Use of mono- and diacylglycerol lipase as immobilized fungal whole cells to convert residual partial glycerides enzymatically into fatty acid methyl esters

The accumulation of partial glycerides such as monoglyceride (MG) and diglyceride (DG) is one of the rate-limiting steps in plant oil methanolysis catalyzed by Rhizopus oryzae producing triacylglycerol lipase. To convert partial glycerides efficiently into their corresponding methyl esters (MEs), we attempted to use a mono- and diacylglycerol lipase (mdlB) derived from Aspergillus oryzae. By considering cost efficiency, R. oryzae and recombinant mdlB-producing A. oryzae were immobilized independently within polyurethane foam biomass support particles and directly utilized as a whole-cell biocatalyst. The mdlB-producing A. oryzae effectively exhibited substrate specificity toward MG and DG and was then used for the methanolysis of intermediate products (approximately 82% ME), which were produced using R. oryzae. In the presence of 5% water, the use of mdlB-producing A. oryzae resulted in less than 0.1% of MG and DG, whereas a considerable amount of triglyceride was present in the final reaction mixture. On the basis of these results, we developed a packed-bed reactor (PBR) system, which consists of the first column with R. oryzae and the second column containing both R. oryzae and mdlB-producing A. oryzae. Ten repeated-batch methanolysis cycles in the PBR maintained a high ME content of over 90% with MG and DG at 0.08–0.69 and 0.22–1.45%, respectively, indicating that the PBR system can be used for long-term repeated-batch methanolysis with partial glycerides at low levels. The proposed method is therefore effective for improving enzymatic biodiesel production.