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Pseudomonas of high yield lipase and enzyme production method by fermentation of pseudomonas

A technology of pseudomonas and fermentation method, which is applied in the field of microorganisms to achieve the effects of reducing fermentation costs, increasing fermentation production capacity, and improving production efficiency

Active Publication Date: 2017-12-19
SHANDONG LONGKETE ENZYME PREPARATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the optimal reaction temperature of lipase in the prior art is generally 40°C or higher, and the inactivation is severe below 30°C. Therefore, subject to the restriction of enzyme activity and applicable conditions of the enzyme, the lipase in the prior art still cannot meet the requirements. Therefore, a high-yield lipase-producing Pseudomonas strain was selected. The fermented product has good alkali resistance and low temperature resistance, and the production cost is low. It is of great significance to promote the mechanization of various industries on a large scale.

Method used

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  • Pseudomonas of high yield lipase and enzyme production method by fermentation of pseudomonas
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  • Pseudomonas of high yield lipase and enzyme production method by fermentation of pseudomonas

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Mutagenesis and breeding of Pseudomonas LD-14

[0041] Take two fresh slopes of the starting strain LA-32, wash the bacteria with sterile water, shake them in a test tube with glass beads to disperse the bacteria, collect the bacteria by centrifugation, resuspend the bacteria with 5% glycerol, and count the cells Count the plate until the bacteria concentration is 10 7 -10 8 Pcs / mL, as the starting bacteria suspension.

[0042] Turn on the normal temperature and normal pressure plasma system, wipe the operation room and outside with alcohol cotton, and turn on the ultraviolet lamp for sterilization for 30 minutes. After the sterilization in the operating room of the system is completed, 10 μL of bacterial suspension is spotted on the rough surface of the slide, and the slide is transferred to the operating room table with tweezers under aseptic conditions. Open the helium valve, set the gas flow rate and mutagenesis time for mutagenesis. The mutagenesis time was ...

Embodiment 2

[0054] Example 2 Pseudomonas LD-14 fermentation and enzyme production

[0055] The fermentation method of Pseudomonas LD-14 mainly includes the following steps:

[0056] Slope culture: Pick a loop of Pseudomonas LD-14 and inoculate it on a solid slant medium, culture it at 35°C for 36 hours to obtain first-class seeds;

[0057] Shake flask culture: Take a ring of first-level seeds into the seed culture medium, culture for 48h at a constant temperature of 35°C and a shaker speed of 200r / min to obtain a second-level seed solution;

[0058] Seed pot culture: Put the secondary seed liquid into the seed pot culture medium at a ratio of 6% (v / v) of the inoculum, and cultivate for 12h at a constant temperature of 35°C and a rotation speed of 200r / min;

[0059] Fermentation tank culture: The seed liquid in the seed tank is connected to the fermentation tank culture medium according to the inoculation amount 6% (v / v), at a constant temperature of 35°C, the rotation speed is 200r / min, and the ven...

Embodiment 3

[0070] Example 3 Verification of Pseudomonas LD-14 fermentation performance

[0071] The 50L fermentor verification experiment was carried out according to the enzyme production method of Pseudomonas LD-14 in Example 2. The fermentation period was 150h, and the enzyme production of 6 batches of fermentation, the average enzyme production level was 25558U / mL, Table 2 shows the strain Not only high-yield lipase, but also its fermentation performance and the enzyme activity of the lipase produced by it have remarkable stability.

[0072] Table 2 Fermentation of 6 batches of high-producing lipase strains

[0073] batch

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Abstract

The invention belongs to the technical field of microorganisms and particularly relates to particularly relates to a pseudomonas of high yield lipase and an enzyme production method by fermentation of the pseudomonas. The pseudomonas is particularly pseudomonas sp. LD-14 and has a strain collection number of CGMCC (China General Microbiological Culture Collection Center) No. 14414. Enzyme activity of fermentation liquid of the lipase produced by fermentation of the pseudomonas sp. LD-14 reaches 25000-26000U / mL; the most suitable pH (potential of hydrogen) scope of the produced lipase is 8.5-10.0; most suitable operative temperature scope is 30-40 DEG C; the residual enzyme activity is 80% after heat preservation at 20 DEG C for 1d; and the lipase has significant low temperature resistance and alkali resistance and can be widely applied in industrial production.

Description

Technical field: [0001] The invention belongs to the technical field of microorganisms, and specifically relates to a strain of Pseudomonas with high lipase production and a method for producing enzyme by fermentation. Background technique: [0002] Lipase (Triacylglycerol acylhydrolases, EC 3.1.1.3) is a general term for a class of enzymes that can hydrolyze triglycerides at the oil-water interface. It can hydrolyze triacylglycerides into fatty acids, diglycerols, monoglycerides and glycerol. The natural substrate is generally water-insoluble long-chain fatty acid acyl ester. [0003] Lipases are mainly derived from plants, animals, and microorganisms. Among them, microbial lipases are widely found in bacteria, yeasts and molds. They have the characteristics of many types, short cycles, fast reproduction, and prone to genetic variation. They are more than animal and plant lipases. With a wide range of temperature, pH and substrate specificity, it can catalyze the hydrolysis, alco...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/20C12R1/38
CPCC12N9/20C12Y301/01003C12N1/205C12R2001/38
Inventor 王兴吉郭庆文刘文龙曹世源佟新伟
Owner SHANDONG LONGKETE ENZYME PREPARATION
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