Screening And Optimization Of Recombinant Lipase Strains For High Expression

Lipase is one of the most important enzymes for industrial application. It is extensively applied in food, chemical engineering, medicine and energy industry. The Pichia pastoris expression system is the most successful eukaryotic expression system which was widely applied and developed in recent years. It possesses the advantage of eukaryotic cell and has the potential to perform many of the post translational modifications typically associated with higher eukaryotes. These include cleavage of signal peptide, disulfide bond formation and glycosylation. The aim of this study was to obtain highly expressed recombinant lipase strains which can be used for large-scale applications. The results are divided into the following two parts:1. The BTLm lipase gene was cloned into the vector pPICZαA to construct a secreted expression plasmid pPICZαA-btlm, and transformed into Pichia pastoris GS115 after being linearized. The multiple integrants secreting BTLm were obtained by zeocin of gradient concent rations. The SDS-PAGE analysis indicated that the molecular mass of the expression protein was in accordance with that of BTLm. The optimal temperature and pH for the recombinant lipase BTLm were 60℃and pH10.0, respectively. The lipase activity of the supernatant from the culture was up to 69.24U/mL under the optimal conditions towards pNPP, which indicated that this P. pastoris recombinant strain had huge productive potential in industry.2. The lipAB gene was cloned from the genome DNA of Pseudomonas aeruginosa strain HS-D38. Primers Pf and Pr were designed based on the nucleotide sequence of the AB008452. The lipAB gene revealed two open reading frames(ORF) of 936bp for the first ORF1, and 849bp for the second ORF2. The ORF1 is lipase A. The ORF2, plays an important role in the expression of the lipAB gene, known as chaperone. The chaperone gene(lipB) which is downstream of lipase gene, is required for the expression of lipase activity as a trans-acting. To construct an expression plasmid pET-lipAB, we cloned the lipAB gene into the vector pET28a, and then it was transformed into E.coli BL21. The recombinated lipase protein was secreted into the cultural broth, both LipA and LipB were detected. Functions of lipB is the act gene for lipA. As a result, the lipAB gene was actively expressed.