Substrate specificity of commercial lipases activated by a hydration–aggregation pretreatment in anhydrous esterification reactions

Substrate specificity in non-aqueous esterification catalyzed by commercial lipases activated by hydration–aggregation pretreatment was investigated. Four microbial lipases from Rhizopus japonicus, Burkholderia cepacia, Rhizomucor miehei, and Candida antarctica (fraction B) were used to study the effect of the carbon chain length of saturated fatty acid substrates on the esterification activity with methanol in n-hexane. Hydration–aggregation pretreatment had an activation effect on all lipases used, and different chain length dependencies of esterification activity for lipases from different origins were demonstrated. The effects of various acidic substrates with different degrees of unsaturation, aromatic rings, and alcohol substrates with different carbon chain lengths on esterification activity were examined using R. japonicus lipase, which demonstrated the most remarkable activity enhancement after hydration–aggregation pretreatment. Furthermore, in the esterification of myristic acid with methanol catalyzed by the hydrated–aggregated R. japonicus lipase, maximum reaction rate (5.43 × 10⁻⁵ mmol/(mg-biocat min)) and Michaelis constants for each substrate (48.5 mM for myristic acid, 24.7 mM for methanol) were determined by kinetic analysis based on the two-substrate Michaelis-Menten model.