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A thermostable neutral protease

A neutral protease and stability technology, applied in the field of neutral protease, can solve the problems of poor thermal stability of neutral protease, and achieve the effect of wide pH value range, enhanced effect and high storage stability

Active Publication Date: 2016-01-20
湖南康捷生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The technical problem solved by the present invention is to overcome the defect of poor thermostability of the existing neutral protease, and use Bacillus subtilis (Bacillus subtilis) 1398-2-12, which produces strong thermostable neutral protease, as the starting strain, and optimize the culture medium And the fermentation process is improved, and a kind of neutral protease with strong thermal stability and high enzyme activity is prepared through liquid submerged mixed fermentation under specific fermentation conditions

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] The mensuration of embodiment 1 neutral protease activity

[0046] (1) Definition of enzyme activity unit: 1 mL of crude enzyme solution, under the conditions of 70°C and pH 7.2, decomposes casein for 1 minute to produce 1 μg of tyrosine, which is defined as 1 enzyme activity unit (1U / ml).

[0047] (2) Enzyme activity assay method: spectrophotometric method

[0048] ① Take 5 test tubes, numbered 1, 2, 3, 4, 5, and add 2ml of casein solution respectively;

[0049] ②Add 1ml of the crude enzyme solution to be tested to test tubes 1, 2, and 3, and react in a water bath at 70°C for 10 minutes;

[0050] ③ Add 1ml of the crude enzyme solution to be tested into test tubes 4 and 5;

[0051] ④Add 2ml of trichloroacetic acid (TCA) solution with a concentration of 0.4mol / L to each of the 5 test tubes, shake well and then bathe in boiling water for 5 minutes;

[0052] ⑤ After cooling in a water bath, measure the absorbance of No. 1, No. 2 and No. 3 tubes at 660 nm with No. 4 and ...

Embodiment 2

[0055] A preparation method for strong thermostable neutral protease, comprising the following steps:

[0056] (1) Activation of strains

[0057] The well-preserved slant strain of Bacillus subtilis 1398-2-12 was inoculated on the slant medium, cultured at 33°C for 30 hours to activate the strain, and activated twice in this way;

[0058] The slant medium consists of: beef extract 6g, sodium chloride 8g, peptone 15g, glucose 4g, (NH) 2 SO 4 4g,K 2 HPO 4 7g, CaCl 2 2g, 18g agar, 8g Chinese herbal powder, 1000mL distilled water, pH 7.2, sterilized at 121°C for 20min;

[0059] The preparation method of described Chinese herbal medicine powder is as follows:

[0060] Weigh 25 parts of Radix Astragali; 16 parts of Codonopsis; 12 parts of Bupleurum; Keep it for 3 hours, then lower the temperature to 50°C, add a mixed enzyme preparation of 8% of the total weight of the mixed material for enzymolysis, adjust the pH value to 6.0 with lactic acid, and perform enzymatic hydrolysis...

Embodiment 3

[0083] Embodiment 3: the thermostability analysis of neutral protease

[0084] The crude enzyme solution of Example 2 was respectively placed at 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, and 75°C, and samples were taken every 10 minutes to determine the enzyme activity. At 40°C, 45°C, 50°C, and 55°C in the crude enzyme solution, the enzyme activity did not decrease for 60 minutes. At 60°C and 65°C, the original enzyme activity is reduced to 95% in 30 minutes, and to 85% in 60 minutes. At 70°C, it will drop to 85% of the original enzyme activity in 30 minutes and 80% in 60 minutes. At 75°C, the enzyme activity will be reduced to 80% of the original enzyme activity in 30 minutes, and to 70% in 60 minutes.

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Abstract

The invention discloses a neutral protease with strong thermal stability and belongs to the preparation technology field of enzymic preparation. The neutral protease with strong thermal stability and high enzyme activity is prepared by using bacillus subtilis 1398-2-12 for generating the neutral protease with strong thermal stability as an original strain to optimize a culture medium and improve a fermenting process, and carrying out submerged mixing fermentation of a liquid. The enzyme activity of a fermenting liquid is 5500-7000U / ml; the optimal reaction temperature is 70 DEG C; the enzyme activity is completely stable when the pH value is 4.5-8.5; the pH value of the optimal reaction is 7.2; more than 80% of the enzyme activity is kept after preserving for 1 hour at the temperature of 70 DEG C, and more than 70% of the enzyme activity is kept after preserving for 1 hour at the temperature of 75 DEG C. Compared with the existing neutral protease, the neutral protease has stronger thermal stability, high thermal stability, wide enzyme action optimal pH value range and high storage stability, and is especially suitable for industrial requirements of a coexisted process of proteolysis, saccharification and liquidation at high reaction temperature.

Description

technical field [0001] The invention belongs to the technical field of enzyme preparation, in particular to a neutral protease with strong thermostability. 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 the protein molecule to...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/56C14C1/08C12R1/125
CPCC12N9/54
Inventor 李海清胡永明辜承兰周玉明封章平刘德凤
Owner 湖南康捷生物科技有限公司
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