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Lipase LIP, gene and application thereof

A lipase gene and lipase technology, applied in the field of lipase and its gene and application, can solve the problems of high production cost and low enzyme activity

Active Publication Date: 2013-05-01
GUANGDONG VTR BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Generally speaking, the enzymatic activity of the lipase recombinant enzymes reported so far is generally low, and the production cost is relatively high

Method used

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  • Lipase LIP, gene and application thereof
  • Lipase LIP, gene and application thereof
  • Lipase LIP, gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Embodiment 1, cloning and sequence analysis thereof of Aspergillus niger lipase gene lip

[0064] Use the RNA extraction kit to extract the total RNA of Aspergillus niger, and follow the reverse transcriptase SuperScript TM III Reverse Transcriptase Instructions Synthesis of first-strand cDNA. Using cDNA as a template, design primers to amplify the lipase gene for PCR amplification, and double-digest the PCR product with restriction endonucleases EcoRI and NotI, and then connect it to the Pichia pastoris expression vector pPICzalphaA that has undergone the same digestion , The ligation product was transformed into Escherichia coli Topl0 competent cells, and positive clones were obtained after screening with the antibiotic Zeocin. Plasmids of positive clones were extracted. The samples were sent to Shanghai Yingjun Biological Co., Ltd. for sequencing. The sequencing results showed that the cloned DNA insert contained the complete open reading frame of the lipase gene....

Embodiment 2

[0069] Embodiment 2, the construction of the Pichia pastoris engineered bacterium that comprises lipase gene lip

[0070] The above recombinant expression vector pPICzαA-lip was linearized with SacI, and the linearized recombinant vector was electroporated to transform Pichia pastoris X33 to obtain a transformant of the recombinant strain of Pichia pastoris, which was transferred to a YPDZ medium plate with a toothpick. Cultivate at 28°C for 2 days, then use a toothpick to transfer the colonies on the YPDZ medium plate to the lipase activity detection function plate, add methanol dropwise for induction culture, and observe the changes of the lipase activity detection function plate regularly. Experimental results such as figure 1 As shown, there are obvious color circles around transformants 1 and 2, indicating that the lipase gene introduced into Pichia pastoris X33 has been functionally expressed and secreted outside the cell.

Embodiment 3

[0071] Embodiment 3, high expression of lipase recombinant strain

[0072] A single colony of the above-mentioned recombinant bacteria was subjected to high-density fermentation culture. Prepare 20L of basic salt medium, sterilize it in a 50L automatic control fermenter, and cool it to room temperature for later use. Adjust the pH value of the fermentation broth to 4.8 with ammonia water and phosphoric acid, control the dissolved oxygen to be greater than 30% by adjusting the rotation speed and air flow rate, and the fermentation temperature is 30°C. The entire fermentation process is divided into three stages: the first stage is the cell culture stage, inoculate the recombinant bacteria into the fermenter according to the inoculation amount of 10%, add 4L of 50% glucose that has been sterilized, and cultivate for 24~30 hours. It is marked by the completion of glucose; the second stage is the starvation stage. After the glucose is replenished, no carbon source is added, and w...

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Abstract

The invention relates to the field of gene engineering, and in particular relates to a lipase, gene and application thereof. The lipase has an amino acid sequence shown as a SEQ ID NO.1. The invention aims to solve the defects of an existing technology, and clones a lipase gene from Aspergillus niger. High expression of the lipase in Pichia pastoris reaches a level higher than that of the existing technique. Therefore, the recombinant lipase provided by the invention can greatly reduce production cost in industrial production, so as to show great application potentials in industries such as washing, papermaking and food.

Description

technical field [0001] The present invention relates to the field of genetic engineering, specifically, the present invention relates to a kind of lipase and its gene and application. Background technique [0002] Lipase (Lipase, E.C.3.1.1.3), the full name of triacylglycerol acylhydrolase (triacylglycerol acylhydrolase), which belongs to the α / β-fold enzyme family, is a serine hydrolase. Lipase catalyzes the hydrolysis of the ester bonds of triacylglycerols, releasing glycerides or glycerol and fatty acids with fewer ester bonds. Lipase has excellent stereoselectivity, mild reaction conditions and does not cause environmental pollution, so it is widely used in many industrial fields such as washing, food, and papermaking. [0003] Lipase widely exists in animals, plants and microorganisms. Compared with animals and plants, the lipase secreted by microorganisms has wider substrate specificity, action temperature and action pH range. Although the lipase secreted by microorg...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/20C12N15/55C12N15/63C12N15/81C12R1/84
Inventor 王建荣李阳源罗长财钟开新陈丽芝
Owner GUANGDONG VTR BIO TECH
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