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Strain for producing organic solvent tolerant lipase, organic solvent tolerant lipase and preparation method of organic solvent tolerant lipase

A technology of lipase and strain, applied in biological field

Active Publication Date: 2013-07-03
WILMAR SHANGHAI BIOTECH RES & DEV CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] At present, there is no relevant report on lipase derived from Cladosporium that is resistant to organic solvents, especially methanol, ethanol, etc.

Method used

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  • Strain for producing organic solvent tolerant lipase, organic solvent tolerant lipase and preparation method of organic solvent tolerant lipase
  • Strain for producing organic solvent tolerant lipase, organic solvent tolerant lipase and preparation method of organic solvent tolerant lipase
  • Strain for producing organic solvent tolerant lipase, organic solvent tolerant lipase and preparation method of organic solvent tolerant lipase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Example 1: Isolation, screening and identification of lipase-producing filamentous fungus Cladosporium sp.WBRD3.10062425

[0072] Take a piece of Tibetan ghee sample as fresh as possible, remove about 1cm thick surface layer, cut about 10g of Tibetan ghee sample and place it in 100mL sterile water containing 1.0% Tween 80. After shaking for 30 minutes, it was diluted to different dilutions according to the traditional dilution coating method, and the samples of each dilution were coated on Rhodamine B plates and placed in a 28°C biochemical incubator for cultivation.

[0073] The making method of the plate of rhodamine B is: formula A solution (ammonium sulfate 0.1%, yeast extract juice 0.5%, tryptone 0.5%, K 2 HPO 4 0.1%, KCl 0.5%, magnesium sulfate heptahydrate 0.05%, ferrous sulfate heptahydrate 0.01%, agar 2.0%) sterilized at 115°C for 30min, and then added formula B solution sterilized in the same way (olive oil and 2% polyvinyl alcohol Mix at a ratio of 1:3, sti...

Embodiment 2

[0081] Embodiment 2: Preparation of organic solvent-resistant lipase derived from Cladosporium

[0082] (1) Fermentation of Cladosporium lipase

[0083] Add about 10 mL of sterile normal saline to a freshly cultured Cladosporium WBRD3.10062425 slant to prepare a spore suspension, and then take 1 mL of the suspension and inoculate it into a 250-mL seed medium with a volume of 30 mL. Cultivate at 28°C and 200rpm for 24h (medium composition: 3% glucose, 0.3% peptone, 0.5% corn flour, NaH 2 PO 4 12H 2 O 0.2%, MgSO 4 12H 2 O 0.1%, CaCl 2 0.05%, pH 6.5).

[0084] After the seeds have been cultured, 3 mL of the seed culture solution is inoculated into multiple 250 mL volume fermentation shake flasks, and the amount of liquid in the shake flasks is 30 mL. The composition of the fermentation medium is: 2% sucrose, 3% peptone, 0.5% corn flour, 1% yeast extract powder, 0.5% Tween-800.5%, 0.1% talcum powder, NaNO 3 0.3%, KH 2 PO 4 0.7%, Na 2 HPO 4 12H 2 O 0.25%, MgSO 4 12H ...

Embodiment 3

[0093] Example 3: Enzymatic Properties of Cladosporium Lipase

[0094] (1) Investigation of the tolerance of lipase to organic solvents such as methanol

[0095] The Cladosporium lipase purified in Example 2 is mixed with organic solvents such as methanol and ethanol according to a certain ratio (0-90%, v / v), and the lipase is detected by the pNPP method after being placed at 4°C for 24h The size of the activity is compared with the sample without organic solvent, the results are shown in figure 2 . Cladosporium lipase has strong tolerance to methanol, ethanol and acetone, especially to methanol, and there is almost no loss of activity after 24h treatment with 50% methanol.

[0096] (2) Determination of temperature-enzyme activity relationship curve and thermal stability curve of lipase:

[0097] In a series of water baths at different temperatures, the activity of Cladosporium lipase was detected by the pNPP method. The results of the enzyme action temperature curve are s...

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Abstract

The invention belongs to the technical field of biology. Lipase widely exists in animal tissues, plants and microorganisms and can catalyze hydrolysis, alcoholysis, esterification and ester interchange of ester compounds, synthesis of compounds and the like. The invention aims to provide cladosporium capable of producing organic solvent tolerant lipase, which is a cladosporium strain (Cladosporium sp.) WBRD3.10062425 and has a preservation number of CGMCC (China General Microbiological Culture Collection Center) No. 4962. The invention also aims to provide the organic solvent tolerant lipase produced by the strain and a preparation method thereof. The lipase produced by the strain disclosed by the invention is the organic solvent tolerant lipase, has strong tolerance for methanol, ethanol and acetone and has wide application value in various industrial fields of biodiesel, medical industry, foods, oleochemical industry, environmental protection, detergents, textile, papermaking and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a strain (Cladosporium sp.) WBRD3.10062425 capable of producing organic solvent-resistant lipase, the preservation number of which is: CGMCCNo.4962, and the organic-resistant lipase produced by the strain. Solvent lipase and the preparation method of the lipase. Background technique [0002] Lipase (lipase, EC3.1.1.3) is a special class of ester bond hydrolase, which widely exists in animal tissues, plants and microorganisms. Lipase can catalyze the reactions of hydrolysis, alcoholysis, esterification, transesterification and compound synthesis of ester compounds, so it can be used in various industries, such as food industry, dairy industry, pharmaceutical industry, detergent industry, textile industry, etc. Industry, biodiesel, paper, leather, cosmetics, material synthesis and environmental industry, etc. (Hasan F, Ali SA, Hameed A. Industrial applications of microbial...

Claims

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Application Information

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IPC IPC(8): C12N1/14C12N9/20C12R1/645
Inventor 徐正军顾思天周美凤陈苗苗戴小军常桂芳
Owner WILMAR SHANGHAI BIOTECH RES & DEV CENT
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