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

1. Speaker:XineYeah,Tang

2. Rhizopusoryzae producing triacylglycerollipase mono- and diacylglycerol lipase (mdlB) derived from Aspergillusoryzae.

3. Introduction R. oryzae, which originally produces a 1,3-position-specific triacylglycerol lipase, is one of the most studied fungi in lipase-catalyzed methanolysis

4. Studies by our group revealed that BSP-immobilized R. oryzae can efficiently catalyze the methanolysis with the addition of a certain amount of water , and that a packed-bed reactor (PBR) using BSPimmobilizedR. oryzae can be used for long-term repeated-batch methanolysis, with the methyl ester (ME) content around 80–90% during 10 batch cycles .

5. Mono- and diacylglycerol lipase is an enzyme that hydrolyzes MGandDG, but doesnothydrolyzetriglyceride (TG). Themono-and diacylglycerollipase-encoding genes have been cloned fromthefilamentous fungi, Penicilliumcamembertii (mdlA) and A. oryzae (mdlB) .

7. Materials and methods Two fungal strains, R. oryzae IFO4697 and A. oryzaeniaD300,were used. Reticulated polyurethane foam (Bridgestone Co. Ltd., Osaka, Japan) with a particle voidageofmore than 97% and a pore size of 50 pores per linear inch (ppi) was cut into 6mm×6mm×3mm cuboids for use asBSPs.

8. Construction of lipase expression vector and transformation of A. oryzae(skip)

9. Results and discussion Substrate specificity of mdlB

10. Effect of water content on methanolysis usingmdlB-producing A. oryzae

11. Composition of reaction mixtures in methanolysis usingmdlB-producing A. oryzae

12. Two-step batch methanolysis in PBR

13. Two-step batch methanolysis in PBR

14. Two-step batch methanolysis in PBR

15. Conclusions In this study, we attempted to use a mono- and diacylglycerol lipase to convert residual partial glycerides efficiently into their corresponding MEs. The newly developed mdlB-producing A.

16. The newly developed mdlB-producing A. oryzaeshowed an effective methanolysis activity toward MG and DG. In the presence of 5% water, the methanolysis of intermediate products using mdlB-producing A. resulting in less than 0.1% of MG and DG.

17. Ten repeated-batch methanolysiscycles in the PBR, containing both R. oryzae and mdlBproducing A. oryzae, maintained a high ME content of over 90% with MG and DG at 0.08–0.69 and 0.22–1.45%, respectively. These results indicate that the use of mdlB-producing A. oryzaeallows maintaining MG and DG at low levels during long-term repeatedbatchmethanolysis. We thus conclude that the proposed method is effective for improving enzymatic biodiesel production.