Preparation Of Recombinant Serratia Marcescens Lipase And Its Application In Resolution Of Racemates

Serratia marcescens lipase is an important biocatalyst that has been widely used to catalyze various reactions for industrial application purpose.For example,it is an important industrially biocatalyst for the stereospecific synthesis of Diltiazem intermediate.In this work,we cloned the lipase gene from Serratia marcescens H30 and constructed three recombinant expression vectors pET28a,pET32a and pGEX-4T-1 to express it in Escherichia coli BL21(DE3).We chose pET32a-lipase for futher study as its soluble expression level is even more than the other two.After optimization,we got a higest enzyme activity of 25000U/L.When the Trx-tag of pET32a was removed by thrombin,enzyme activity was unexpectedly improved to 65000U/L.We also tried to use lactose to induce lipase expression instead of IPTG.After optimizing the medium composition and fermentation conditions,we got an enzyme activity of 23000U/L,which was a little lower than the lipase induced by IPTG.When the lipase fermentation was carried out in a 7-L fermenter,the lipase production reached 19000U/L?The lipase was immobilized on amino-support,epoxy-support and ion exchange resin.After comparing,LH-EP was the best carrier chose to immobilize this lipase.The optimal temperature and pH of immobilized lipase were 35? and 8.0,and the temperature and pH stabilities were improved through immobilization.The capacity of lipase-catalyzed the kinetic resolution of chiral alcohols was investigated,and the transesterification resolution of 4-phenyl-2--butanol was used as a model reaction.After optimization,the conversion and ees attained to 49.5%and 99.4%respectively,suggested the lipase had good enantioselectivity.We also investigated the lipase catalyzed resolution of MPGM,and the resolution effectiveness achieved the current level reported in the literatures.