Construction Of Thermotolerant Lipase Mutants From Burkholderia Sp.ZYB002 Using Site-directed Mutangenesis

Compared with the mesophilic lipase,The thermophilic lipase has the following advantages:It catalyzes various types of chemical reactions at higher temperatures,increases the reaction rate,enhances the stability of the reaction system,and reduces the possibility of contamination of the reaction system with other microorganisms.Based on the comprehensive analysis of LipA 3D structure of Burkholderia sp.ZYB002 lipase,this project uses computer software to construct LipA's electronic mutation library.After screening,The corresponding LipA mutants were constructed by using site-directed mutagenesis for some structures with reasonable energy and less than-0.5 Kcal/mol mutants.and Its thermostability was characterized.The specific experiment content and results are as follows:1.Use Castp,Voronoia,Cave and other software to predict the cavities present in the LipA molecule and the amino acids that make up the cavities;Compare the composition of the amino acid residues analyzed by the above three softwares,and construct mutations to amino acid residues at the same site.Screening the above mutant library(screening criteria:free energy less than-0.5 Kcal/mol and volume reduction of the cavity before and after the mutation)to construct an electron mutant library of 58 LipA mutants.2.Further in-depth analysis of the above 58 electron mutants,and selecting 20 electron mutants,using the site-directed mutagenesis technology to construct,screen and characterize LipA mutants,and finally select two positive mutants(LipA-Hl”P,LipA-A210V).The half-life of mutation at 55 °C was 9.6-fold and 2.9-fold higher than that of wild-type LipA,respectively;superimposition mutations were performed on the two above-mentioned two positive mutants,and a lipase mutant with a half-life increase of 23.1 times compared to wild-type LipA was finally obtained(LipA-H15 P/A210V).3.Consurf software(http://consurf.tau.ac.il)was used to select 219 protein sequences with 30%to 95%homology with LipA polypeptide sequences from the Clean Uniprot database database.After the deletion,105 sequences were obtained.These 105 sequences were constructed into an evolutionary tree.A representative sequence was selected from each branch.35 sequences were finally selected for homologous sequence alignment with LipA.A mutant library consisting of 20 mutants was screened;after mutation analysis,10 mutants were constructed by site-directed mutagenesis,and characterized by enzymatic properties,A total of 2 heat stability and enzyme activities were improved.The T5012 of LipA-L243 P and LipA-Y175F was increased by 5 ? and 1 ?,Over the wild type,and the half-life was 5.7 and 1.8 times that of the wild type.The lipase activity U/OD600 was 4.1 times and 5.7 times that of the wild type.