Reverse micelles in organic solvents: a medium for the biotechnological use of extreme halophilic enzymes at low salt concentration
FRUTOS C. MARHUENDA-EGEA,1,2 SONSOLES PIERA-VELÁZQUEZ,1,3 CHIQUINQUIRÁ CADENAS,1 and EDUARDO CADENAS 1
1 División de Bioquímica, Facultad de Ciencias, Universidad de Alicante, Apartado 99, 03080 Alicante, Spain
2 Author to whom correspondence should be addressed ([email protected])
3 Thomas Jefferson University, BLSB 511, 233 South 10th Street, Philadelphia PA 19107, USA
Received December 19, 2001; accepted March 8, 2002; published online April 10, 2002
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+.
alkaline phosphatase, archaea, CTAB, Halobacterium.