerate hybrid oligonucleotide primers) program to select two highly conserved amino acid sequences and design degenerate primers.

[0020] Using the soil genomic DNA extraction kit from MP bio ( Spin Kit for Soil) Soil Genome Extraction Kit to extract the total genomes of 50 soil samples from different sources.

[0021] In the 50μl system, add upstream primer: 5′-gcaccggattccgacgacag-3′, and downstream primer: 5′-tcgccgcaggccgactctag-3′, the final concentration is 0.8μM, the final concentration of dNTPs is 0.2mM, and the total DNA of soil genome is 10ng, 2U Pfu DNA polymerase. The amplification program was 94°C for 5min; 35×(94°C for 30s, 55°C for 60s, 72°C for 60s); 72°C for 10min. The PCR products were sent to Shanghai Sangon Bioengineering Company for sequencing.

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Embodiment 2

[0023] Embodiment 2: Novel MTG gene synthesis and construction of prokaryotic expression vector

[0024] According to the MTG gene sequence obtained by sequencing, BamHI recognition sequences were added to the 5' and 3' ends respectively ( GGATCC ), commissioned Shanghai Sangon Bioengineering Company to synthesize the gene. The synthesized gene and vector pET-23a were digested with appropriate amount of the same restriction endonuclease BamHI, respectively, and connected with T4DNA ligase. The recombinant vector transformed Escherichia coli DH5α. Randomly pick a single colony from the transformation plate, insert it into LB liquid medium, shake culture, extract a small amount of plasmid, electrophoresis, use the plasmid with electrophoresis delay as a template for PCR verification, and send it to Shanghai Sangon Bioengineering Co., Ltd. after confirming that the connection is successful sequencing.

Embodiment 3

[0025] Embodiment 3: the expression of novel MTG enzyme gene in Escherichia coli

[0026] Transform Escherichia coli expression host strain BL21(DE3)pLysS containing MTG expression plasmid pET-23a-MTG, pick small colony after culturing at 37°C for 10-11 hours, insert into 50ml LB liquid medium containing ampicillin, 70- Cultivate overnight at 90rpm at 30°C, take the seed liquid at a volume ratio of 1:40 and add it to 100ml LB liquid medium containing ampicillin, shake at 35°C and 180rpm for 2-3 hours until the OD600 is about 0.6, add IPTG (final concentration 100μg / ml) induction. After 1.5 hours, the cells were collected by centrifugation. Break the bacteria, centrifuge to collect the supernatant, which is the recombinant MTG crude enzyme solution

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Abstract

The invention discloses a thermal-stable microbial transglutaminase, and a coding gene thereof. The amino acid sequence of the thermal-stable microbial transglutaminase is represented by SEQ ID NO.1.The nucleotide sequence of the gene used for coding the thermal-stable microbial transglutaminase is represented by SEQ ID NO.2. The half life of the thermal-stable microbial transglutaminase at 60 DEG C is 10min or longer. The thermal-stable microbial transglutaminase is capable of enriching microbial transglutaminase gene resources, and providing foundation of enlargement of application range ofmicrobial transglutaminase in food processing.

Description

technical field [0001] The invention belongs to the field of food industry biotechnology, and relates to a thermostable microbial transglutaminase and its coding gene. Background technique [0002] Microbial Transglutaminase (Microbial Transglutaminase, EC 2.3.2.13 MTG for short) is a thiolase that catalyzes intramolecular or intermolecular crosslinking of proteins, connections between proteins and amino acids, and intramolecular glutamine The hydrolysis of the amide group improves the functional properties of the protein, improves the structure, and improves the nutritional value of the protein by introducing lysine. [0003] MTG can change the functional properties of food proteins mainly due to: (1) it can change the physical properties of proteins such as casein, myosin and lactoglobulin; Amino acids are combined with various food proteins in the form of covalent bonds to make up for the loss or destruction of essential amino acids during processing and improve the nutr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54A23L29/00
Inventor 步国建张充张晓健程伟
Owner TAIXING DONGSHENG FOOD TECH
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