Method for producing high-activity chitosanase preparation

A technology of chitosanase and high activity, which is applied in the field of microbial fermentation technology and enzyme engineering, can solve the problems of long enzymatic hydrolysis time, low concentration of enzymatic hydrolysis substrate, low enzyme activity, etc., and achieve the improvement of enzyme activity and enzymolysis The effect of high process efficiency

Inactive Publication Date: 2008-03-26
WUHAN EAST ANGEL BIOENG
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AI Technical Summary

Problems solved by technology

There are a large number of chitosan enzymes, but there are generally problems such as low enzyme acti...

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Breeding of TQK strains highly expressing chitosanase.

[0027] Based on the TQS6 strain obtained from CCTCC (preserved by the China Center for Type Culture Collection), through UV (ultraviolet mutagenesis) and NTG (nitrosoguanidine mutagenesis) compound mutagenesis, the conditions for ultraviolet mutagenesis are: UV lamp power 15 ~30w, irradiation distance 20~50cm, irradiation time 1~8min. The conditions for NTG mutagenesis are: NTG concentration 1-3 mg / ml, in tris buffer solution with pH 6.0, treatment for 90-120 min. The strains were inoculated on plate medium containing 0.3% Congo red, induced and cultured at 35°C for 30 hours, and transparent hydrolysis circles could be seen. The medium formula is calculated by mass percentage: 2% agar, 0.5% chitosan, 1% tryptone, 1% yeast extract powder, 0.5% NaCl, and 0.3% Congo red.

[0028] Select the strain with the larger H / C colony diameter ratio of the hydrolysis circle diameter, carry out shake flask re-scr...

Embodiment 2

[0029] Embodiment 2 fermentation produces chitosanase

[0030] 1): The TQK strain (preservation number is CCTCC No: M207129) was inoculated by streaking on a flat surface containing 1% yeast extract powder, 1% tryptone, 0.5% sodium chloride, 0.5% chitosan, and 2% agar Incubate at 33°C for 24 hours to use.

[0031] 2) First-level liquid culture: use 1000ml triangular flask to pack 200ml liquid medium containing 1% yeast extract powder, 1% tryptone, 0.5% sodium chloride, 0.5% chitosan, sterilize for 20 minutes at 10 pounds, and inoculate one In a ring, culture on a shaker (260r / min) at 34°C for 27 hours.

[0032] 3) Use a 7-liter fermenter to pack 5 liters of liquid medium containing yeast extract powder, tryptone, chitosan, inorganic salt composite components, and corn dregs. After the same sterilization as above, connect 200 mL of shaker flask seeds and ventilate and stir at 35 ° C. 18-24 hours out of the tank.

[0033] 4) Low-temperature centrifugal separation of enzyme ...

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PUM

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Abstract

The present invention is production process of UV and NTG mutagenizing Bacillus subtilis TQS6 to culture high efficiency expressing chitosanase strain TQK and fermentation producing high activity chitosanase. The chitosanase strain is first slant cultured and then fermentation cultured while introducing oxygen-rich air to increase dissolved oxygen to 30-80 %; and after culturing for 18-24 hr, average enzyme activity may reach 718 mu/ml. High activity chitosanase may be obtained through fermentation in optimized enzyme producing condition. The process of the present invention can obtain chitosanase with high yield, high stability and high activity, and the produced chitosanase is applied in converting chitosan into chitin oligose effectively and economically.

Description

technology field; [0001] The invention relates to microbial fermentation technology and enzyme engineering technology, in particular to a production method of a bacillus TQK strain highly expressing chitosanase and a high-activity chitosanase preparation. Background technique [0002] At present, domestic and foreign methods for preparing chitosan oligosaccharides can be roughly divided into three categories: enzymatic degradation, oxidative degradation and acid degradation. The process of preparing chitosan oligosaccharides by bioenzyme degradation is obviously better than the other two chemical degradation methods. This is because the enzymatic degradation process makes the molecular weight distribution of the degradation products easier to be controlled, and can monitor the degradation process to produce effective (2 -10 sugar) chitosan oligosaccharides with higher content, and the degradation conditions are relatively mild, without adding a large amount of reaction reage...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N13/00C12N15/01C12N9/24C12R1/125
Inventor 黄代勇黄芳万霞
Owner WUHAN EAST ANGEL BIOENG
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