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Method for sifting and cultivating bacillus thuringiensis of high-yield thermostable proteinase

A technique for screening Bacillus aureus and a screening method, which is applied in the field of screening and culturing high-yield, high-temperature resistant protease Bacillus thuringiensis, which can solve the problems of heavy workload and long cycle, and achieve the effect of wide application

Active Publication Date: 2008-04-16
LIFECOME BIOCHEM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Usually the fermentation optimization process requires a long cycle and a large workload

Method used

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  • Method for sifting and cultivating bacillus thuringiensis of high-yield thermostable proteinase
  • Method for sifting and cultivating bacillus thuringiensis of high-yield thermostable proteinase
  • Method for sifting and cultivating bacillus thuringiensis of high-yield thermostable proteinase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment one: a kind of Bacillus thuringiensis screening method of high-yielding thermostable protease comprises the following steps:

[0040] Production of the casein substrate agar identification plate: the agar identification plate formula (weight percentage) is 2% casein, 2% agar, the rest is water, pH is 7.0-7.2, and is prepared into an agar plate with a thickness of 3mm.

[0041] Primary screening 1: Separating 34 strains of Bt strains on a flat plate, after the growth of sparse small colonies, use a puncher to take out a small piece of agar with a single colony, take 3 agar pieces for each strain, and place the 34 agar pieces in the On the same identification plate, a total of three identification plates were placed at 37 ° C for 24 hours. According to the large proteolytic circle formed around the agar block, the strain with high transparency was a high-yielding strain.

[0042] Preliminary screening 2: use a hole puncher to punch 34 holes of 1.5 mm in size on...

Embodiment 2

[0045]Embodiment two: Bt140 produces enzyme fermented liquid preparation

[0046] Bt140 fermentation broth preparation, including strain selection, slant culture, primary seeds, slant medium is beef extract peptone medium. The Bt140 slant culture was placed at 37°C, and after 2 days of culture, it was stored in a refrigerator at 4°C. Connect a loop from the inclined plane to a 250mL Erlenmeyer flask, the medium capacity is 35mL / 250mL Erlenmeyer flask, the medium is beef extract peptone, under the condition of 31°C, place it on a shaker at 250r / min, cultivate it for 15h, and obtain a grade seeds. By counting the primary seeds, the inoculum size of the controlled fermentation was 0.75mL (bacteria suspension concentration 7×10 6 Individual / mL), the fermentation medium formula (percentage by weight) is: soybean cake powder 1.8%, yeast powder 0.36%, glucose 0.14%, KH 2 PO 4 0.25%, MgSO 4 ·7H 2 O 0.035%, CaCO 3 0.05%, the rest is water. The culture conditions for fermentati...

Embodiment 3

[0047] Embodiment three: Utilize Bt140 to produce enzyme fermented liquid to prepare thermostable protease and Bt insecticide

[0048] 1. Preparation of Bt insecticide: Take 1000L of Bt140 enzyme-producing fermentation broth as described in Example 1, adjust the pH to 5.0, and add flocculant Al in a ratio of 3% under stirring at 50r / min 2 (SO 4 ) 3 .18H 2 030kg, after precipitation 2h, adopt butterfly centrifuge, centrifuge speed is 8000r / min, centrifugal operation 15-20min, carry out bacterial liquid separation, obtain wet thalline and remove the enzyme-producing fermented liquid after thalline. The wet bacteria were collected and vacuum-dried for 8 hours, and crushed to obtain Bt insecticide; the enzyme-producing fermentation liquid after removing the bacteria was used to prepare high-temperature-resistant protease.

[0049] 2. Preparation of food-grade high-temperature-resistant protease: take 1000L of the enzyme-producing fermentation liquid after removing the bacteria,...

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Abstract

A screening and cultivation method for Bacillus thuringiensis of high-yield high temperature resisting protease includes the following steps: high-yield high temperature resisting protease Bacillus thuringiensis Bt140 is obtained through adopting casein substrate agar to identify flat primary screen and fermenting enzyme activity measuring secondary screening; Plackett-Burman design and response surface analytical method are adopted to obtain optimal fermentation zymogenic formula and fermentation condition for Bt140 quickly; finally, shaking fermentation level reaches to 918.56U / mL. During adopting Bt140 to produce high temperature resisting protease, spore collection for producing pesticide after fermentation is not influenced; therefore, the Bacillus thuringiensis combines the production capacity of protease and pesticide. For example, when 1 ton Bacillus thuringiensis is used for fermentation, corresponding Bt pesticide is obtained along with 60 to 90 Kg / megaunit enzyme powder.

Description

[0001] Technical field The present invention relates to a method for screening and cultivating high-yielding protease strains, in particular to a method for screening and cultivating high-yielding thermostable protease Bacillus thuringiensis. Background technique [0002] Protease is an important enzyme preparation widely used in food, medicine, detergent, textile and leather industries. High temperature resistant protease has the advantages of heat resistance, denaturant resistance, organic solvent resistance, etc., and its research is particularly important. People have isolated various types of high-temperature proteases from high-temperature bacteria such as Thermus aquatics, Bacillusthermoproteolyticus, B. stearothermophilus, Thermoactinomycesvulgaris, Sulfolobus solfataricus, and Pyrococus furiosus. At present, the output of high-temperature proteobacteria is low and the production cost is high. Bacillus thuringiensis (Bt) is a very important entomopathogenic bacterium ...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N9/56A01N63/02C12R1/125
Inventor 关雄郑毅黄志鹏黄必旺黄天培沙莉李今煜张群林吴昌标余洁张灵玲
Owner LIFECOME BIOCHEM
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