A low-temperature protease-producing Exiguobacterium siberia and its application

A low-temperature protease and microbacteria technology, applied in bacteria, microorganisms, microorganisms, etc., can solve the problems of poor thermal stability and poor low temperature adaptability, and achieve the effects of good thermal stability, expansion of application scope, and easy control of fermentation conditions.

Active Publication Date: 2022-07-05
QUFU NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the problems of poor low-temperature adaptability and poor thermal stability of medium and high-temperature proteases in the prior art, the present invention provides a low-temperature protease-producing Exobacterium siberia and its application. According to the screening, the optimum growth temperature and the optimum enzyme production temperature are close to the temperature in the natural environment, so the heating or cooling process can be omitted during the fermentation process, and the optimum reaction temperature of the protease produced by the strain is around 37°C , and has good catalytic activity between 0-60°C (more than 60% of the highest catalytic enzyme activity), low temperature adaptability and good thermal stability

Method used

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  • A low-temperature protease-producing Exiguobacterium siberia and its application
  • A low-temperature protease-producing Exiguobacterium siberia and its application
  • A low-temperature protease-producing Exiguobacterium siberia and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0028] The low-temperature protease-producing Exiguobacterium siberia of the present invention is derived from Antarctic krill, obtained after repeated screening, and is classified and named as Exiguobacterium siberia ( Exiguobacterium sibiricum ). The strain was deposited in the General Microbiology Center of the China Microbial Culture Collection Management Committee on July 14, 2020. The preservation address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No. 20358.

[0029] Strain screening: including sample collection, sample processing, primary screening, rescreening, determination of enzyme activity, identification of high-yielding strains, and strain preservation.

[0030] 1) Collect samples: buy Antarctic krill from a seafood market in Shandong.

[0031] 2) Sample treatment: Grind Antarctic krill with a mortar at low temperature until ground, weigh 3 g, add...

Embodiment 2

[0062] Low temperature protease enzyme activity assay

[0063] (1) Determination of enzyme activity

[0064] As a method for measuring the enzymatic activity of alkaline protease, the Folin-phenol colorimetric method is generally used. One unit of enzyme activity U This experiment sets the amount of enzyme required to hydrolyze casein to generate 1 μmol of tyrosine per minute at a constant temperature of 37 °C. Calculate the enzyme activity value according to the following formula

[0065] U=A*B*C / D

[0066] U protease activity value;

[0067] The difference between the absorbance value of the sample A and the blank absorbance value is compared to the tyrosine release amount after the standard curve (μg / mL);

[0068] B reaction magnification;

[0069] C enzyme dilution factor;

[0070] D reaction time.

[0071] Prepare the liquid culture medium shown in Table 1 of Example 1, put 100 mL of fermentation medium in a conical flask with a capacity of 250 mL, and sterilize it ...

Embodiment 3

[0077] Optimum enzyme activity assay

[0078] The fermentation and enzymatic treatment steps of Exiguobacterium siberia are the same as those in Example 2, the difference is that the temperature gradient (0-60°C, one reaction per 5°C) is set for 10 minutes, then 2ml of TCA is added, centrifuged for 1 minute, and sodium carbonate solution is added. and Folin phenol reagent, color development 10min, 660nm measurement value. The relative enzyme activities are shown in Table 6 below.

[0079] It was determined that the optimum enzyme activity temperature of this strain was 37°C, and there was also high activity at 0-60°C, indicating that this low-temperature protease was resistant to both high temperature and low temperature, indicating that the low-temperature protease had better thermal stability.

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Abstract

The invention discloses a low-temperature protease-producing Microbacterium siberia ( Exiguobacterium sibiricum ), preserved in the China General Microorganism Culture Collection and Management Center with the preservation number of CGMCC No.20358. The low-temperature protease-producing Exiguobacterium siberia was screened from Antarctic krill, and its optimum growth temperature and optimum enzyme-producing temperature are both close to the temperature in the natural environment, so the heating or cooling process can be omitted during the fermentation process. The optimal reaction temperature of the protease produced by this strain is about 37°C, and it has good catalytic activity (more than 60% of the highest catalytic enzyme activity) between 0-60°C, with low temperature adaptability and good thermal stability. sex.

Description

technical field [0001] The invention belongs to the field of microorganism screening and application, in particular to a low-temperature protease-producing Exiguobacterium siberia and its application. Background technique [0002] Enzymes generally refer to a class of enzymes that can catalyze the hydrolysis of peptide bonds at room temperature or even low temperature. Low-temperature protease refers to a class of proteolytic enzymes whose optimal catalytic temperature is below 40 °C, and can still maintain a high enzyme activity (above 50%) between 20 and 30 °C, because its optimal catalytic temperature is close to the natural environment. Therefore, the heating or cooling process can be omitted in the application process. Compared with medium and high temperature protease, it has the characteristics of energy saving and time saving, and has wide application prospects in the food and washing industry. The low-temperature protease-producing strains that have been found so f...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N9/52C12R1/01
CPCC12N9/52C12R2001/01C12N1/205
Inventor 杨革谷松鹤
Owner QUFU NORMAL UNIV
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