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Bacillus subtilis capable of producing neutral protease with strong heat stability and application of bacillus subtilis

A Bacillus subtilis, neutral protease technology, applied in the direction of enzymes, bacteria, microorganism-based methods, etc., can solve the problems of limited application scope, complex production and use conditions, poor thermal stability of neutral protease, etc. Wide value range, high temperature tolerance, stable enzymatic activity

Active Publication Date: 2014-03-26
HUNAN HONGYING BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the properties and advantages of fast catalytic reaction speed, no industrial pollution, and wide adaptability to catalytic reaction conditions, neutral proteases are used in industries such as food, pharmaceuticals, cosmetics, detergents, silk spinning, and fur softening. The thermal stability of protease is poor, and the production and use conditions are complicated, which limits the scope of its further application. Further optimization and breeding of excellent strains producing neutral protease is of great significance to the industrial production of neutral protease, especially the thermal stability of neutral protease Enzymatic properties such as resistance to acid and alkali are still one of the most concerned issues for technicians in this industry

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] The Bacillus subtilis strain producing neutral protease of the present invention is specifically 1398-2-12. The strain was deposited in the China Center for Type Culture Collection (CCTCC for short, address: School of Life Sciences, Wuhan University, Luojia Mountain, Wuchang District, Wuhan City, Hubei Province, Zip Code: 430072) on November 3, 2013, and the preservation number is CCTCC NO: M2013539, classified as Bacillus subtilis (Bacillus subtilis) 1398-2-12.

[0020] The bacillus subtilis 1398-2-12 provided by the invention is obtained from a neutral protease-producing bacillus subtilis 1398-2 preserved in a laboratory through ultraviolet-lithium chloride-diethyl sulfate compound mutagenesis screening.

Embodiment 2

[0021] Embodiment 2 produces the screening method of strong thermostable neutral protease strain, comprises the following steps

[0022] (1) Preparation of bacterial suspension

[0023] Inoculate the single colony of Bacillus subtilis 1398-2 slant strain separated on the plate by streaking and inoculate the seed at 100r / min. After culturing at 35°C for 12h, take 1mL of the culture medium, centrifuge it, wash it twice with normal saline, and resuspend it. In 9mL saline.

[0024] (2) Ultraviolet-Lithium Chloride-Diethyl Sulfate Compound Mutagenesis

[0025] The bacterial suspension was placed on a sterile plate, stirred and irradiated for 100s under a UV lamp with a distance of 30cm and a power of 15w. The irradiated bacteria solution was diluted and spread on the lithium chloride plate after gradient dilution, and the bacteria solution without ultraviolet irradiation was diluted and spread on the plate as a control. Wrap the uniformly coated plate above with black cloth or n...

Embodiment 3

[0037] Embodiment 3: the optimization of the screening medium for strains that produce strong thermostable neutral protease

[0038] (1) Strains

[0039] The starting strain was the 1398-2-12 strain with the best screening results, and the neutral protease activity could reach 6000U / mL after 72 hours of fermentation.

[0040] (2) Culture medium optimization

[0041] The lithium chloride plate medium, seed medium and fermentation medium in the process of strain screening were optimized to ferment for 72 hours under the same fermentation conditions before optimization. After testing, the neutral protease activity of the crude enzyme liquid in the fermentation broth can reach 7000U / mL.

[0042] Lithium chloride optimized plate medium consists of: starch 10g, peptone 10g, (NH) 2 SO 4 4g,K 2 HPO 4 8g, CaCl 2 2g, lithium chloride 9g, Chinese herbal medicine powder 5-20g, agar 20g, distilled water 1000mL, pH value 7.2, sterilized at 121°C for 20min;

[0043] The composition o...

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Abstract

The invention discloses a bacillus subtilis capable of producing a neutral protease with strong heat stability and application of the bacillus subtilis, and belongs to the technical field of preparation of enzyme preparations. The bacillus subtilis 1398-2-12 capable of producing the neutral protease with strong heat stability is used as an original strain, culture medium optimization and fermentation process improvement are performed, the neutral protease prepared through liquid submerged and mixed fermentation under special fermentation conditions is 5500-7000 U / mL in enzyme activity, 30-75 DEG C in applicable temperature range, 70 DEG C in optimum reaction temperature, completely stable in enzyme activity at the temperature of 75 DEG C, 4.5-8.5 in applicable reaction pH value and is 7.2 in optimum reaction pH value. Compared with the conventional neutral protease, the neutral protease produced by the bacillus subtilis has stronger heat stability and higher enzyme activity, and meets industrial demands.

Description

technical field [0001] The invention belongs to the technical field of microbial fermentation, in particular to a bacillus subtilis producing strong heat-stable neutral protease. Background technique [0002] Proteases are a class of enzymes (enzymes) in living organisms that break down proteins. Breaking down works by breaking the peptide bonds that link amino acids into polypeptide chains. Small molecule compounds that inhibit the activity of proteases are called protease inhibitors. Inhibitors of many viral proteases are very effective antivirals. Protease is a general term for a class of enzymes that hydrolyze peptide bonds in proteins. According to their way of hydrolyzing polypeptides, they can be divided into two types: endopeptidases and exopeptidases. Endopeptidase cuts off the inside of protein molecules to form peptones and peptones with smaller molecular weights. The exopeptidase hydrolyzes the peptide bonds one by one from the free amino or carboxyl end of ...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N9/56C12R1/125
Inventor 李洪兵张锦杰朱永明孙明芳陈香刘德凤
Owner HUNAN HONGYING BIOTECHNOLOGY CO LTD
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