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Aspergillus usamil high in yield of acid protease and application of aspergillus usamil high in yield of acid protease

A technology of acid protease and Aspergillus Usami, applied in the field of Aspergillus Usami, can solve the problems of complicated production process and low fermentation activity

Inactive Publication Date: 2016-02-10
湖南新鸿鹰生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the fermentation activity of 3.350 acid protease strain is low, and the production process is complicated

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Aspergillus Usami 801-2 provided by the invention is successively subjected to ultraviolet compound lithium chloride mutagenesis, nitrosoguanidine mutagenesis, and diethyl sulfate mutagenesis by a strain of acid protease-producing Aspergillus Usami 801 preserved in a laboratory, and to Mutant strains were isolated and screened, and finally the excellent strains were screened through fermentation performance tests to obtain Aspergillus usami 801-2, which produced high-activity acid protease.

[0024] The high-activity acid protease-producing bacterial strain provided by the present invention is specifically Aspergillus samil 801-2. The strain was deposited in the China Center for Type Culture Collection (CCTCC for short, address: China. Wuhan. Wuhan University Zip Code: 430072) on November 24, 2013. The preservation number is CCTCC NO: M2013601, and the classification is named Aspergillus Usami 801- 2Aspergillususamil801-2.

Embodiment 2

[0026] Produce the screening method of high activity acid protease bacterial strain, comprise the following steps

[0027] 1) Slant culture: the original Aspergillus usami strain 801 was inoculated into PDA slant medium by streaking, and cultured at 31-32°C for 3-4d until the mycelia matured and sporulated.

[0028] 2) Preparation of spore suspension: when Aspergillus Usami produces spores, add 5 mL of sterile water to the inclined surface of the test tube, scrape off the spores, pour into a sterilized triangular flask and add Tween-80 with 0.5-2% spore mass, Place the flask in a shaker and vibrate for 1.5h to activate the spores; then centrifuge at 3500r / min for 15min, wash once with pH7.2 phosphate buffer, then wash with 5mL of buffer solution and transfer to a small flask, add 5-10 capsules Sterile glass beads with a diameter of 3-5 mm were shaken for 10 minutes to disperse the spores; the dispersed spores were filtered with four layers of sterilized lens-cleaning paper to ...

Embodiment 3

[0054] Embodiment 3 produces the culture medium optimization of high-activity acid protease screening starting strain

[0055] (1) Strains

[0056] The starting strain was the 801-2 strain with the best screening results, and the acid protease activity could reach 12000U / mL after 96 hours of fermentation.

[0057] (2) Culture medium optimization

[0058] The medium was optimized for the seed medium and fermentation medium in the process of screening strains, and fermented for 96 hours under the same fermentation conditions before optimization. After testing, the enzyme activity of acidic protease in the crude enzyme solution of the fermentation broth can reach 13000-14000U / mL.

[0059] The seed optimization medium consists of: bran 60-80g, corn flour 50-60g, bean cake powder 35-40g, trehalose 10-15g, fish meal 6-10g, ammonium chloride 10-12g, calcium chloride 5- 10g, casein 5-10g, Chinese herbal medicine extract 5-10g, magnesium sulfate 2-4g, dipotassium hydrogen phosphate 1...

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Abstract

The invention discloses aspergillus usamil 801-2 high in yield of acid protease and belongs to the technical field of microbial fermentation. The aspergillus usamil is preserved with the preservation number CCTCC NO: M2013601 in China Center for Type Culture Collection on November 24, 2013. The aspergillus usamil 801-2 is obtained by subjecting aspergillus usamil 801 yielding the acid protease, preserved by a laboratory, to ultraviolet and lithium chloride compound mutagenesis, nitrosoguanidine mutagenesis and diethyl sulfate mutagenesis sequentially and subjecting a mutant to isolation and screening, and grows faster than a starting strain, the diameter of a colony is 82mm after the aspergillus usamil 801-2 grows at the temperature of 28 DEG C for four days, and enzyme activity of the acid protease in fermentation liquor can be up to 13000-14000U / mL which is 8 times of that of the starting strain. The acid protease secreted by the aspergillus usamil 801-2 is tolerable to high temperature, and under the same conditions, in order to reach same enzyme activity, tolerable temperature of the acid protease is averagely increased by 8-12 DEG C than that of the starting strain.

Description

technical field [0001] The invention belongs to the technical field of microbial fermentation, in particular to aspergillus usami producing high-activity acid protease and its application. Background technique [0002] Acid protease is a kind of aspartic acid protease with the optimum pH value of 2.5-5.0, and the relative molecular mass is 30000-40000. Acid proteases mainly come from animal organs and microbial secretions, including pepsin, rennet and some microbial proteases. According to the different producing bacteria, microbial protease can be divided into mold acid protease, yeast acid protease and basidiomycete acid protease. According to the mode of action, it can be divided into two categories: one is similar to pepsin, and the main enzyme-producing microorganisms are Aspergillus, Penicillium and Rhizopus, etc.; the other is similar to rennet, and the main enzyme-producing microorganisms are Mucor and so on. Because acid protease has better acid resistance, it is ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/14C12N9/62C12R1/66
Inventor 李洪兵李海清张锦杰朱永明胡永明易继云
Owner 湖南新鸿鹰生物工程有限公司
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