A kind of phospholipase c expressed by Cladosporium and its producing strain

A technology of phospholipase and strain, applied in the field of enzyme degumming, can solve problems such as single substrate, and achieve the effect of wide range of action and good degumming effect

Active Publication Date: 2019-07-12
WILMAR SHANGHAI BIOTECH RES & DEV CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the above reports on extracellular phospholipase C, most of the extracellular PLCs can only act on a relatively single substrate, such as some acting on phosphatidylcholine / phosphatidylethanolamine, and some can only act on Phosphatidylinositol, etc.

Method used

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  • A kind of phospholipase c expressed by Cladosporium and its producing strain
  • A kind of phospholipase c expressed by Cladosporium and its producing strain
  • A kind of phospholipase c expressed by Cladosporium and its producing strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] Example 1: Isolation and identification of Cladosporium sp. WBRD00050

[0089] Take a fresh Tibetan ghee sample, remove about 1 cm thick surface layer, cut about 10 g of Tibetan ghee sample and place it in 100 mL of sterile aqueous solution containing 1.0% mixed phospholipids (purchased from Beijing Meiyasi Phospholipid Technology Co., Ltd.). After fully shaking for 30 min, the solutions were diluted 10 1 、10 2 、10 3 、10 4 Times, and the samples of each dilution were spread on the screening plate and incubated at 28°C.

[0090] Observe whether there are colonies on the screening plate every 24 hours. After culturing for 96 hours, the microorganisms with larger and more obvious turbidity circles were picked for further streak separation to obtain pure strains. The extracellular phospholipase of these purebred strains was detected on a screening plate, and it was found that there was a strain with a very significant turbidity circle named WBRD00050.

[0091] The str...

Embodiment 2

[0097] Example 2: Preparation and Identification of Cladosporium Extracellular Phospholipase C

[0098] (1) Fermentation of Cladosporium extracellular phospholipase C

[0099] Cladosporium WBRD00050 was inoculated into PDA slant medium, and cultured at 28°C for 120 hours to obtain Cladosporium WBRD00050 slant. Take 2 Cladosporium WBRD00050 slopes and add 10ml of sterile water to each, wash thoroughly to obtain a spore suspension, and combine all the spore suspensions. The spore suspension was inoculated into the Cladosporium fermentation medium with an inoculation amount of 1 ml / bottle, and cultured at 28° C. and 200 rpm for 110 h.

[0100] Take Cladosporium WBRD00050 fermentation culture liquid and centrifuge at 12000g for 10min, collect and obtain about 400ml of centrifuged supernatant, detect the activity of phospholipase C in the centrifuged supernatant, the result shows that the activity of phospholipase C in the centrifuged supernatant is about 0.042u / ml .

[0101] (2...

Embodiment 3

[0105] Embodiment 3: Cladosporium phospholipase C catalyzes the hydrolysis of soybean mixed phospholipids

[0106] Prepare 2% aqueous solution of soybean mixed phospholipids (purchased from Beijing Meas Phospholipid Technology Co., Ltd., the total phospholipid content is not less than 95%), divide into two tubes, each tube is 4.45ml and marked as tube A and tube B, of which Tube is the control sample, and tube B is the enzyme action sample.

[0107] Add 0.05ml of 250mM CoCl2 aqueous solution to tubes A and B respectively, and mix well. Add 0.5ml of distilled water to tube A, and add 0.5ml of the phospholipase C concentrated enzyme solution prepared in Example 2 to tube B. Mix well and react in a water bath at 40°C for 24 hours. After the reaction is over, add equal volumes of chloroform to extract respectively, centrifuge, get about 3ml of the lower organic phase of the two tubes of A and B and place them in a fume hood for air-drying, and then use 31 The P-NMR method (Glon...

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Abstract

The invention provides an isolated Cladosporium strain expressing phospholipase C, which is preserved in the General Microorganism Center of China Microbiological Culture Collection Management Committee, and the preservation number is CGMCC No.7508. The phospholipase C prepared by using the bacterial strain and the preparation method provided by the invention can be used for oil refining, has good degumming effect, and can be widely used in the fields of oil refining, additives, medicine and the like.

Description

technical field [0001] The invention belongs to the field of enzyme degumming, in particular to a phospholipase C expressed by Cladosporium and its producing strain. Background technique [0002] Phospholipase (Phospholipase, PL) is an enzyme that can hydrolyze glycerophospholipids in organisms. The hydrolyzed products are various phosphatidic acids and amino alcohols, such as cholamine, choline, serine, ethanolamine, etc. According to the different sites of phospholipase hydrolyzing glycerophospholipids, phospholipases can be divided into phospholipase A (Phospholipase A, PLA), phospholipase B (Phospholipase B, PLB), phospholipase C (Phospholipase C, PLC) and phospholipase D (Phospholipase D, PLD), such as figure 1 shown. [0003] Phospholipase can be widely used in oil refining, phospholipid modification, feed improver, food industry and medicine. Degumming is an important link in the refining process of vegetable oil, which is crucial to improving the quality of oil. D...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/14C12N9/16C11B3/00C12R1/645
CPCC11B3/003C12N9/16C12Y301/04003C12N1/145C12R2001/645
Inventor 徐正军周美凤许骏
Owner WILMAR SHANGHAI BIOTECH RES & DEV CENT
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