Separation and purification method of microbial transglutaminase

A technology for separation and purification of glutamine, which is applied in the field of separation and purification of biotechnology, can solve the problems of complex purification process, low recovery rate and high cost of transglutaminase, and achieve industrial production convenience, low cost and stable properties Effect

Active Publication Date: 2016-07-13
EAST CHINA NORMAL UNIV +1
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Problems solved by technology

[0006] The purpose of the present invention is to provide a microbial transglutaminase purification method with low cost, high recovery rate and high purity, which is suitable for industrial production and meets the requirements of pharmaceutical grade, so as to solve the problem of complicated purification process of transglutaminase at present. Problems of high cost, low recovery rate and low purity

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  • Separation and purification method of microbial transglutaminase
  • Separation and purification method of microbial transglutaminase
  • Separation and purification method of microbial transglutaminase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1 alcohol precipitation obtains MTGase crude extract

[0032] (1) Centrifuge 8,000g of the fermented broth at 4°C for 10 minutes to remove the precipitate; slowly adjust the pH to 5.0 with phosphoric acid, and centrifuge at 8,000g for 10 minutes at 4°C to remove the precipitate; Add, place in an ice box for 1 hour, centrifuge at 8000g for 10 minutes at 4°C, and remove the supernatant.

[0033] (2) SDS-PAGE detection results such as Figure 4 As shown in middle lane 2, the molecular weight of MTGase is about 38KDa. From lane 2, it can be seen that the crude extract obtained by alcohol precipitation contains a large amount of miscellaneous proteins. The total enzyme activity of the fermentation broth before alcohol precipitation was 2760.6U, and the total enzyme activity after alcohol precipitation was 2484.5U. The specific results are shown in Table 1. The enzyme activity assay results showed that the total enzyme activity recovery rate in this step was 90%....

Embodiment 2

[0034] The detection of embodiment 2MTGase crude extract temperature stability

[0035] (1) The MTGase (experimental group) obtained by alcohol precipitation in Example 1 is lyophilized and dissolved in the phosphate buffer of pH6.0 with 3U / mL, and two arbitrary two MTGases (control group) that have been reported at present are selected simultaneously. 1: 40587 and the control group 2: 40847 (both from Taixing Dongsheng Food Technology Co., Ltd.) were used as controls, and were also dissolved in phosphate buffer at pH 6.0 at 3 U / mL.

[0036] (2) Place the three MTGases in water baths at 40°C, 50°C, 60°C and 70°C respectively, and press figure 2 Store MTGase at intervals under various temperature conditions shown, and measure the enzymatic activity of MTGase after a certain period of time. A is the result of different MTGase stability at 70°C, B is the result of different MTGase stability at 60°C, C is the result of different MTGase stability at 50°C, D is the result of differ...

Embodiment 3

[0038] Example 3 Anion-exchange chromatography method obtains high-purity MTGase after purification

[0039] (1) The purification instrument selected is from General Corporation of the United States avant25, the chromatographic column used for purification is a prepacked anion exchange column HiprapQSepharose FF (1 mL) purchased from General Corporation of the United States.

[0040] (2) Appropriate amounts of 20 mM sodium dihydrogen phosphate and 20 mM disodium hydrogen phosphate were prepared respectively, and the two buffer solutions were mixed in proportion to finally prepare a phosphate buffer solution with a pH of 6.5, and the conductance was about 2.5 mS / cm. After preparation, use a 0.22um filter membrane to filter, and then degas in an ultrasonic instrument for 20-30min. Store in refrigerator at 4°C for later use.

[0041] (3) Add 1M sodium chloride on the basis of the equilibrium buffer, and after fully dissolving, adjust the pH to 6.5 with phosphoric acid, and the...

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Abstract

The invention discloses a separation and purification method of new microbial transglutaminase. The method comprises the following steps of carrying out alcohol precipitation on new streptomyces mobaraensis fermentation liquor with the strain number being CGMCC No.10804 obtained through natural screening, carrying out anion exchange chromatography, desalting, freezing and drying to obtain the microbial transglutaminase. The obtained transglutaminase has the purity being larger than or equal to 90 percent, the plasma endotoxin level being less than or equal to 0.02EU/mL, the specific enzyme activity being 25 to 30U/mg, and the recovery rate being larger than or equal to 70 percent. The separation and purification method of the new microbial transglutaminase is simple in process, low in cost, and high in enzyme recovery; the obtained transglutaminase is high in purity; the microbial transglutaminase obtained through fermenting the strain is stable in property, so that the industrial production is convenient; a feasible method is provided for satisfying medical grade MTGase scale production.

Description

technical field [0001] The invention belongs to the field of separation and purification of biotechnology, and in particular relates to a separation and purification technology of transglutaminase produced by a new bacterial strain. Background technique [0002] Transglutaminase (Transglu t ami na se, TGase for short, EC2.3.2.13) is a transferase that catalyzes acyl transfer, which can catalyze the formation of ε-(γ-glutaminyl)lysine covalent bonds in protein molecules and intermolecular molecules, thereby catalyzing the intramolecular, molecular Cross-linking occurs between proteins, thereby changing the properties and functions of proteins. [0003] Transglutaminase widely exists in plants, animals and microorganisms. TGase was first obtained by scholars in the liver of guinea pigs, which has been used as a source of commercial TGase in Europe for a long time. Scarcity of resources and complex separation and purification process of TGase from animal sources lead to ver...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/10C12R1/625
CPCC12N1/20C12N9/1044C12Y203/02013C12N1/205C12R2001/625
Inventor 金明飞常忠义高红亮易正芳王志珍沈迎辉陈旭宋佳雯步国建
Owner EAST CHINA NORMAL UNIV
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