Expression Of Pro-and Mature Rhizopus Chinensis Lipases In E.coli And Their Characteristics

Rhizopus lipase is a typical kind of Microbial Lipase, which has a important role in theoretical research and practical application, because of their particular catalytic characteristics. Rhizopus chinensis lipase (RCL) has good application in industrial practice, such as synthesis of esters, preparation of biodiesel and chiral compounds. Expression Rhizopus chinensis lipases and research their characteristics was the basic of further research for theoretical and application of RCL.To reach relative comprehensive knowledge of Rhizopus chinensis lipases, expression the lipase gene in accurate way and guide the industrial application, the prolipase and mature lipase genes were amplified by PCR method using the genomic DNA isolated from Rhizopus chinensis CCTCCM201021. The target genes were then inserted into the expression vector pET-28a and expressed in E.coli BL21 with IPTG induction. SDS-PAGE analysis showed the relative molecular weight of pro- and mature Rhizopus chinensis lipases (ProRCL and MRCL) were about 43 kD and 33 kD, respectively. Under the control of conditon, pro- and mature Rhizopus chinensis lipases expressed in E.coli could obtain active forms. The target proteins were purified by the affinity chromatograph.The ProRCL and MRCL showed similar characteristics, optimal temperature at 35℃,stable below 40℃, optimal pH at 8.0 and 8.5 respectively. These two recombinant lipases were specific to short chain fatty acid of p-nitrophenyl esters. The Km and Kcat for the hydrolysis of p-nitrophenyl palmiate and p-nitrophenyl propionate of ProRCL and MRCL explained that the hydrolytic activity of both enzyme using p-nitrophenyl propionate as substrate were higher than that of p-nitrophenyl palmiate. Both lipases were stimulated by 10 mmol/L Mg2+, while Hg2+,SDS and PMSF had strong inhibition on their activities. The results showed prosequence had no obvious effects on the characteristics of RCL, which was different from Rhizopus oryzae lipase (ROL).