Water may be a “lubricant” that maintains enzymes in theiractive conformation [23]. During the transesterification reaction,water is necessary to maintain the enzyme configuration and toincrease the available interfacial area between water and oil [20].With increasing water addition, the amount of water available foroil to form oil–water droplets increases, which helps to maintainlipase activity [17]. The reaction rate can be reduced by a water content that is either too low, which results in a decreased contactarea between the lipase and substrate, or too high, which results ina dilution of the lipase concentration [41]. In addition, it was found that the highest catalytic activity of enzyme could be achieved atthe optimum water content, which depends on the enzyme, there action medium composition, the feedstock oil and the type ofenzyme-catalyzed reaction [20,23]. There fore, appropriate water content is an essential parameter in enzymatic biodiesel synthe-sis. Thus, the optimal water content (quality ratio of water to oil)for biodiesel production was determined (Fig. 7e). By increasing the water content from 2.5% to 12%, the biodiesel yield increasedfrom 55.32% to 90.66%, while decreased slightly from 88% to 87.03% when the water content increased to 20%, and decreased substantially to 37.36% at a water content of 35%. The optimal water content(12%) of lipase SL-4 is slightly higher than those of P. aeruginosa42A2 lipase (10%) [21] and soluble Aspergillus niger lipase NS81020(10%) [26], and obviously lower than that of liquid recombinant C.rugosa lipase 2 (30–50%) [20]. The subsequent decrease in yield athigher water contents may be caused by diffusive limitations of thesubstrates. Furthermore, the presence of excessive water can bring about the accumulation of catalysts, which increases the biodiesel viscosity and reduces the reaction efficiency [23]. More water couldalso increase the hydrolysis of FAMEs and decrease the biodiesel conversion rate [45]. Based on this analysis, 12% water content wasused in the following experiments.