High-level Expression And Application Of Aspergillus Oryzae Lipase In Pichia Pastoris

Currently,more than 500 proteins have been successfully expressed in the Pichia pastoris,and the Pichia pastoris expression system is recognized as a mature and efficient eukaryotic expression system.Microbial lipase is an important source for industrial application.A lipase-producing strain of Aspergillus oryzae WZ007 that was screened in our laboratory has been successfully expressed in the E.coli expression system,but the lipase activity was not improved obviously.Therefore,the AOL was expressed in Pichia pastoris expression system in order to obtain high lipase activity and the enzymatic properties and catalytic applications were investigated in this study.The results of research are shown as follows:Pichia pastoris X-33/?A-aol engineering strains were successfully constructed.The pPICZ?A expression vector containing aol was electroporated into P.pastoris X-33 competent cells,and the positive transformant X-33/?A-aol engineering strains were screened.Positive recombinants were incubated with methanol at 30?for 4 days.The crude lipase activity was 341 U/mL.SDS-PAGE analysis showed that the molecular weight is about 28 kDa.The 58#strain with the highest lipase activity was screened by Rhoda mine B plate method.To optimize the fermentation condition,the 58#strain was cultivated in 500 mL shake flasks.the optimal fermentation conditions were shown as follows:methanol induced 192 h,the amount of methanol added was 2.0%every 24 h,the initial pH of the culture medium was 7.0,the shaking liquid volume was 50 mL,and the temperature of methanol-induced was 28°C,the enzyme activity was 793 U/mL,which was 2.34 times that of the initial enzyme.The 5 L fermentor was used to scale up the engineered strain by fed batch culture.During the whole fermentation process,the dry weight of the cells reached 311 g/L,and the highest enzyme activity was 4240 U/mL.The protein concentration is15.21 mg/mL and the specific activity of AOL reached a maximum of 432U/mg at 84 h of fermentation.The separation and purification of recombinant lipase AOL were studied.After ultrafiltration,exchange chromatography and hydrophobic interaction chromatography the collected AOL solution volume is 100?L,the protein concentration was 7.50 mg/mL,the total protein amount was 0.75 mg,the total enzyme activity was 4153.99 U and the specific activity was 5,509.27 U/mg.The purification factor reached 72.33-fold,but the recovery of enzyme activity was only 3.77%.A single band of SDS-PAGE showed that the target protein was successfully isolated and purifiedThe enzyme has a certain hydrolysis activity on the substrate of the triacylglyceride with carbon chain length?C2-C18?,the results have shown that the optimum pH was 8.0,and the enzyme had higher activity in the pH range of 7.5-9.0.The optimum temperature was 40°C,and it was stable at 30-50°C.The Kinetic parameter V_max and K_m were determinated as6841.98 U/mg and 1.23 mM.In order to study the effect of metal ions on enzyme activity,different concentration of metal ions were researched.When the concentration at 1 mM,Cu²⁺and Zn²⁺metal ions had the most obvious inhibitory effects.When the metal ion concentration is 5 mM,Zn²⁺almost completely inhibits the lipase activity,and the activity was lower than 20%of the original enzyme activity.Metal ions such as Ca²⁺,Mg²⁺,and Mn²⁺had no significant activation and inhibitory effects on AOL.Theimmobilizedrecombinantlipasewasprepared by covalent-bonding method and applied to the chiral resolution reaction of the R,S-phenoxypropionic acid methyl ester.The optimized enzyme immobilization process was as follows:the ratio of primary amino resin activated by 0.25%glutaraldehyde and enzyme protein was 1:0.2?g/mg?,the concentration of the enzyme solution was 0.04 mg/mL,and the volume of the enzyme solution corresponding to 1 g of the resin was 5 mL,.and then cultured at 150 rpm and 30°C for 1 h in the water bath shaker.The K_m and V_max of the immobilized lipase AOL were 1.97 mM and 4839.35U/mg,respectively.The prepared immobilized enzyme still retained relative activity of more than 85%after repeated use of 15 times.The optimal conditions for the reaction of immobilized enzymatic resolution of R,S-phenoxypropionic acid methyl ester were as follows:the catalytic reaction system included 0.2 mol/L pH 7.5 sodium phosphate buffer,0.5mol/L substrate concentration,the reaction was carried out at 200 rpm and30°C in a water bath shaker for 160 min,the conversion of the R,S-phenoxypropionic acid methyl ester reached 50%,and the e.e._s reached97.2%.