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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  • Abstract
  • Description
  • Claims
  • Application Information

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 re

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
Comparison scheme
Effect test

Example Embodiment

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

[0040] The wild-type Candida antarctica lipase B gene was synthesized by Nanjing GenScript Company through the upstream primer 5’-AATA CCATGG CTCTACCTTCCGGTTCG-3' (underlined base is the recognition site of restriction enzyme NcoI) and downstream primer 5'-TAA CTCGAG GGGGGTGACGATGCCGGA-3' (underlined bases are restriction enzyme XhoI recognition sites) to amplify the target gene. This PCR reaction uses Takara's PrimeSTAR polymerase. The PCR reaction conditions are: 98℃ for 2min, then 98℃ for 10sec, 55 ℃15sec, 72℃1min, a total of 25 cycles; the last 72℃10min. After the reaction, the PCR amplified product was detected by 1% agarose gel electrophoresis, and a 1kb band was obtained, which was consistent with the expected result. DpnI digests the template, recovers, and purifies the target fragment. After double-enzyme digestion with restriction enzymes NcoI and XhoI, it is ligated with the plasmid pET22b (Novag...

Example Embodiment

[0041] Example 2 Structural analysis and hot spot determination of Candida antarctica lipase B

[0042] The crystal structure of lipase B of Candida antarctica was analyzed (ID: 1TCA). The catalytic triad of lipase is: serine at position 104, aspartic acid at position 187, and position 224 from the N-terminus. Histidine. Select the amino acid residues around the catalytic serine for factor B analysis, and determine a series of higher factor B residues (from the N-terminus, 278th leucine, 285th valine, 277th leucine, Glycine at position 281 and aspartic acid at position 223) serve as hot spots for saturation mutations.

Example Embodiment

[0043] Example 3 Establishment of saturation mutant library and screening of mutants

[0044] The following method is used to screen variants of Candida antarctica lipase B with better thermal stability. The specific process includes the following steps:

[0045] 1. Construct a library of saturation mutants based on hot spots and import them 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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