Reverse micelles in organic solvents: a medium for the biotechnological use of extreme halophilic enzymes at low salt concentration

Abstract

Alkaline p-nitrophenylphosphate phosphatase (pNPPase) from the halophilic archaeobacterium Halobacterium salinarum (previously halobium) was solubilized at low salt concentration in reverse micelles of hexadecyltrimethylammoniumbromide in cyclohexane with 1-butanol as cosurfactant. The enzyme maintained its catalytic properties under these conditions. The thermodynamic “solvation–stabilization hypothesis” has been used to explain the bell-shaped dependence of pNPPase activity on the water content of reverse micelles, in terms of protein–solvent interactions. According to this model, the stability of the folded protein depends on a network of hydrated ions associated with acidic residues at the protein surface. At low salt concentration and low water content (the ratio of water concentration to surfactant concentration; w₀), the network of hydrated ions within the reverse micelles may involve the cationic heads of the surfactant. The bell-shaped profile of the relationship between enzyme activity and w₀ varied depending on the concentrations of NaCl and Mn²⁺.