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Lipase mutant with improved heat stability, and construction method thereof

A technology for thermostability and lipase, applied in the field of lipase mutants and their construction, can solve the problems of limited application scope, poor thermostability, restricting the development and application of lipase, etc., achieving broad market prospects and improving thermostability , the effect of high practical application value

Active Publication Date: 2012-09-12
SHANGHAI JIAO TONG UNIV
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AI Technical Summary

Problems solved by technology

However, the contradiction between the harsh conditions required for industrial production and the stability of the enzyme has been restricting the development and application of lipase for a long time.
For example, in order to ensure catalytic efficiency, many reactions need to be carried out at higher temperatures, and Candida antarctica lipase B belongs to mesophilic lipase. Its poor thermal stability not only limits its application range, but also makes the enzyme easy to inactivate, increasing the Cost of production

Method used

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  • Lipase mutant with improved heat stability, and construction method thereof
  • Lipase mutant with improved heat stability, and construction method thereof
  • Lipase mutant with improved heat stability, and construction method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Embodiment 1 Cloning of wild-type Candida antarctica lipase B gene

[0040] The wild-type Candida antarctica lipase B gene was synthesized by Nanjing GenScript, through the upstream primer 5'-AATA CCATGG CTCTACCTTCCGGTTCG-3' (underlined base is the recognition site of restriction endonuclease NcoI) and downstream primer 5'-TAA CTCGAG GGGGGTGACGATGCCGGA-3' (the underlined base is the restriction endonuclease XhoI recognition site) to amplify the target gene. The PCR reaction uses Takara's PrimeSTAR polymerase. The PCR reaction conditions are: 98°C for 2min, then 98°C for 10sec, 55°C 15sec at ℃, 1min at 72℃, a total of 25 cycles; the last 10min at 72℃. After the reaction, the PCR amplification product was detected by 1% agarose gel electrophoresis, and a band with a size of 1 kb was obtained, which was consistent with the expected result. DpnI digests the template, recovers, and purifies the target fragment. It is double-digested with restriction enzymes NcoI and XhoI ...

Embodiment 2

[0041] Example 2 Structural Analysis and Hot Spot Determination of Candida Antarctica Lipase B

[0042] The crystal structure of Candida antarctica lipase B (ID: 1TCA) was analyzed. The catalytic triad of lipase is: serine at the 104th position from the N-terminus, aspartic acid at the 187th position, and histidine. Select the amino acid residues around the catalytic serine to analyze the B factor, and determine a series of higher residues of the B factor (from the N-terminus, the 278th leucine, the 285th valine, the 277th leucine, Glycine at position 281, aspartic acid at position 223) as a hotspot for saturation mutation.

Embodiment 3

[0043] Example 3 Establishment of Saturation Mutant Library and Screening of Mutants

[0044] Use the following method to screen the variant of Candida antarctica lipase B with better thermal stability, and the specific process includes the following steps:

[0045] 1. Construct a saturated mutant library according to hotspots and import it into host cells

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Abstract

The invention provides a lipase mutant with improved heat stability and a construction method thereof. The lipase mutant with improved heat stability is obtained by subjecting Candida Antarctica lipase B gene to several rounds of fixed point saturation mutagenesis, the Candida Antarctica lipase B having an amino acid residue sequence expressed by SEQ ID No:1, and the lipase mutant having amino acid residue sequences expressed by SEQ ID No:2, SEQ ID No:3 or SEQ ID No:4, each of which consists of 317 amino acids. A method of semi-rational design is utilized in the invention, and a lipase mutantis obtained by subjecting Candida Antarctica lipase B gene to several rounds of fixed point saturation mutagenesis, wherein the mutant includes amino acid mutations D223G, L278M and combination of the two. Expressed by T50<15> and half-life t1 / 2 at the temperature of 48 DEG C respectively, the heat stability of the lipase mutant is improved, and a high actual application value and broad market prospects are also provided.

Description

technical field [0001] The invention relates to biotechnology, in particular to a lipase mutant with improved thermostability and a construction method thereof. Background technique [0002] Lipase (Lipase) is a biocatalyst with a wide range of uses. It can not only catalyze the hydrolysis of oil in the aqueous phase, but also catalyze ester synthesis and transesterification in the non-aqueous phase. It is often used in detergent additives, Food, pharmaceutical industry, paper industry and bio-energy etc. Wherein, Candida antarctica lipase B is one of the most effective biocatalysts at present, is widely used in the chiral resolution of medicine and the production of biodiesel (Kirk O, Anderson EM, Karin M (1998) One biocatalyst-many applications : The use of Candida antarctica b-lipase in organic synthesis. Biocatal Biotransform 16(3):181-204). However, the contradiction between harsh conditions required for industrial production and enzyme stability has long restricted t...

Claims

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Application Information

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IPC IPC(8): C12N9/20C12N15/55C12N15/63C12N1/21
Inventor 冯雁谢渊杨广宇
Owner SHANGHAI JIAO TONG UNIV
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