Effect of Water Content on the Thermal Stability of Chromobacterium viscosum Lipase in AOT/isooctane Reverse Micelles

Abstract

According to the different reaction systems such as aqueous media, glycerol pool, and reverse micelles, changes in thermal stability and enzyme-deactivation kinetic parameters were investigated in this study, employing glycerolysis and hydrolysis catalyzed by Chromobacterium viscosum lipase and two-step series-type deactivation model. Thermal stability of the lipase (triacylglycerol hydrolase, EC 3.1.1.3) was found to be increased by the enzyme-entrapment in AOT/isooctane reverse micelles. The half-life (6.81 hr) of Chromobacterium viscosum lipase entrapped in reverse micelles at 70°C was 9.87- and 14.80-fold longer than those solubilized in a glycerol pool and in 50 mM Tris-HCl buffer (pH 8.0), respectively. The enzyme deactivation model considering a two-step series-type was employed and deactivation constants for the first and second step (k1 and k2) at all temperatures were drastically decreased after the lipase was entrapped in reverse micelles. In particular, k1 (3.84 hr-1) at 70°C in reverse micelles was 1.57-fold lower comparing to that in the aqueous buffer (6.03 hr-1). The deactivation energies (from k1, k2) for the lipase entrapped in the reverse micelles, solubilized in a glycerol pool, and in the aqueous buffer were (12.80 kcal/mol, 32.98 kcal/mol), (11.93 kcal/mol, 32.24 kcal/mol), and (11.65 kcal/mol, 28.10 kcal/mol), respectively. Based on the experiments in hydrolysis, it was also found to be consistent with these phenomena indicate the lipases in reverse micelles were more stable against thermal treatment than those in aqueous media. From the structural analysis of thermally-deactivated lipase through fluorescence spectrometry to detect the conversion of aromatic residues, the amount of excited forms of tryptophan and tyrosine increased markedly in the case of thermal-treatment in the aqueous buffer, whereas there was no significant fluctuation in the reversed micellar system.