High-activity chitosanase and preparation method thereof

A chitosanase, high-activity technology, applied in the field of bioengineering, can solve the problems of difficult commercialization, long enzyme production cycle, large enzyme production and other problems, achieve a wide range of temperature and pH, reduce processing costs, The effect of high biological activity

Active Publication Date: 2012-12-12
CHAMBROAD CHEM IND RES INST CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In short, through the continuous efforts of domestic and foreign scholars, many microbial strains producing chitosanase have been discovered in recent years, and some important strains of chitosanase production have also been studied in depth, but overall, the enzyme activity is still generally low. The enzyme production cycle is long, and it is difficult to achieve commercialization. There are no reports of strains that actually produce large amounts of enzymes and are suitable for industrial large-scale production.

Method used

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  • High-activity chitosanase and preparation method thereof
  • High-activity chitosanase and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Enzyme activity detection: DNS method.

[0040] Chitosanase preparation: take 1g of the prepared chitosanase, dissolve it in 80ml of distilled water, and set the volume to 100ml to obtain the chitosanase solution.

[0041] Accurately weigh 0.1g of chitosan powder into a test tube, add 10mL of 0.2M acetic acid solution to make a 1% chitosan solution, shake for 10min, and keep warm in a water bath at 50°C for 10min. Accurately measure 1mL of the above-prepared chitosan enzyme solution, add it to the test tube of chitosan solution, keep it warm at 50°C for 30min, adjust the pH value to 8 with NaOH solution, and centrifuge to remove the precipitate. Aspirate 1mL of the supernatant, add 9mL of 2M / mL hydrochloric acid, hydrolyze at 100°C for 2h, measure the reducing sugar content by DNS method, and calculate the enzyme activity. The enzyme activity unit U is the amount of enzyme required to produce 1 μmol of reducing sugar per minute.

Embodiment 2

[0042] Embodiment 2 solid fermentation

[0043] Determination of the Composition and Culture Conditions of Medium for Enzyme Production in Solid Fermentation

[0044] Using single factor test and orthogonal test, by changing the temperature, initial pH value, inoculum size, loading amount and culture time, the optimal culture conditions of liquid seeds and the best enzyme production conditions of solid fermentation were determined; Cross experiment, by changing the carbon and nitrogen source and inorganic salt composition of the culture to determine the optimal composition of the medium for liquid seeds and the composition of the optimal enzyme-producing medium for solid fermentation of the strain.

[0045] Solid fermentation medium composition and enzyme production conditions determined by the above-mentioned test method:

[0046] The composition of the solid medium, in parts by weight, is: 65-80 parts of bran, 10-20 parts of soybean meal, 8-15 parts of corn flour, 1-3 parts...

Embodiment 3

[0053] Embodiment 3 liquid fermentation

[0054] Determination of the Composition and Culture Conditions of Medium for Enzyme Production in Liquid Fermentation

[0055] Using single factor test and orthogonal test, by changing the temperature, initial pH value, inoculum size, loading amount and culture time, the optimal culture conditions of liquid seeds and the best enzyme production conditions of solid fermentation were determined; Cross experiment, by changing the carbon and nitrogen source and inorganic salt composition of the culture to determine the optimal composition of the medium for liquid seeds and the composition of the optimal enzyme-producing medium for solid fermentation of the strain.

[0056] The composition of the seed liquid culture medium determined by the above-mentioned test method and the fermentation conditions are as follows:

[0057] Activation of strains: move the slant strains of CGMCC No.6129 test tubes stored on nutrient agar medium at 4°C to roo...

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Abstract

The invention belongs to the technical field of biological engineering, and provides a separation and purification process of high-activity chitosanase. The strain of microorganism involved is waxy Bacillus cereus JBSH-003 with the collection number being CGMCC (China General Microbiological Culture Collection) No. 6129. The strain is used as an original strain, crude chitosanase is prepared by liquid or solid fermentation, and the high-purity and high-activity food-grade chitosanase is prepared by separation and purification under certain conditions. The chitosanase can be applied to hydrolyze chitosan to prepare pharmaceutical-grade and food-grade chitooligosaccharide. The activity of the enzyme of the purified chitosanase preparation is more than 25000U / g. The preparation process is simple, the conditions are easy to control, the yield of enzyme activity is high, and the process is suitable for industrial production.

Description

technical field [0001] The invention belongs to the technical field of bioengineering and provides a high-activity chitosanase and its separation and purification process. Background technique [0002] Chitin has the reputation of "the sixth element of human life", and it is the second largest natural polymer compound on the earth after cellulose. Chitosan is the product of chitin deacetylation and contains free amino groups. It is the only basic polysaccharide among natural polysaccharides. The scope of application of sugar. In recent years, it has been found that chitosan degradation product chitosan oligosaccharide has unique physiological functions, and its biological activity is 10 times that of chitosan with equal mass. Therefore, the research on chitosan oligosaccharide has been highly valued by governments and researchers in various countries. Although the research in this area in our country started late, after more than ten years of hard work, great achievements ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12R1/085
Inventor 徐泽平马韵升史庆苓张心青王秀芝虞凤慧高洪奎
Owner CHAMBROAD CHEM IND RES INST CO LTD
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