Glutamine transaminase high-yielding strain and application thereof

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 achieves the effects of good genetic stability, great social significance and economic value.

Inactive Publication Date: 2014-08-13
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 TG e

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) Bacterial suspension preparation: 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 to 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 on th...

Embodiment 2

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

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

[0053] The results of the subculture fermentation test are shown in Table 1

[0054] 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

[0055] From the results of the genetic stability experiment, it can be seen that after 6 consecutive passages, the mutant strain Streptomy...

Embodiment 3

[0057] This example illustrates the production of transglutaminase by fermentation of the mutant strain Streptomyces hygroscopicus NYU-70 in a 250 ml shake flask.

[0058] The culture medium formula described in the present embodiment:

[0059] 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;

[0060] 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

[0061] 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

[0062] 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 glutamine transaminase high-yielding strain and application thereof. The strain is named as streptomyces hygroscopicus NYU-70 according to classification and naming, and has been preserved in China Center for Type Culture Collection and preservation number of CCTCCM2014121. Streptomyces hygroscopicus NY-1 is used as a starting strain for low energy ion implantation mutagenesis, activity of the enzyme of the strain after mutagenesis is measured through casein gel method and colorimetric method, mutant strains with high activity in producing glutamine transaminase are screened out as the starting strains for the next round of mutagenesis, and the above steps are repeated to obtain the glutamine transaminase high-yielding strain. The strain has high activity and good genetic stability in fermentation production of glutamine transaminase. The mutant strain can be used in liquid shake flask fermentation to obtain glutamine transaminase, and has enzyme activity of 16.9U / 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) modify var...

Claims

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Application Information

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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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