Characterization of an immobilized recombinant lipase from Rhizopus oryzae: Synthesis of ethyl-oleate

In recent years there has been a surge of interest in using lipases to catalyze the synthesis of biodiesel, as the biocatalytic route has several advantages over the chemical route. The principal objective of this work was to study the synthesis of ethyl-oleate, as a model ester of biodiesel, using a recombinant lipase of Rhizopus oryzae (rROL) that had been expressed in Pichia pastoris. This lipase was immobilized on the hydrophobic support Accurel MP 1000. Best results were obtained with a protein to support ratio of 15 mg g−1, giving a p-nitrophenyl palmitate-hydrolyzing activity in n-heptane of 220 U g−1. This immobilized enzyme (I-rROL) showed good stability in n-heptane, with a residual activity of 77% after 24 h incubation at 40 °C. The best result for the synthesis of ethyl-oleate, in terms of the specific productivity of esters (i.e. per mass of catalyst preparation), was 10,664 mg h−1 g−1. This result corresponded to 79% conversion of the fatty acid in 30 min and was obtained with 560 mM oleic acid in n-heptane, with a molar ratio of ethanol to oleic acid of 1.4:1, with the ethanol being added in 6 equal aliquots at 5-min intervals. This reaction was done with 30 mg mL−1 of I-rROL at 30 °C. The high productivities obtained in the presence of n-heptane indicate that the recombinant lipase of R. oryzae has a good potential for application in biocatalytic processes undertaken in organic media, however, it appears to be susceptible to denaturation by the alcohol.