Lipase mutant with improved heat stability, and construction method thereof

[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 (Novagen) double-enzyme-cut with the same enzyme, and the ligation product is transformed into E. coli Rosetta (DE3) Among competent cells, spread the transformed cells on an LB plate containing 100ug/ml ampicillin to screen positive clones, extract plasmids, and sequence them. The sequencing results show that the cloned Candida antarctica lipase B gene sequence is correct , And correctly connected to pET22b, the recombinant plasmid was named pET22b-CALB.

[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.

[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