Low temperature protease production strain, low temperature protease produced by the same and gene of enzyme

A low-temperature protease and bacteria-producing technology, applied in genetic engineering, plant genetic improvement, microorganism-based methods, etc., can solve problems such as poor stability, and achieve the effects of short fermentation cycle, fast growth, and easy control of fermentation conditions.

Inactive Publication Date: 2015-08-05
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the optimum catalytic temperatures of low-temperature proteases produced by these strains are between 30 and 40°C, and only a few of them have optimum catalytic temperatures lower than 20°C, but their stability is very poor.

Method used

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  • Low temperature protease production strain, low temperature protease produced by the same and gene of enzyme
  • Low temperature protease production strain, low temperature protease produced by the same and gene of enzyme
  • Low temperature protease production strain, low temperature protease produced by the same and gene of enzyme

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preparation example Construction

[0037] Five, the preparation method of low temperature protease crude product

[0038] The low-temperature protease produced by the strain is a secreted protein, which mainly exists in the supernatant of the fermentation broth. First, the method of filtration or centrifugation is used to obtain the supernatant or filtrate from the culture medium, and then the method of salting out, conventional desalination, freeze-drying or spray-drying is used to obtain the crude low-temperature protease.

Embodiment 1

[0041] Fermentation of Low Temperature Protease-Producing Bacillus Motility Coccus TCCC11813

[0042] ⑴ Inoculate the slant of the fermentation strain into 50mL (250mL Erlenmeyer flask) 2216E liquid medium (5g peptone, 1g yeast extract, 0.01g ferric phosphate, 1000ml seawater, pH value 7.6-7.8), 20°C, 200rpm shaking culture 24 hours,

[0043] (2) Then inoculate 50mL (500mL Erlenmeyer flask) fermentation medium (5g of peptone, 1g of yeast extract, 1000mL of seawater, pH value 7.0-8.0) at a volume ratio of 2%, respectively, at different temperatures (15°C, 20°C, 25°C, ℃), shake culture at 200r / min for 72h, take samples regularly, and measure the enzyme activity.

[0044] The results showed that the optimum growth temperature of the strain was consistent with the optimum enzyme production temperature, both at about 20°C + 5°C, and the enzyme production peak could be reached in about 48 hours of fermentation culture, and the enzyme production amount was about 280U / mL.

Embodiment 2

[0046] Low temperature protease activity assay method

[0047] Take 1mL of appropriately diluted enzyme solution (50mmol / L Tris.HCl, pH8.0), incubate at 37°C for 2min, add the substrate at the same temperature (prepared with 50mmol / L Tris.HCl, pH8.0 buffer 1.0% casein) 1 mL, reacted at 37° C. for 10 min, and added 2 mL of 10% trichloroacetic acid to terminate the reaction. Centrifuge statically, take 1mL supernatant, add 5mL0.4mol / LNa 2 CO 3 Solution, 1mL Folin’s phenol reagent, mix well, keep warm at 40°C for 20min, measure the absorbance A 680 . The inactivated enzyme solution reaction system was used as a blank control.

[0048] The enzyme activity unit is defined as: per milliliter of enzyme solution, under the conditions of 37°C and pH 8.0, the amount of enzyme required to produce 1 μg of tyrosine per minute is 1 enzyme activity unit (U / mL)

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Abstract

The invention relates to a low temperature protease production strain, low temperature protease produced by the same and a gene of the enzyme. A strain of planococcussp. producing low temperature protease is separated from the southern Indian Ocean deep-sea sediment for the first time, the preservation number of the strain is CGMCC No.8088, the optimum growth temperature and the optimum enzyme production temperature are both 20DEG C, and the fermentation level protease yield can reach 280U/mL. The optimum operation temperature of the protease produced by the strain is 30-37DEG C, the optimum pH is 7.5-9.0, under the condition of 30DEG C, over 80% of the highest catalytic activity can be maintained, and in the range of 20-25DEG C, the protease still has high catalytic activity. The protease produced by the strain belongs to typical low temperature alkaline protease and has good catalytic activity at normal temperature, thus having high research value in food processing industry, cold water detergent development and other aspects.

Description

technical field [0001] The invention relates to the field of microorganisms and microbial enzyme preparations, in particular to a strain of low-temperature protease-producing bacteria derived from deep-sea sediments, the protease produced and a method for producing low-temperature protease by using the strain. Background technique [0002] Low-temperature protease refers to a class of proteolytic enzymes whose optimum catalytic temperature is below 40°C and which can still maintain high enzyme activity (more than 50%) between 20 and 30°C. Because its optimum catalytic temperature is close to that in the natural environment Therefore, the heating or cooling process can be omitted in the application process. Compared with the medium and high temperature protease, it has the characteristics of energy saving and time saving, and has broad application prospects in the food and washing industries. Most low-temperature proteases are derived from low-temperature-resistant or low-tem...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N15/57C12N9/52C12R1/01
CPCC12N9/52C12N1/205C12R2001/01
Inventor 张会图路福平莫清珊
Owner TIANJIN UNIV OF SCI & TECH
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