Mechanism of adaptation of an atypical alkaline p-nitrophenyl phosphatase from the archaeon Halobacterium salinarum at low-water environments.

Enzymes suspended in organic solvents represent a versatile system for studying the involvement of water in catalytic properties and their flexibility in adapting to different environmental conditions. The extremely halophilic alkaline p-nitrophenylphosphate phosphatase from the archaeon Halobacterium salinarum was solubilized in an organic medium consisting of reversed micelles of hexadecyltrimethylammoniumbromide in cyclohexane, with 1-butanol as cosurfactant. Hydrolysis of p-nitrophenylphosphate was nonlinear with time when the enzyme was microinjected into reversed micelles that contained substrate. These data are consistent with a kinetic model in which the enzyme is irreversibly converted from an initial form to a final stable form during the first seconds of the encapsulation process. The model features a rate constant (k) for that transition and separate hydrolysis rates, v(1) and v(2), for the two forms of the enzyme. The enzyme conversion may be governed by the encapsulation process.

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

Alkaline p-nitrophenylphosphate phosphatase (pNPPase) from the halophilic archaeobacterium Halobacterium salinarum (previously halobium) was solubilized at low salt concentration in reverse micelles of hexadecyltrimethyl-ammoniumbromide in cyclohexane with 1-butanol as co-surfactant. 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+.

An extreme halophilic enzyme active at low salt in reversed micelles.

Possible biotechnological applications of extreme halophilic enzymes are strongly determined by their high salt requirement of around 4 M NaCl. Consequently, the use of these in organic media seemed to be unlikely. However, we have succeeded in dissolving a halophilic enzyme, p-nitrophenylphosphate phosphatase from the archaeon Halobacterium salinarum, in an organic medium by creating a reverse micellar system with very low salt concentration. The enzyme retained its catalytic properties in reversed micelles made with an anionic surfactant (dioctyl sodium sulphosuccinate) or with a cationic surfactant (hexadecyltrimethylammonium bromide) in cyclohexane plus 1-butanol as co-surfactant. The dependence of the rate of hydrolysis of p-nitrophenylphosphate phosphate on the molar water/surfactant ratio (w(0) value) showed a bell-shaped curve for each surfactant system. Kinetic parameters were determined in each system. The enzymatic reaction appeared to follow Michaelis-Menten kinetics with the anionic surfactant only. The kinetic behaviour was determined at different concentrations of Mn(2+) in reversed micelles of dioctyl sodium sulphosuccinate as surfactant.