The reaction kinetics on esterification were assumed to follow the Ping Pong Bi Bi mechanism [13] and [14]. The Ping Pong Bi Bi mechanism with alcohol inhibition is depicted following Cleland’s notation in Fig. 1. In this reaction mechanism, the free fatty GSK461364 (A) first binds to the free enzyme (E) and forms a non-covalent enzyme-acid complex (EA), which releases the first product, water (P) and acylated enzyme (E∗). Next, the second substrate, alcohol (B) reacts with E∗ to give the complex E*B, which is transformed to the complex EQ, and gives the ester (Q) as the final product and the free enzyme (E). At the same time, B can also form a complex [EiB] by binding with free enzyme [E]. The reaction rate on enzymatic esterification of palmitic acid with methanol was fitted to Eq. (1):equation(1)ν=νm[PA][MeOH]Km,MeOH[PA]+Km,PA[MeOH]1+[MeOH]Ki,MeOH+[PA][MeOH]where v is the reaction rate; vm is the maximum reaction rate; Km,PA and Km,MeOH are the Michaelis–Menten’s constants for palmitic acid and methanol, respectively; Ki,MeOH is the inhibition constant for methanol; and [PA] and [MeOH] are the concentrations of palmitic acid and methanol, respectively.