Thermophilic esterase/phosphatidase gene, engineering bacteria, enzyme and use thereof

A technology of phospholipase and thermophilic ester, which is applied in the fields of genetic engineering, application, plant gene improvement, etc., can solve the problems of poor heat resistance of porcine pancreatic phospholipase, production efficiency and cost of degumming process not reaching the optimum level, etc., to speed up Kinetic response, low requirement standard, strong resistance to high temperature and chemical substance denaturation effect

Inactive Publication Date: 2010-02-03
JILIN UNIV
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main problem is that the porcine pancreatic phospholipase used has poor heat resistance (the best catalytic activity is within 60°C), and the oil degumming process requires a higher temperature (70-80°C)
The production efficiency and cost of the degumming process are not optimal due to the limitation of the reaction temperature of conventional enzymes

Method used

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  • Thermophilic esterase/phosphatidase gene, engineering bacteria, enzyme and use thereof
  • Thermophilic esterase/phosphatidase gene, engineering bacteria, enzyme and use thereof
  • Thermophilic esterase/phosphatidase gene, engineering bacteria, enzyme and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1: Construction of thermophilic enzyme engineering bacteria and its enzyme expression

[0036] (1) The cultivation of the thermophilic archaea Aeropyrum pernix K1 and the extraction of chromosomal DNA.

[0037] Take 37.4g Bacto marine broth 2216 (Difco) (culture medium) and dissolve it in 990ml distilled water, and sterilize with moist heat; take 1.0g Na 2 S 2 o 3 ·5H 2 O (nutrient salt) was dissolved in 10ml of distilled water, sterilized by 0.45m filter membrane and added to the sterilized medium. Add 0.5ml of medium to the stem cells of the archaea Aeropyrum pernix K1, dissolve and transfer to a test tube containing 5mL of the above medium, mix well; take 0.5ml from it and transfer to a test tube containing 5ml of fresh medium, and culture at 90°C 7 days. Then transfer to Erlenmeyer flask with 100ml of culture medium and shake culture at 90°C for 3 days, and store the cells at -40°C for future use.

[0038] Take 1.5ml of archaeal culture to collect t...

Embodiment 2

[0051] Embodiment 2: recombinant thermophilic esterase / phospholipase A 2 characteristics

[0052] (1) Optimum reaction temperature

[0053] Reaction temperature is an important factor affecting the catalytic activity of enzymes. Generally, thermophilic esterase / phospholipase A 2 The reactivity at high temperature is much higher than at low temperature. Considering the stability of the substrate, the maximum temperature of the enzyme catalytic activity detection here is only 100°C.

[0054] With NBD-PC as the reaction substrate, phospholipase A was determined in the temperature range of 50-100°C 2 vitality. It can be seen from Figure 4(a) that its activity increases with the increase of temperature in the temperature range of 50-90°C, and decreases above 90°C, and the optimum temperature is 90°C.

[0055] Using p-nitrophenol propionate as the substrate, we also studied the esterase activity of the recombinant enzyme at different temperatures. The results are shown in Fig...

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Abstract

A gene of the thermophilic lipase / phosphatidase A2, the engineering bacterium expressed by its recombinant vector in host, the thermophilic lipase / phosphatidase constructed with said engineering bacterium and the industrial use of said enzyme are disclosed. Said enzyme A2 is prepared from the thermophilic Aeropyrum pernix K1 through constructing engineering bacterium, fermenting, centrifugal separation, collecting deposit, freeze thawing, ultrasonic breaking, heat treating, and affinity chromatography.

Description

Technical field: [0001] The invention belongs to the field of biological engineering, in particular to a thermophilic esterase / phospholipase A 2 The gene of the gene, the engineered bacterium expressed in the normal temperature host by the gene recombinant vector, the thermophilic esterase / phospholipase A constructed by the engineered bacterium 2 and the application of the enzyme in industry. Background technique: [0002] Since TaqDNA polymerase, the first thermophilic enzyme, was successfully used in polymerase chain reaction (PCR) in 1985, microorganisms growing in special high-heat environments have attracted increasing attention. Many thermophilic enzymes (thermophilic enzymes, 55-80℃) with hydrolase activity have been developed successively and have played an important role in many fields. In recent years, people have isolated hyperthermophilic enzymes (hyperthermophilic enzymes, 80-113°C) from thermophilic archaea in seabed heat flow, which has shown new application...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/55C12N9/16C12N15/63C12N15/70C12N1/00
Inventor 冯雁曹淑桂卢冬梅高仁钧韩四平任晓东
Owner JILIN UNIV
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