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Method for preparing alkaline protease by fermentation method

A technology of protease and fermentation method, applied in the field of microorganisms, can solve the problems that the enzymatic properties of alkaline protease cannot meet the requirements of the practical application field, insufficient screening, etc., and achieve the effects of easy large-scale production, simple operation, and high product yield

Inactive Publication Date: 2013-09-11
BEIJING RENFENG TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the activity level of alkaline protease researched and produced in our country is constantly improving, the screening of alkaline protease starting strains in our country is still not enough, and the enzymatic properties of alkaline protease still cannot meet the requirements of practical application fields. , especially in the field of food applications, most of them use imported alkaline protease preparations, and large-scale industrial alkaline proteases also mainly rely on imports

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1 Fermentation method produces a kind of method of alkaline protease, carries out successively according to the following steps:

[0026] 1. Screening of high-yield strains

[0027] Insert the SVF176 strain that was screened out into solid slant medium, and after cultivating at 30°C for a period of time, wash the bacterial lawn with normal saline to make a bacterial suspension, the number of which is about 10 8pieces / ml. Take 6ml of the bacterial suspension and put it into a petri dish with a diameter of 9cm, and irradiate it with an ultraviolet lamp (20w, distance 30cm) in the dark for 20s, 30s, 40s, 50s, 60s respectively. NTG was added to the bacterial suspension to make the final concentrations 40, 100, 200, and 400 g / mL, respectively, treated in a 37°C water bath for 30 minutes, shaken intermittently, and centrifuged to remove the supernatant containing nitrosoguanidine. The treated bacterial solution was diluted and spread on the casein plate separatio...

Embodiment 2

[0038] Embodiment 2 Fermentation method produces a kind of method of alkaline protease, carries out successively according to the following steps:

[0039] 1 with embodiment 1.

[0040] 2. Fermentation in 50L fermenter

[0041] The bacterial strains were picked to prepare 0.1 mL of bacterial suspension, and the seeds were gradually expanded and cultivated, and transferred to a 50 L fermenter for fermentation according to 10% inoculum amount. The composition of the fermentation medium is: cottonseed cake powder (100 mesh) 5%, yeast extract powder 1.00%, maltodextrin (DE=30%) 10%, sodium citrate 0.1%, CaCl 2 0.1%, K 2 HPO 4 1.5%. The fermentation conditions are: loading coefficient 0.50, temperature 30°C, rotation speed 800r / min, ventilation rate 1:2.0, and dissolved oxygen maintained at 35%.

[0042] 3. Extraction and purification

[0043] ① Fermentation broth pretreatment: The fermentation broth was pre-cooled at 4°C, centrifuged at 5000r / min for 20min, and the superna...

Embodiment 3

[0050] Embodiment 3 Fermentation method produces a kind of method of alkaline protease, carries out successively according to the following steps:

[0051] 1 with embodiment 1.

[0052] 2. Fermentation in 50L fermenter

[0053] Pick the strains to prepare 0.1mL bacterial suspension, expand the cultivation step by step through the seeds, and transfer to a 50L fermenter for fermentation according to 15% inoculum amount. The composition of the fermentation medium is: cottonseed cake powder (80 mesh) 1%, yeast extract powder 1.75%, maltodextrin (DE=30%) 15%, sodium citrate 0.5%, CaCl 2 0.5%, K 2 HPO 4 1.0%. The fermentation conditions are: loading coefficient 0.80, temperature 37°C, rotation speed 600r / min, ventilation rate 1:0.5, dissolved oxygen maintained at 40%.

[0054] 3. Extraction and purification

[0055] ① Fermentation broth pretreatment: The fermentation broth was pre-cooled at 4°C, centrifuged at 5000r / min for 20min, and the supernatant was taken to obtain the ...

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PUM

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Abstract

The invention provides a method for preparing an alkaline protease by a fermentation method. The method comprises the following steps of: obtaining a high-yield bacillus alcalophilus strain Svf4-21-7 stable in heritability through ultraviolet-NET compound mutation treatment; carrying out the following treatment steps on the fermentation broth: salting out by using ammonium sulfate, dialysis and desalination, ion-exchange column chromatography, polyethylene glycol embedment and enrichment and gel filtering chromatography, thus obtaining the alkaline protease having activity greater than or equal to 100000 u / g after being freeze-dried. The alkaline protease prepared by the method provided by the invention is high in yield, and the preparation process is relatively simple; therefore, the alkaline protease is suitable for industrial production.

Description

technical field [0001] The invention relates to the mutagenesis and breeding of a high-yield alkaline protease strain and a method for preparing alkaline protease by fermenting the strain, and relates to the field of microorganisms. Background technique [0002] Alkaline protease (Alkaline Protease) refers to the enzyme that hydrolyzes the peptide bond of protein under the condition of alkaline pH value, and its optimum pH value is generally 9-11. In addition to catalyzing the hydrolysis of proteins into amino acids, alkaline protease can also catalyze the synthesis of polypeptides in organic solvents. [0003] Alkaline protease was first found in the pancreas of pigs. In 1913, Rohm first used trypsin as a washing soak. In 1945, Dr. Jaag in Switzerland discovered microbial alkaline protease (Pose A H, 1980), which made it possible for protease to be widely used in the detergent industry. In 1963, Novo Nordisk (now Novozymes) discovered the alkaline protease Alcalase, whic...

Claims

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Application Information

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IPC IPC(8): C12N9/54C12N13/00C12N15/01C12N1/20C12R1/07
Inventor 王鹏丛倩千
Owner BEIJING RENFENG TECH
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