Design and mechanistic insights of water-regulated lipase activation in promoting esterification process for the bio-production of diacylglycerol: Experimental optimization and molecular dynamics simulations

Initial water additions, which serve as a critical variable for regulating the synthesis of diacylglycerol (DAG) from oleic acid esterification reaction, have not been sufficiently investigated regarding the molecular mechanism by which lipase is affected. This study elucidated that the initial water additions effectively modulated the conformational flexibility of the lipase at the interface, promoting the esterification process and enhancing the glycerol dioleate yield. Subsequently, a multi-scale research methodology was employed, gradually revealing the water-mediated catalytic mechanism. The results demonstrated that a 2 wt.% initial water addition significantly increased both esterification conversion rate and esterase activity. Moreover, secondary structure (α-helix increased) and tertiary structure (fluorescence intensity enhanced) indicated that an appropriate amount of water contributed to maintaining the conformational integrity of the enzyme molecules. Additionally, molecular dynamics (MD) simulations revealed that DAG synthesis was influenced by the initial water additions through the promotion of further opening of the lipase lid at the oil-water interface. This article systematically showed how water-regulated interfacial activation of lipase controlled the esterification process, thereby providing a theoretical foundation for the directed synthesis of structured acylglycerols.