Study On Construction Of Candida Antarctica Lipase B Surface Display System And Fermentation Process Optimization

Candida antarctica lipase B(CALB),an excellent lipase which shows great substrate specificity and strong catalytic activity in a number of synthetic reactions,such as hydrolyzation,esterification and transesterification,is widely used in the resolution of chiral compounds and organic synthesis.Low expression of CALB and the complicated immobilization process are the main reasons for the high price of CALB.Many researches based on Pichia pastorisshowsthe surface displayof CALB is a promising way to solve this problem.Due to its shorter fermentation period,E.coli has more advantages than Pichia pastorison cell surface display of CALB.Ag 43(Antigen 43)is a member of the monomolecular protein family secreted by the Va secretion system of Escherichia coli.Ag43 is an auto-transporter protein,which contains all information required for transporting to the outer membrane and secretion through the cell envelope.By fusing foreign protein with Ag43,Ag43 auto-transport system has been developed as an efficient and versatile tool for surface display of heterogeneous protein.In this study,Ag43 auto-transporter system is used as the expression system to display CALB on the E.coli cell surface by fusing CALB with Ag43 through genetic engineering method.To increase the yield of CALB and reducing its preparation costs,optimization of fermentation process was also performed.Firstly,the gene encoded CALB was constructed into vector with Ag43.The recombinant plasmids pTrc99a-Ag43’-WT-CALB,pTrc99a-Ag43’-NH-CALB,pbad99a-Ag43’-D551-CALB and pET28a-Ag43’-WT-CALB were successfully constructed by gene cloning technology and were transformed into different E.coli competent cells.SDS-PAGE analysis of recombinant expression sample showed that pET28a-Ag43’-WT-CALB/BL21(DE3)could successfully express the fusion protein Ag43’-WT-CALB.After the expression of recombinants,whole cells were treated with trypsin and SDS-PAGE analysis.It was proved that the Ag43 surface display system could display CALB on the BL21(DE3)surface.Then,the activity of CALB in the fusion protein was analyzed.The tributyl butyrate ester plate has hydrolysis circle,indicating that CALB in the fusion protein has its natural activity.In addition,the optimum substrate of pET28a-Ag43’-WT-CALB/BL21(DE3)whole cell catalyst was found to be pNPA,and the optimum pH and temperature of this whole cell catalyst were 8.0 and 45℃.Secondly,our research optimized the culture medium and the fermentation conditions of the recombinant strain pET28a-Ag43’-WT-CALB/BL21(DE3)by single factor experiment.The optimum medium was composed of(g/L):glycerol 15.0,tryptone 32.0,yeast powder 16.0,(NH4)2SO4 2.5,MgSO4 1.2,KH2PO4 2.3,K2HPO4.3H2O 16.4,trace element solution 0.5 mL/L.Trace element solution contained(g/L in 1 mol/L HCl):FeS04-7H20 2.8,MnCL2· 4H20 2,CoS04-7H2O 2.8,CaCl2.2H2O 1.5,CuCl2·2H2O 0.2,ZnSO4·7H2O 0.3.The optimal fermentation conditions were as follows:the time of adding IPTG was 7 h,the induction time was 4 h,the final concentration of IPTG was 0.1 mM,the optimum induction temperature was 30 °C,the initial pH of culture medium was 7.6,the inoculation quantity was 1.00%(v/v),the working volume was 35 mL in a 250 mL flask.The biomass(measured by the value of OD600)was increased from 6.32 to 8.36,and the enzyme activity increased from 107.2 U/mL to 468.4 U/mL after optimization.