Method for producing high temperature resistant neutral protease

A technology of neutral protease and high temperature resistance, applied in the field of bioengineering, can solve the problems of poor thermal stability, restriction of industrial production and wide application, and achieve stable performance, high activity of neutral protease, and low manufacturing cost

Active Publication Date: 2020-01-31
SHANDONG LONGKETE ENZYME PREPARATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Among them, neutral protease has mild action conditions and can be used in pharmaceutical, food, leather, textile

Method used

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  • Method for producing high temperature resistant neutral protease
  • Method for producing high temperature resistant neutral protease
  • Method for producing high temperature resistant neutral protease

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The mutagenesis selection of embodiment 1 bacterial strain

[0036] 1. Microwave mutagenesis: take 5mL of the bacterial suspension grown to the logarithmic growth phase in a test tube, put the test tube in a beaker containing ice cubes, use a Midea microwave oven with a frequency of 2450MHz, and an output power of 700W, according to different time The test tubes were irradiated one by one with a dilution gradient of 10 -1 ~10 -6 . Bacterial solutions treated for different times were taken and diluted respectively to obtain 3 gradients (10 -4 ~10 -6 Interval) 0.1mL of bacterial solution, spread on the screening plate medium, culture at 30°C for 16h, count the number of colonies, and draw the lethality curve. Pick a single colony with a larger transparent circle and inoculate it into a seed bottle, and then measure the neptase activity of each strain by liquid medium. Select a strain with high neutral protease production and stable inheritance for more than 3 generat...

Embodiment 2

[0043] The mutant strain was continuously cultured for 10 generations, the experimental results can be seen from Table 1, the genetic stability of the mutant strain is good. Embodiment 2 neutral protease activity assay method

[0044] 1. Definition of enzyme activity: 1 g of solid enzyme powder (or 1 mL of liquid enzyme), under certain temperature and pH conditions, hydrolyzes casein for 1 min to produce 1 μg of tyrosine, which is 1 enzyme activity unit, expressed in U / g ( U / mL) expressed.

[0045] 2. Draw the standard curve:

[0046] Prepare a series of L-tyrosine standard solutions (0, 10, 20, 30, 40, 50μg / mL) with different concentrations, take 1.00mL respectively (parallel experiments are required), add 5mL of 0.4mol / L sodium carbonate solution to each , 1 mL of Folin reagent solution, put it in a water bath at 40±0.2°C for 20 minutes, take it out, measure its absorbance with a spectrophotometer at a wavelength of 680nm, and use the "0" tube without tyrosine as the blank...

Embodiment 3

[0064] Embodiment 3 bacterial strain SJ-215 liquid fermentation production neutral protease and its extraction

[0065] 1. Cultivation of strains

[0066] The composition of medium mass volume percentage is as follows:

[0067] a. Plate separation medium: glucose 2%, skimmed milk powder 1%, sodium chloride 2%, yeast powder 1%, agar 1.5-2.0%. b. Seed bottle medium: glucose 2%, peptone 5%, sodium chloride 1%, beef extract 5%, pH 7.0.

[0068] c. Fermentation shake flask culture medium: 8% corn starch, 4% corn flour, 5% cottonseed protein, 0.05% calcium chloride, 0.1% magnesium sulfate, 4.5% corn steep liquor, adjusted to pH 6.3 after liquefaction.

[0069] d. Culture conditions

[0070] Separation plate: culture at 30°C for 16 hours; Seed bottle: culture at 30°C for 5-6 hours, shaker speed 240r / min; Fermentation shaker flask: culture at 30°C for 72h, shaker speed 240r / min.

[0071] 2. Expansion of seed tank cultivation

[0072] The composition of medium mass volume percenta...

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Abstract

The present invention belongs to the technical field of biological engineering and particularly relates to a method for producing high temperature resistant neutral protease. A production strain usedin the method is specifically bacillus subtilis SJ-215 and has a deposit number of CGMCC No.18645. At the same time, the present invention also optimizes a corresponding fermentation mechanism. The fermentation mechanism has higher fermentation vigor, higher extraction yield, lower manufacturing cost, and average fermentation enzyme activity above 65,000 U/mL. The produced neutral protease is highin enzyme activity, good in thermal stability and stable in performance, and has a wide range of application prospects.

Description

Technical field: [0001] The invention belongs to the technical field of bioengineering, in particular to a method for producing high-temperature-resistant neutral protease. Background technique: [0002] Protease is a class of enzymes that catalyze the hydrolysis of peptide bonds, which can hydrolyze macromolecular proteins into products such as amino acids and small peptides. According to their way of hydrolyzing polypeptides, they can be divided into two types: endopeptidases and exopeptidases. Exopeptidase hydrolyzes the peptide bonds one by one from the end of the free amino acid or carboxyl group of the protein molecule, and then releases the amino acid. The former is called aminopeptidase and the latter is called carboxypeptidase. According to its active center, it can be divided into sulfhydryl protease, serine protease, aspartic acid protease and metalloprotease. According to the optimum pH value of its reaction, it can be divided into acid protease, neutral protea...

Claims

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Application Information

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IPC IPC(8): C12N9/50C12N1/20C12R1/125
CPCC12N1/20C12N9/50C12N1/205C12R2001/125
Inventor 钱娟娟刘文龙王克芬王帅王兴吉王金余张杰
Owner SHANDONG LONGKETE ENZYME PREPARATION
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