Kinetic studies of an extremely halophilic enzyme entrapped in reversed micelles

Alkaline p-nitrophenylphosphate phosphatase (pNPPase) from the halophilic archaeobacterium Halobacterium salinarum (earlier halobium) was solubilised in reversed micelles of hexadecyltrimethylammoniumbromide (CTAB) in cyclohexane, with l-butanol as co-surfactant. The hydrolysis of p-nitrophenylphosphate (pNPP) appeared to follow Michaelis-Menten kinetics. K-m and V-max depended on the method of reaction initiation. The kinetic parameters of halophilic pNPPase in CTAB reversed micelles with high salt concentration (0.85 M NaCl) were determined. pNPPase showed the same dependency on the buffer ionic strength in reversed micelles as in aqueous macrosolution. The dependence of the maximum reaction rate (V-max) on the molar water/surfactant ratio two value) showed a bell-shaped curve for NaCl and KCl, with a maximum reaction rate being found at omega(0) = 10.27. The pH value for the maximum reaction rate was 9.0. The optimum temperature for enzyme activity was around 45 degrees C.