Cloning And Expression Of Lipase Gene From Rhizopus Oryzae

Lipase is a typical enzyme which catalyzes the hydrolysis of long chain triglycerides. They constitute the most important group of biocatalysts for biotechnological applications. Lipase from Rhizopus oryzae has 1,3 positional specificity, it can hydrolyze triacylglycerol producing fatty acid and monoglyceride (MG), MG is an important non-ionic surfactants, widely used as emulsifier in the food stuff and other industries. As a result, today people focus on the construction of gene recombinant strain with high enzyme activity by gene measuses.Besides,. There is still no uniform method to measure the activity of lipase exactly , while it is important in reasearching of characters and amplication of lipases.In This thesis , the gene which encoding lipase ROL from Rhizopus oryzae was amplified by PCR and cloned into vector pGM-T was saved in our lab.The Recombinant plasmid Is named pGMT-ROL, and I designed primer pair 4 and 4.2 to clone the lipase gene ROL from pGMT-ROL,separated connect with pET-28a and pET-11c. The recombinant plasmid are named pET-28a-ROL and pET-11c-ROL, and pET-28a-ROL was transferred into E.coli.BL21 and pET-11c-ROL was transferred into E.coli.Origami DE3. Expression Recombinant strains were obtained separated by Kana and Amp plates screening ,the Expression Recombinant strains were farther filtered by strain PCR, and The engineered strain was expressed the gene ROL by the induction of IPTG. The expression products of ROL gene was analysis by SDS-PAGE, the result indicated that ROL gene was functionally expressed in E.coliBL21 and Origami DE3. Recombinant lipase had a molecular mass of 35kD.On the other side,This thesis discusses three assays in common use,including the spectrophotometry assay with the synthetic substrate p-NP palmitate, the spectrophotometric assay using the formation of copper soaps and the alkaly titrition assay.We compare the consuming time of reaction, vericity and repeatity of methods.We have found an effective method to terminate the reaction for the spectrophotometry assay with the substrate p-NP palmitate,this modified method shows good repeatity and takes less time relatively.In addition,we also improved the copper soaps assay by using toluene insead of benzene,it has merits of less toxicity and good repeatity. Through discussing merits and demerits of these assays and improvement of them ,This thesis provides chosen basises of assays for lipase activity measure.