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A high-producing bacterium of transglutaminase and its application

A technology for glutamine and transaminase, which is applied in the field of high-producing glutamine transaminase bacteria, can solve the problems of low recovery yield and the like, and achieve the effects of good genetic stability, great social significance and economic value.

Inactive Publication Date: 2016-01-13
虞龙
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In view of the low recovery yield of extracting TG from animals and plants, the cost

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] This example illustrates the mutagenic selection method of Streptomyces hygroscopicus NYU-70.

[0041] Specific steps are as follows:

[0042] (a) Preparation of bacterial suspension: take Streptomyces hygroscopicus NY-1 cultured at 32°C for 5 days, wash the spores on the spore-forming solid medium plate with sterile physiological saline, and pour them into a sterile triangle filled with glass beads. In the bottle, shake for 20 minutes, filter with absorbent cotton, centrifuge at 4000rpm for 10 minutes, discard the supernatant, take the precipitate and wash it, and obtain a spore suspension. Count directly with a hemocytometer and adjust the spore concentration to about 10 6 cells / mL, take 0.1ml of the bacterial suspension and evenly spread it on a 100cm Petri dish.

[0043] (b) Low-energy nitrogen ion implantation mutagenesis: take 0.1mL of the bacterial suspension in step (a) and evenly spread it on a sterile plate, and perform low-energy nitrogen ion implantation o...

Embodiment 2

[0051] Example 2 This example illustrates the biochemical characteristics and genetic stability of Streptomyceshygroscopicus NYU-70

[0052] The morphological, physiological and biochemical characteristics of Streptomyceshygroscopicus (streptomyceshygroscopicus) NYU-70 are: colony color: white; colony size: 2mm-5mm; colony shape: surface dry, protruding, irregular; The center of the colony in the late stage is gray; the mycelia in the base: gray; , The spore filaments are tight spirals; aerobic mode: aerobic; nutritional mode: chemoheterotrophic; suitable growth temperature: 30-40 ℃.

[0053] The results of the subculture fermentation test are shown in Table 2

[0054] Table 2 Genetic stability of Streptomyces hygroscopicus NYU-70

[0055] Number of passages Activity of transglutaminase in fermentation broth (U / ml) 1 16.8 2 16.9 3 16.7 4 16.5 5 16.4 6 16.4

[0056] From the results of the genetic stability experiment, it can b...

Embodiment 3

[0058] This example illustrates the production of transglutaminase by fermentation of the mutant strain Streptomyces hygroscopicus NYU-70 in a 250 ml shake flask.

[0059] The culture medium formula described in the present embodiment:

[0060] Plate medium and slant medium are (g / L): wort juice 50, yeast extract 5, dipotassium hydrogen phosphate 2, light calcium carbonate 5, agar 18, the rest is water, initial pH 7.2;

[0061] Seed liquid medium: glycerin 15, glucose 10, fish meal peptone 20, yeast extract 5, anhydrous magnesium sulfate 2, dipotassium hydrogen phosphate 2, potassium dihydrogen phosphate 2, light calcium carbonate 5, pH7.2

[0062] The fermentation medium is: glycerin 20, glucose 15, fish powder peptone 25, yeast extract 5, anhydrous magnesium sulfate 2, dipotassium hydrogen phosphate 2, potassium dihydrogen phosphate 2, light calcium carbonate 5, pH7.2

[0063] The screened mutant Streptomyces hygroscopicus NYU-70 was inoculated on the basic medium of the pl...

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Abstract

The invention discloses a strain of high-yield transglutaminase and its application, which is classified as a Streptomyces hygroscopicus NYU-70, and has been preserved in CCTCC, China's Type Culture Collection Center, with the preservation number CCTCC? M? 2014121. Using Streptomyces? hygroscopicus NY-1 as the starting strain, low-energy ion implantation mutagenesis was carried out. After mutagenesis, the enzyme activity of the strain was further measured by casein gel method and colorimetry, and the activity of producing glutamine transaminase was screened out. Higher mutant strains, as the starting bacteria for the next round of mutagenesis, were obtained by repeating the above steps for screening. The strain produces high activity of transglutaminase through fermentation and good genetic stability. The mutant strain was used for liquid shake flask fermentation to obtain transglutaminase, and the enzyme activity was 16.9 U / mL.

Description

technical field [0001] The invention relates to a high-yield transglutaminase bacterium and its application, belonging to the technical field of microbial fermentation. Background technique [0002] Transglutaminase (protein-glutamic acid-glutaminase, Transglutaminase, referred to as TG, EC2.3.2.13), also known as transglutaminase, is a catalyzed glutamine residue in protein or polypeptide A transferase for the acyl transfer reaction. Because it can catalyze the covalent cross-linking and polymerization of many proteins and improve the functional properties of various proteins, it has broad application prospects and advantages in the food industry, textile industry and pharmaceutical industry, and is considered to be the most important method for the production of new protein foods. One of the enzymes, it has attracted great attention and interest from researchers at home and abroad. The main functions of transglutaminase in food industry processing include: (1) modifying ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N9/10C12R1/55
Inventor 虞龙李玉燕李市场刘媛黄思思
Owner 虞龙
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