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Pseudomonas aeruginosa strain and application thereof in producing proteinase

A Pseudomonas aeruginosa, protease technology, applied in microorganism-based methods, bacteria, microorganisms, etc., to achieve the effects of strong protease ability, simple conditions, and stable genetic properties

Inactive Publication Date: 2016-05-04
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the Pseudomonas aeruginosa found to be used so far mainly comes from land, but there are few reports on strains that can produce proteases with high enzyme activity and high salt-tolerant stability from deep-sea mud.

Method used

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  • Pseudomonas aeruginosa strain and application thereof in producing proteinase
  • Pseudomonas aeruginosa strain and application thereof in producing proteinase
  • Pseudomonas aeruginosa strain and application thereof in producing proteinase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] The isolation, screening and identification of bacterial strain SWJSS3 comprises the following steps:

[0034] Enrichment: Weigh 1g of sea mud into a 50mL sterilized centrifuge tube with glass beads, add 10mL of 0.9% sterile normal saline, place in a shaker and culture for 30min to fully disperse, take 1mL of supernatant In enriched medium, culture at 37°C, 180r / min constant temperature for 24h.

[0035] Primary screening: Take 1 mL of the culture solution enriched twice and dilute it to 10 with 0.9% sterile saline -1 、10 -2 、10 -3 100 μL of each of the three concentrations was added to the solidified casein plate, spread evenly, and incubated upside down in a constant temperature incubator at 37°C for 24 hours. Pick a single colony with an obvious hydrolysis circle, streak it three times, inoculate it into the slant medium of the test tube, and store it in a refrigerator at 4°C.

[0036] Re-screening: activate the above-mentioned pure culture stored on the slant of...

Embodiment 2

[0053] The application of bacterial strain SWJSS3 in producing protease comprises the following steps:

[0054] (1) Optimizing the fermentation temperature. Using the above fermentation medium, the initial pH value is 7.5, and the liquid volume is 10%, under the conditions of 1% inoculum, after 48 hours of cultivation, the enzyme activity was measured by centrifugation, and the results were as follows: figure 1 , it can be seen from the figure that the strain has a certain amount of enzyme production at a temperature of 20°C to 40°C, but the enzyme production is the highest at 25°C.

[0055] (2) Fermentation time optimization, at 30°C, pH value is 7.5, liquid filling volume is 10%, fermentation under the condition of 180rpm, from 0-80h, continuous sampling every 6h, measure the biomass and protease activity in it with the fermentation time trend of change.

[0056] The result is as figure 2 , as can be seen from the figure: the strain has entered a stable period after 20 h...

Embodiment 3

[0058] Study on Genetic Stability of Strain SWJSS3

[0059] Strain SWJSS3 was continuously subcultured for 5 times, and its protease activity was measured as an index for evaluating genetic stability.

[0060] The results are shown in Table 4. The protease activity was maintained at about 750 U / ml after subcultured for 5 times, indicating that the protease had good genetic stability.

[0061] Table 4 strain passage enzyme activity

[0062]

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Abstract

The invention discloses a Pseudomonas aeruginosa strain SWJSS3 and application thereof in producing proteinase. The strain is collected by General Microorganism Center of China Committee for Culture Collection of Microorganisms on June 10th, 2015; the collection address is Institute of Microbiology in Chinese Academy of Sciences, Yard 1, Beichenxi Road, Chaoyang District, Beijing City; and the collection number is CGMCC No.10973. The application method of the strain for producing proteinase comprises the following steps: adding a fermentation culture medium into a fermentation tank, wherein the liquid filling amount is 8-12%, the initial pH value is 7.0-7.5, the strain inoculum size is 0.8-1.2%, and the initial temperature in the tank is 20-40 DEG C; and fermenting under oscillating conditions for 30-80 hours to generate abundant proteinase in the tank, and separating and extracting to obtain the proteinase. The strain SWJSS3 has high proteinase production capacity; and the obtained proteinase has the characteristics of high enzyme activity and high salt tolerance. The fermentation method disclosed by the invention has the advantages of simple conditions and stable hereditary property, and is suitable for industrial production.

Description

technical field [0001] The invention belongs to the field of industrial microorganisms, and in particular relates to a strain of Pseudomonas aeruginosa derived from deep sea mud and its application in the production of protease. Background technique [0002] The ocean is the birthplace of life, covering 70% of the earth's surface area and possessing 80% of the earth's biological resources. The ocean generally has the characteristics of high salinity, high pressure, oligotrophy, and low temperature. In order to adapt to these extreme environments, some metabolites and active substances formed by marine microorganisms during the long-term evolution process are somewhat different from those of terrestrial microorganisms. [0003] Microbial protease is a kind of hydrolytic enzyme characterized by hydrolyzing protein peptide bonds, which plays a very important role in human production and life. With the rapid development and wide application of genetic engineering and fermentati...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/52C12R1/385
CPCC12N9/52C12N1/205C12R2001/385C12N1/20
Inventor 赵谋明苏国万舒会赵强忠
Owner SOUTH CHINA UNIV OF TECH
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