Dependency of water concentration on ethanolysis of trioleoylglycerol by lipases

The effects of water concentration on ethanolysis of trioleoylglycerol catalyzed by four different lipases were studied. The target product of the ethanolysis was 2-monooleoylglycerol (2-MO). Novozym 435 (a commercially available preparation of immobilized Candida antarctica lipase B, CALB) exhibited both the highest product yield and the reaction rate at very low (less than 1 wt.%) free water concentration. Its catalytic activity did not drop even in dry state, i.e. in the system of dry CALB in dry ethanol (water concentration was ca. 0.1 wt.%). In contrast, other three immobilized lipases tested (Rhizomucor miehei lipase, Burkholderia cepacia lipase and Thermomyces lanuginosus lipase) required larger amounts of free water (ca. 7–9 wt.%) for their best performance and exhibited no ethanolysis reaction at low free water concentrations. The CALB’s anomalous behavior was also observed in other two different preparations of CALB; i.e., free CALB powder and silica-immobilized CALB as well as Novozym 435. Thus, it was confirmed that no extra water requirement of CALB was an intrinsic property of CALB itself. No extra water requirement of CALB implies that this enzyme is able to keep water molecules tightly to retain its catalytic activity even in dry ethanol (a water-depriving solvent). It is suggested that some structural features, such as a carbohydrate molecule, a lid-like domain, or very tight bonding of the essential water molecule(s), might be involved in this very unique property.