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Transglutaminase producing strain

A technology of transglutaminase and production bacteria, which is applied in the field of bioengineering, can solve the problems of low fermentation level of production strains and achieve the effect of effective accumulation

Active Publication Date: 2020-08-28
上海东之汇生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] For the defect of low fermentation level of existing transglutaminase production strains, the present invention combines chemical mutagenesis, physical mutagenesis and cross breeding to screen mutant strains of Streptomyces mobara, and obtains high-yield transglutaminase production strains

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: UV mutagenesis and screening

[0040] 1.1 Preparation of spore suspension: add 5ml of sterile water to the Streptomyces mobara DSM40587 culture plate (petri dish), gently scrape the bacteria with a sterile toothpick, blow and mix 5 times with a 1ml pipette, and then put it into a 100ml sterile cone In a shaped bottle (with 2g of glass beads), seal the bottle with a sealing film, put it in a constant temperature shaker at 200r / 5min to oscillate to break up the bacteria, use a 1ml pipette to draw the bacteria solution, and use a sterile funnel with 0.45μm filter paper Filter into a new sterile Erlenmeyer flask, draw the filtrate into a sterile 2ml EP tube (2 tubes), centrifuge at 3000g / min for 10min, pipette the supernatant and discard it.

[0041] 1.2 Mutagenesis (the following operations need to be protected from light): add 1ml of sterile water to each tube, blow and mix 5 times with a 1ml pipette, then use a 1ml pipette to draw 1ml of the bacterial solution...

Embodiment 2

[0050] Embodiment 2: produce transglutaminase strain fermentation and yield determination

[0051] 2.1 Strain shake flask fermentation

[0052] Seed culture: After the fermentation medium is prepared and mixed, it is divided into 50ml / bottles into 250ml shake flasks, and a quarter of the spores on the culture plate obtained in Example 1.4 are scraped with an inoculation loop and inoculated in shake flasks. C, 220rpm and cultivate for 24h to obtain the seed culture solution.

[0053] Shake flask fermentation: Prepare and mix the fermentation medium and distribute 30ml / bottle into a 250ml shake flask, inoculate 3ml of seed culture solution into the fermentation shake flask, and culture at 30°C and 220rpm for about 28h. The fermentation broth was centrifuged at high speed, and the supernatant was taken to detect the enzyme activity.

[0054] 2.2 Determination of enzyme activity of transglutaminase

[0055] 2.2.1 Principle

[0056] The principle of the enzyme activity assay of...

Embodiment 3

[0071] Example 3: Nitrosoguanidine mutagenesis of mutant strain DSHO0618

[0072] In order to obtain a strain with further improved production of transglutaminase as much as possible, we continued to carry out chemical mutagenesis on DSHO0618, using nitrosoguanidine (NTG) as a mutagen. NTG is a very strong chemical mutagen, and the mutations induced by it are mainly GC-AT conversion, and there are also small excision, frameshift mutation and GC pair loss.

[0073] Since nitrosoguanidine is a strong carcinogenic mutagenic substance, in order to prevent harm to the human body, rubber gloves, work clothes, and a mask should be worn during operation. It must be processed in time, soak the used consumables in 1M sodium hydroxide solution (8g sodium hydroxide dissolved in 200mL distilled water) overnight, and use 0.5% sodium thiosulfate (7.9g sodium thiosulfate dissolved in 100mL distilled water) Wipe down the safety cabinet.

[0074] 3.1 Activation of the strain DSHO0618 and prep...

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Abstract

A transglutaminase producing strain is obtained through mutagenesis, is collected in the China Center for Type Culture Collection, and has a collection number of CCTCC NO:M 2020147 or CCTCC NO:M 2020148. Compared with an original strain, the transglutaminase producing strain disclosed by the invention has the advantages that the transglutaminase yield is increased by more than 4 times, the enzymeactivity reaches 7.5 U / mL or above, and the transglutaminase producing strain can be industrially applied.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a transglutaminase-producing bacterium. Background technique [0002] Transglutaminase (EC 2.3.2.13, Transglutaminase, TGase), also known as glutaminase transaminase (Glutamine transaminase), referred to as TGase, is a monomeric protein with an active center with a molecular weight of about 38,000 composed of 331 amino groups. It widely exists in human body, advanced animals, fish, plants and microorganisms in nature, for example, it exists in various tissues and organs of mammals (such as liver, hair follicle, epidermis, prostate, blood). It is an acyltransferase that catalyzes the conjugation reaction between the ε-amino group on lysine and the γ-hydroxylamide group on glutamic acid in proteins (see figure 1 ), causing covalent cross-linking of protein polypeptides within and between molecules. As a polymerase that forms covalent compounds through transglutamination,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/10C12R1/465
CPCC12N9/1044C12Y203/02013C12R2001/465C12N1/205
Inventor 钱源
Owner 上海东之汇生物科技有限公司
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