Overexpression of lipase from aureobasidium pullulans var. melanogenum sry14-3 and fusarium solani in pichia pastoris and application for wastewater pretreatment

Abstract

The purpose of this study was to clone a lipase gene from Aureobasidium melanogenum and Fusarium solani for increasing lipase production via Pichia pastoris expression system, determine the properties of lipase and apply the recombinant P. pastoris expressing lipase for lipid-containing wastewater pretreatment. The genes of A. melanogenum lipase (AML) and F. solani lipase (FSL) were successfully expressed in P. pastoris using inducible expression system and constitutive expression system under the control of alcohol oxidase 1 promoter (pAOX1) and glyceraldehyde-3-phosphate dehydrogenase promoter (pGAP), respectively. Both of them produced more lipase than the wild-type. The recombinant Pichia expressing AML obtained using the inducible promoter (P. pastoris/pPICZαA-AML) showed specific lipase activity of 9.9 U/mg after 6 days of 2% v/v methanol induction. Optimal lipase activity was observed at 35–37 °C and pH 7.0 using p-nitrophenyl laurate as the substrate. Lipase activity was enhanced by Mg2+, Mn2+, Li+, Ca2+ and Ni2+ (1 mM), DTT and EDTA (5 mM) but was inhibited by Hg2+, Ag+, SDS, Tween 20 and Triton X-100. The recombinant Pichia expressing FSL obtained using the constitutive promoter (P. pastoris/pGAPZαA-FSL) showed the highest specific activity of 18.8 U/mg after 3 days. Optimal lipase activity was observed at 35°C and pH 7.0 using p-nitrophenyl laurate as the substrate. Lipase activity was enhanced by Mn2+, Ba2+, Li+, Ca2+, Ni2+, CHAPS (1 mM) and Triton X-100 (0.1% and 1% v/v) but was inhibited by Hg2+, Ag+ and SDS. Recombinant lipases from P. pastoris/pPICZαA-AML and P. pastoris/pGAPZαA-FSL were stable in both water-miscible and water-immiscible organic solvents. The addition of 10% v/v of octanol and p-xylene increased activity of both lipases. For application in wastewater pretreatment, cultivation of P. pastoris/pGAPZαA-FSL in synthetic wastewater containing 1% w/v palm oil resulted in degradation of 87% of the oil within 72 h. KM and Vmax value of P. pastoris/pGAPZαA-FSL for palm oil degradation were 1% w/v and 38.4 µM TAG degraded/h/OD600, respectively. From the results, P. pastoris expressing FSL by constitutive expression system could be used as an alternative microorganism for the wastewater pretreatment.