Protein Engineering Of Lipase For Improved Detergent Tolerance

Lipase from Thermomyces lanuginosus(TLL)is a thermostable alkaline lipase,which has extensively been employed in industrial applications,such as,cosmetics and detergent industry.However,the expression level of TLL is relatively low in original organism Thermomyces lanuginosus.To solve this problem,in this study,the gene of TLL was cloned into expression vector p ET22 b and successfully transformed into Escherichia coli.After isopropyl-?-D-thiogalactopyranoside(IPTG)inducing about 52 h,it reached highest activity at 0.322 U/m L.For the directed evolution of TLL for higher enzyme activity and detergent tolerance,high-throughput screening system based on microfluidic technology using Fluorescein Dicaprylate as substrate was constructed.By directed evolution,one TLL variant7-D11 with 3 fold increased activity and 2.07 fold increased stability towards SDS was obtained.The mutation of 7-D11 is on the leader peptide.The other variant 84R/H with 7.15 fold increased stability towards AEO-9 was obtained.The mutation of 84R/H is on the lid area of active pocket of TLL.In addition,to improve the expression level of TLL,the gene of TLL was also transferred into Pichia pastoris.The expresson level of recombinant pichia pastoris is 139-fold higher than recombinant E.coli(44.71 U/ml vs 0.322 U/ml).Thereafter,the application of the TLL in the synthesis of short-chain aromatic esters and de-inking industry is improved.