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A strain of Pseudomonas with high lipase production and method for fermenting and producing enzyme

A technology of Pseudomonas and lipase, which is applied in the field of microorganisms, to achieve the effects of reducing fermentation costs, increasing fermentation production capacity, and increasing application value

Active Publication Date: 2020-01-14
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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  • A strain of Pseudomonas with high lipase production and method for fermenting and producing enzyme
  • A strain of Pseudomonas with high lipase production and method for fermenting and producing enzyme
  • A strain of Pseudomonas with high lipase production and method for fermenting and producing enzyme

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Experimental program
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Effect test

Embodiment 1

[0040] Example 1 Mutation Breeding of Pseudomonas LD-14

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

[0042] Turn on the plasma system at normal temperature and pressure, wipe the inside and outside of the operating room with alcohol cotton, and turn on the ultraviolet lamp to sterilize for 30 minutes. After the sterilization in the operating room of the system is completed, take 10 μL of the bacterial suspension and spot it on the rough surface of the slide, and transfer the slide to the table of the operating room with tweezers under aseptic conditions. Open the helium valve, set the gas flow and mutagenesis time for mutagene...

Embodiment 2

[0054] Example 2 Fermentation of Pseudomonas LD-14 to produce enzyme

[0055] The fermentative enzyme production method of Pseudomonas LD-14 mainly comprises the following steps:

[0056] Slant culture: pick a ring of Pseudomonas LD-14 and inoculate it on a solid slant medium, and culture at a constant temperature of 35°C for 36 hours to obtain first-grade seeds;

[0057] Shake flask culture: take a ring of the first-grade seeds and insert them into the seed medium, and cultivate them for 48 hours at a constant temperature of 35°C and a shaker speed of 200r / min to obtain the second-grade seed liquid;

[0058] Seed tank culture: put the secondary seed solution into the seed tank culture medium at a ratio of 6% (v / v) of the inoculum, and cultivate for 12 hours at a constant temperature of 35°C and a rotational speed of 200r / min;

[0059] Fermentation tank culture: The seed liquid in the seed tank is inserted into the culture medium of the fermenter according to the ratio of the...

Embodiment 3

[0070] Example 3 Pseudomonas LD-14 fermentation performance verification

[0071] Carry out 50L fermenter verification experiment according to the fermentative enzyme production method of Pseudomonas LD-14 of embodiment 2, fermentation cycle 150h, its 6 batches of fermentative enzyme production situation, average enzyme production level is 25558U / mL, table 2 illustrates bacterial strain Not only high-yield lipase, but also the fermentation performance and the enzymatic activity of the lipase produced by it have remarkable stability.

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

[0073] batch Fermentation period (h) Fermentation activity (U / mL) 1 150 25825 2 150 25560 3 150 25025 4 150 25590 5 150 25400 6 150 25950

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Abstract

The invention belongs to the technical field of microbes, and in particular relates to a high-yield lipase-producing pseudomonas strain and a fermentative enzyme production method thereof. The Pseudomonas is specifically Pseudomonas sp. LD-14, and the strain preservation number is CGMCC No.14414. Pseudomonas LD‑14 fermented to produce lipase, the enzyme activity of the fermentation broth reached 25000U / mL to 26000U / mL; the optimum pH range of the lipase produced was 8.5‑10.0, and the optimum action temperature range was 30°C‑40°C, After being incubated at 20°C for 1 day, the remaining enzyme activity is 80%, has remarkable low temperature resistance and alkali resistance, and can be widely used in industrial production.

Description

Technical field: [0001] The invention belongs to the technical field of microbes, and in particular relates to a high-yield lipase-producing pseudomonas strain and a fermentative enzyme production method thereof. 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. Its natural substrates are generally water-insoluble long-chain fatty acid acyl esters. [0003] Lipases are mainly derived from plants, animals, and microorganisms. Among them, microbial lipases are widely found in bacteria, yeasts, and molds. Wide range of action temperature, action pH and substrate specificity, it can catalyze the hydrolysis, alcoholysis, acidolysis, transesterification and synthesis of ester compounds without coenzymes, with mild catalytic conditions, low energy consumption, and side effects The product is less, and it has the characteristics of high efficiency,...

Claims

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Application Information

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Patent Type & Authority Patents(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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