Indirect Surface Display Of Lipase On Pichia Pastoris Cells

Lipase has a wide range of substrate selectivity,as well as substrate specificity,it can react under mild conditions,resistant to organic solvents,and does not produce too many side reactions,has been widely used in industry.However,the free lipase is expensive,difficult to recover,and the enzyme is complicated in production and purification.The technique of displaying enzymes on the cell surface solves this problem very well,the enzymes display on the cell surface do not need too many purification steps and are easy to recycle,while the individual of yeast cells is large,and the number of displayed enzymes will increase accordingly.The small peptide CL7 derived from E.coli is a DNAase-free mutant of E.coli E7(CE7),which can produce highly specific strong interactions with its immune protein7(Im7),respectively.Lipase K80 is an alkaline lipase derived from denaturing bacilli(Proteus sp.),which has high hydrolysis activity and high stability in organic solvents and has a broad application prospect.This study first achieved the soluble expression of fusion protein CL7-K80 in Escherichia coli,the fusion protein CL7-K80 cell lysates expressed in E.coli incubated with yeast displayed Im7 protein directly.The incubated yeast cells were detected on olive oil substrate plate and yellow fluorescence was observed under blue light instrument.And the display efficiency of purified fusion protein CL7-K80 and unpurified cell lysates were 49.8%and 44%,respectively.There's no big difference in efficiency between them.The retention activity was 8% and 7.2%,respectively.Indicating that direct incubation with yeast cells through unpurified CL7-K80 cell lysates is feasible.The enzymatic properties of free lipase K80 and displayed lipase K80 were compared.The optimum temperature of free lipase k80 and displayed lipase k80 was 30 ° C,and the optimum p H was 8.The thermal stability and p H tolerance of the displayed lipase K80 are improved compared with that of free lipase.With 0.5h in 40 °C,free lipase K80 residual enzyme activity has dropped to 14%,but the lipase displayed on the yeast has more than 50% residual enzyme activity with 3 h in p H9.Free lipase k80 residual enzyme activity has dropped to 30%,and the residual enzyme activity of displayed lipase K80 is more than 40%.The tolerance of the displayed lipase to free lipase K80 organic solvent was enhanced.When these two forms of lipase were treated in methanol for 3 h,the free lipase was basically inactive.However,the displayed lipase K80 still retained50% of the residual enzyme activity.After the successfull display of lipase k80 on the yeast surface,we also studied an unknown functional protein HaPL from Halobacteriales archaeoon and we displayed unknown functional protein HaPL on the surface of Pichia pastoris.And we also achieved soluble expression of fusion protein CL7-HaPL in Escherichia coli.The fusion protein CL7-HaPL cell lysates expressed in E.coli and directly incubated with yeast displayed Im7 protein.The incubated yeast cells were detected on the olive substrate plate,yellow fluorescence under the blue light,indicating that the incubated yeast cells have the activity of hydrolyzing oil.But the cell lysates of fusion protein CL7-HaPL expressed in E.coli and the purified fusion protein CL7-HaPL can only detect weak hydrolytic activity on the olive oil tablet,suggesting that this unknown functional protein is a potential lipase.Therefore,based on the highly specific strong interaction between CL7-Im7,this study directly used Escherichia coli cell lysates expressing fusion protein to incubate with Pichia pastoris cells display Im7 without protein purification.The successful display of lipase on Pichia pastoris surface is simple and rapid,which lays an important foundation for further application.