Expression And Optimization Of A Lipase K80 From Proteus Sp.in Pichia Pastoris GS115

Lipase is an important industrial enzyme widely used in the fields of food production,fine chemical processing,and biodiesel production.Previous studies have shown the presence of a Proteus-derived lipase K80 was found in oil waste liquids.This study is for the first to express Proteus-derived K80 lipase in Pichia pastoris.In this study,the full-length lipase K80 gene was synthesized with a total length of 864 base pairs(bp)and 288 amino acids.The K80 plasmid was constructed into the Pichia pastoris expression vector to obtain the pHBM905BDM-K80 plasmid which was linearized by sal I and electroporated into Pichia pastoris GS115 competent cells.The single colony on the MD auxotrophic plate was transferred to the Rhodamine B plate for screening.The positive strain released lipase to decompose the olive oil in the Rhodamine B plate to generate a hydrolyzed circle,and the recombinant lipase strain was screened.Meanwhile through point mutations,the lysine K at position 285 is mutated to arginine R,and the lysine K at position 286 is mutated to glutamine Q,which disrupts the endoplasmic reticulum retention signal sequence increased expression.In this study,Proteus-derived lipase K80 gene was heterologously expressed in Pichia pastoris.It was fermented in BMGY shake flask.After methanol induction,the expression level of K80 supernatant in 100 mL/L was as low as 119U/mL The secretion of the lipase K80 was improved by disrupting the retention signal of the carboxyl-terminus of the protein,and finally,the expression level was increased to 214U/mL,which was nearly 2 times higher than that of the wild-type strain.I purified the K80 using a nickel column and analyzed the enzymatic properties of the purified enzyme.The results showed that the optimum reaction temperature was 40? and the optimum pH was 8.0.The recombinant lipase K80 is sensitive to acid and high temperature,and the enzyme activity decreases rapidly when the pH is lower than 6.0 or the temperature is higher than 50°C.In the range of 30-45 °C and pH 6.010,K80.0 residual enzyme activity remained 80%and 70%,respectively.Mn2+ and Mg2+ promote the activity of lipase.Surfactants SDS and Tween 20 have a strong inhibitory effect on lipase.However,0.05%TrisonX-100 has a catalytic effect on lipase K80.K80 showed tolerance in organic alcohols,especially in isopropanol,which laid a good foundation for the preparation of lipase K80 in biodiesel.