Biodiesel Synthesis in a Solvent‐Free System by Recombinant Rhizopus oryzae Lipase. Study of the Catalytic Reaction Progress

Abstract The recombinant 1,3‐positional selective Rhizopus oryzae lipase (rROL) was used to synthesize biodiesel and monoacylglycerols simultaneously. The reaction was carried out in a solvent‐free system with the enzyme immobilized on octadecyl‐Sepabeads. Using response surface methodology, the methyl ester yield was optimized by means of the study of the effect of water, substrate molar ratio (methanol:olive oil) and methanol stepwise addition. It was concluded that in order to prevent enzyme inactivation by methanol, alcohol should be added slowly; otherwise a large amount of water would be present. Taking the best conditions, a 50.3 % yield was achieved in 3 h, which corresponds to 75.4 % of the acyl groups at the 1,3‐position undergoing transesterification. It was also concluded that methyl esters result from the esterification of the free fatty acid hydrolyzed by the enzyme and also from a direct transesterification of oil. In addition, the fatty acid selectivity of rROL was found not to favor one fatty acid in olive oil over another.