Efficient Expression Of Lipase K80 In Pichia Pastoris

Pichia pastoris expression system is one of the most commonly used systems for the exogenous expression of recombinant proteins because of its complete post-translational modification system in eukaryotes,less endogenous proteins secreted,and strict regulation of its alcohol oxidase promoter（AOX）by methanol.The lipase derived from proteus has the advantages of high catalytic activity and resistance to organic solvents,which makes it potentially valuable in application.Lipase K80 from Proteus sp.is an alkaline lipase.It has been reported that it has a good application prospect in biodiesel synthesis.At present,the secreted expression of lipase K80 based on P.pastoris expression system has not been reported.In this study,the efficient expression of lipase K80 in P.pastoris was achieved by a series of means.Firstly,the expression of wild-type lipase K80 in P.pastoris was realized in this study.The enzyme activity of the shaking flask fermentation supernatant was 38.48 U/mL,and the protein secretion level was 0.179 mg/mL.In order to improve its expression level in P.pastoris,the software analysis in this study found that,There is a highly conserved amino acid sequence KKL in the carboxyl terminal of this enzyme,which may affect its transport and secretion in P.pastoris cells.Through targeted mutagenesis,the mutant K286Q was constructed and secreted in P.pastoris.The enzyme activity and protein secretion in the supernatant of the flask fermentation reached 74.56 U/mL and 0.357 mg/mL,respectively,and the enzyme activity and secretion levels were 1.93 times and 1.99 times higher than those of the wild-type lipase K80.N-acetyltransferases（MPR1）are enzymes that catalyze the transfer of acetyl groups between acetyl-Co A and amines,which can catalyze the acetylation of toxic intermediates generated by intracellular ROS（Reactive oxygen species）,thus reducing ROS production.In order to further improve the expression level of the mutant K286Q in P.pastoris,the recombinant P.pastoris strain K286Q-MPR1 co-expressing MPR1 was constructed in this study.Finally,the enzyme activity of K286Q-MPR1 in the shaker fermentation supernatant reached 121 U/mL,and the protein secretion level reached 0.520 mg/mL.Enzyme activity and secretion levels increased by 64%and 46%,respectively.Through the above two optimization strategies,the enzyme activity and secretion of K286Q-MPR1 in P.pastoris flask fermentation supernatant were 3.14 and 2.99 times of that of wild-type lipase K80,respectively.Finally,the enzymatic properties of recombinant lipase K80 were determined.The results showed that the most suitable substrate for the enzyme was C12,the optimal reaction temperature was 30℃,and the optimal pH was 8.0.The enzyme showed good stability at medium temperature,such as 25℃and 30℃,and its half-life was 3 h.At the same time,it has good tolerance to alkaline conditions.For example,after 6 h treatment at pH9-10,it can still maintain more than 40%activity.Metal ions K⁺,Ca²⁺and Mg²⁺can slightly increase the activity of lipase K80,while Fe²⁺,Cu²⁺and Zn²⁺can significantly inhibit the activity of lipase K80.In addition,the glycosylated lipase K80expressed in P.pastoris showed higher tolerance to organic solvents than the unglycosylated lipase K80 expressed in Escherichia coli.The glycosylated lipase K80 had the best tolerance to methanol and ethanol at 80%,but the lipase K80 expressed by E.coli had no tolerance to methanol,while the tolerance to ethanol was the best at 60%.The lipase K80 expressed by P.pastoris showed strong stability in 80%organic solvents such as acetone,isopropanol and ethanol,and had obvious activation effect at 3 h and 6 h treatment time.When the treatment time was 3 h,the enzyme activity reached 136%,115%,134%and 171%,respectively.In conclusion,this study successfully realized the efficient expression of lipase K80 in P.pastoris,and the recombinant enzyme produced by the expression showed excellent resistance to organic solvents,which laid an important foundation for further research and industrial application of lipase K80.