Gene cloning, expression and characterization of lipase from Fusarium Solani for the production of biodiesel

Abstract

Biodiesel is the product derived from triglycerides by transesterification catalyzed by acid or base. However, biological catalyst such as lipase has become more attractive. The screening for natural microbial lipases with the transesterification activities together with gene technology can therefore be applied to develop and increase the production of large quantities of the enzyme. In this research, the lipase gene was cloned from lipase producing fungus namely, Fusarium solani into the pPICZαA. The result revealed that the gene was composed of 1,002 bp and could be deduced into 333 residues of amino acids. The lipase gene was then transformed and expressed in Pichia pastoris strain KM71. It was found that the incubation in the presence of 3 % of methanol at 30 °C for 5 days was optimal for the induction of the expression. The expressed recombinant lipase was subsequently purified and 2.5 purification folds with the molecular weight of approximately 40 kDa were obtained. The study of substrate specificity showed that the enzyme was specific towards the substrates with carbon chain lengths between 4-14. The optimal conditions were found to be pH 9 at 35-40 °C while the enzyme was stable at pH between 5.0-10.0 and the temperature below 35 °C. The effect of chemicals revealed that the activity of recombinant lipase was not influenced by most studied metal ions and detergents but strongly inhibited by SDS and reducing agent, β- mercaptoethanol. When the recombinant lipase was tested for the hydrolysis of 7 types of plant oils including coconut, rambutan, palm, papaya, olive, physic nuts and safflowers, the highest specific activity of 1.14 μmol/min/mg protein was obtained from the coconut oil as substrate. Finally, the transesterification of 4 types of oils with high hydrolytic activities ie. coconut, rambutan, palm and papaya was investigated. The results indicated that approximately 45% of biodiesel were obtained from rambutan oil which was higher than papaya, palm and coconut respectively. From this study, it could therefore be concluded that lipase gene from Fusarium solani was successfully cloned and expressed. The obtained recombinants could catalyze both the hydrolysis and transesterification for the production of biodiesel.