Comparative Study On The Expression Of Lipase From Proteus Sp. In Pichia Pastoris

Lipases (triacylglycerol acylhydrolases EC3.1.1.3) are a class of hydrolase which catalyze the hydrolysis of triglycerides to glycerol and free fatty acids over an oil-water interface. In addition, lipases catalyze the hydrolysis and transesterification of other esters as well as the synthesis of esters. The ability of lipases to perform very specific chemical transformation (biotransformation) has made them increasingly popular in the food, detergent, cosmetic, organic synthesis, and pharmaceutical industries. The gene used in this study is got from one strain belonging to Proteus sp.. The gene encoding287amino acids is named as LipA with the size of864bp. The conclusions are as follows:1. Construction of expression vectorThe gene of lipase was amplified by PCR technique using plasmid pPIC9k-LipA as a template. Two different plasmids pHBM905A and pHBM905BDM were similarly digested with restriction endonucleases and then connected with LipA to construct recombinant plasmids pHBM905A-LipA and pHBM905BDM-LipA.2. Transformation of P. pastoris GS115and selectionThe two recombinant plasmids were linearized by restriction enzyme SalI and transformed into competent cell P. pastoris GS115prepared by PEG1000and positive transformants were screened by utilizing the screening plate containing olive oil and rhodanmine B.3. Induction of recombinant strains and SDS-PAGE analysis of the expression of LipAThree positive transformants were chosen from each of the screening plate randomly then inoculated in BMGY medium. Harvest the cells and resuspend cell pellet in BMMY medium to start induction. Supernatants after induction were analyzed by SDS-PAGE. The highest expression level of AL strains (strains containing plasmid pHBM905A-LipA) achieved60.01mg/L,64.97mg/L,58.13mg/L respectively in shake flask fermentation and the recombinant protein of BL strains (strains containing plasmid pHBM905BDM-LipA) could be secreted to the culture medium at a level of156.99mg/L,149.14mg/L,180.88mg/L respectively in shake flask fermentation. Under the same colony density and culture condition, the expression level of BL strains were2time higher than AL strains. The result suggested that the expression content of the lipase was enhanced by optimizing the promoter and signal sequence.