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Bacillus and method of producing hyaluronidase by employing the same

A technology of hyaluronidase and production method, applied in microorganism-based methods, biochemical equipment and methods, enzymes, etc., can solve the problem of high cost of hyaluronidase, low enzyme activity of hyaluronidase, and few production processes, etc. problem, to achieve the effect of facilitating large-scale industrial production, high pH stability, and high cost of solution

Inactive Publication Date: 2012-07-11
BLOOMAGE BIOTECHNOLOGY CORP LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to solve the problem that the existing hyaluronidase production process is few, and the obtained hyaluronidase enzyme activity is low, the present invention provides a kind of strain that produces highly active hyaluronidase—Bacillus ( Bacillus )A50
[0006] The present invention also provides the use of the bacillus ( Bacillus ) A50 method for producing hyaluronidase, producing hyaluronidase with this strain has high enzyme activity, can be used for large-scale production, and solves the problem of high cost of animal-derived hyaluronidase, which is convenient for large-scale industrial production

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Slant medium composition (100ml): peptone 0.2g, yeast powder 2.0g, K 2 HPO 4 ·3H 2 O 0.05g, MgSO 4 ·7H 2 O 0.05g, glucose 0.5g, agar powder 2g, adjust the pH to 6.0 with hydrochloric acid.

[0032] Seed medium composition (100ml): peptone 0.2g, yeast powder 2.0g, K 2 HPO 4 ·3H 2 O 0.05g, MgSO 4 ·7H 2 O 0.05g, glucose 0.5g, adjust the pH to 6.0 with hydrochloric acid.

[0033]Fermentation medium composition (100ml): peptone 0.2g, yeast powder 2.0g, K 2 HPO 4 ·3H 2 O 0.05g, MgSO 4 ·7H 2 O 0.05g, glucose 0.5g, Tween80 (Tween 80) 0.05ml. All media use water as solvent.

[0034] Take the slant bacteria (Bacillus ( Bacillus ) A50 CGMCC NO. 5744) inoculated in the sterilized seed medium, 25°C, 150rpm cultured for 24 hours, then inoculated the seed liquid into the sterilized fermentation medium, the inoculation amount was 10%, 25°C, Cultivate at 200rpm for 24 hours. During the fermentation process, sulfuric acid is used to maintain the pH at 6.0. Fermentation ...

Embodiment 2

[0036] Slant medium composition (100ml): peptone 1.0g, yeast powder 1.0g, K 2 HPO 4 ·3H 2 O 0.1g, MgSO 4 ·7H 2 O 0.1g, glucose 1.0g, agar powder 2g, adjust the pH to 7.0 with phosphoric acid.

[0037] Seed medium composition (100ml): peptone 1.0g, yeast powder 1.0g, K 2 HPO 4 ·3H 2 O 0.1g, MgSO 4 ·7H 2 O 0.1g, glucose 1.0g Adjust the pH to 7.0 with phosphoric acid.

[0038] Fermentation medium composition (100ml): peptone 1.0g, yeast powder 1.0g, K 2 HPO 4 ·3H 2 O 0.1g, MgSO 4 ·7H 2 O 0.1g, glucose 1.0g, Tween80 0.05ml,.

[0039] Take the slant bacteria (Bacillus ( Bacillus ) A50 CGMCC NO. 5744) inoculated in the sterilized seed medium, 30°C, 100rpm cultured for 15 hours, then inoculated the seed liquid into the sterilized fermentation medium, the inoculation amount was 10%, 35°C, Cultivate at 300rpm for 16 hours, maintain the pH at 7.0 with sulfuric acid during the fermentation process, and produce hyaluronidase by fermentation. The activity of hyaluronidase ...

Embodiment 3

[0041] Slant medium composition (100ml): peptone 1.5g, yeast powder 1.5g, K 2 HPO 4 ·3H 2 O 0.15g, MgSO 4 ·7H 2 O 0.15g, glucose 1.5g, agar powder 2g, adjust the pH to 8.0 with sulfuric acid.

[0042] Seed medium composition (100ml): peptone 1.5g, yeast powder 1.5g, K 2 HPO 4 ·3H 2 O 0.15g, MgSO 4 ·7H 2 O 0.15g, glucose 1.5g, adjust the pH to 8.0 with sulfuric acid.

[0043] Fermentation medium composition (100ml): peptone 0.5g, yeast powder 1.5g, K 2 HPO 4 ·3H 2 O 0.1g, MgSO 4 ·7H 2 O 0.05g, glucose 1.5g, Tween80 0.05ml.

[0044] Take the slant bacteria (Bacillus ( Bacillus ) A50 CGMCC NO. 5744) inoculated in the sterilized seed medium, 35°C, 200rpm for 13 hours, then inoculated the seed liquid into the sterilized fermentation medium, the inoculum amount was 10%, 40°C, Cultivate at 100rpm for 12 hours, maintain the pH at 7.0 with hydrochloric acid during the fermentation process, and produce hyaluronidase by fermentation. The activity of hyaluronidase in the ...

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Abstract

The invention discloses a (Bacillus) A50 CGMCC No. 5744 and a method of producing hyaluronidase by employing the same. The method is that slant cultivation, seed cultivation and fermentation cultivation are carried out for the (Bacillus) A50 CGMCC No. 5744 so that hyaluronidase is prepared. The hyaluronidase generated by the Bacillus is highly active, up to 105IU / m1. As compared with the prior art, the hyaluronidase prepared from the Bacillus of the invention is high in activity, high in thermal stability and high in pH stability, and can be used for large scale production. Meanwhile, the obtained hyaluronidase can allow high-molecular hyaluronic acid to be degraded, can be used for preparing low-molecular hyaluronic acid and oligomeric hyaluronic acid, enables the cost to be substantially reduced, helps to solve the problem of high cost for hyaluronidase of animal sources, and exhibits a wide application prospect in the field of biochemistry research and for the production of low-molecular hyaluronic acid and oligomeric hyaluronic acid.

Description

technical field [0001] The invention relates to a bacillus and a method for producing hyaluronidase by using the bacillus, belonging to the technical field of biological fermentation. Background technique [0002] Hyaluronidase is a type of enzyme that can degrade hyaluronic acid. According to the mechanism of action of hyaluronidase, Karl Meyer divides hyaluronidase into three categories: (1) Endo-β-N-acetylglucosaminidase It is a hydrolase that acts on β-1,4 glycosidic bonds, and the final product is mainly tetrasaccharides. It can also act on chondroitin or chondroitin sulfate, and has transglycosidase activity, testis, bee venom and lysosome hyaluronic acid Enzymes fall into this category. (2) Hyaluronidase derived from leeches and duodenum worms is an endo-β-glucuronidase, which acts on β-1,3 glycosidic bonds and is also a hydrolase. The main degradation product is tetrasaccharide, which is specific Degrades hyaluronic acid. (3) Bacterial hyaluronidase, also known as...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/42C12R1/07
Inventor 郭学平刘爱华栾贻宏石艳丽李海娜薛蔚
Owner BLOOMAGE BIOTECHNOLOGY CORP LTD
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