Effect of the addition of a nonaqueous polar solvent (glycerol) on enzymatic catalysis in reverse micelles. Hydrolysis of 2-naphthyl acetate by alpha-chymotrypsin.

The kinetics of hydrolysis of 2-naphthyl acetate (2-NA) catalyzed by alpha-chymotrypsin (alpha-CT), in reverse micellar solutions formed by glycerol (GY)-water (38% v/v) mixture/sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/n-heptane has been determined by spectroscopic measurements. To compare the efficiency of this reaction with that observed in micelles with water in the core, as well as in the corresponding homogeneous media, the reaction was also studied in water/AOT/n-heptane reverse micellar solutions and in both homogeneous media (water and GY-water, 38% v/v mixture). In every media, alpha-CT was characterized by the absorption and emission spectra, the fluorescence lifetimes, and the fluorescence anisotropy of its tryptophan residues. The effect of AOT concentration on the kinetic parameters obtained in the micellar systems was determined, at a constant molar ratio of the inner polar solvent and surfactant. Moreover, the data obtained allowed the evaluation of the 2-NA partition constant between the organic and the micellar pseudophase. It is shown that the addition of GY to the micelle interior results in an increase in the catalytic properties of alpha-CT. The fluorescence anisotropy studies in the different media show that the addition of GY increases the viscosity as compared with the aqueous systems. It seems that the GY addition to the reverse micellar aggregates results in a decrease of the conformational mobility of alpha-CT, which leads to an increase of the enzyme stability and activity.

[1]  E. Lissi,et al.  Exploratory study of the effect of polar solvents upon the partitioning of solutes in nonaqueous reverse micellar solutions , 2003 .

[2]  Sajja Hari Krishna,et al.  Developments and trends in enzyme catalysis in nonconventional media. , 2002, Biotechnology advances.

[3]  N. M. Correa,et al.  Acid−Base and Aggregation Processes of Acridine Orange Base in n-Heptane/AOT/Water Reverse Micelles , 2002 .

[4]  S. M. Andrade,et al.  Structural changes of α-chymotrypsin in reverse micelles of AOT studied by steady state and transient state fluorescence spectroscopy , 2001 .

[5]  E. Lissi,et al.  A procedure for the joint evaluation of substrate partitioning and kinetic parameters for reactions catalyzed by enzymes in reverse micellar solutions. I. Hydrolysis of 2-naphthyl acetate catalyzed by lipase in sodium 1,4-bis(2-ethylhexyl) sulphosuccinate (AOT)/buffer/heptane. , 2001, Archives of biochemistry and biophysics.

[6]  Luzia P. Novaki,et al.  Solubilization of Pure and Aqueous 1,2,3-Propanetriol by Reverse Aggregates of Aerosol−OT in Isooctane Probed by FTIR and 1H NMR Spectroscopy , 2001 .

[7]  E. Lissi,et al.  A General Treatment for Meaningful Comparison of Rate Parameters of Enzyme-Catalyzed Reactions in Aqueous and Reverse Micellar Solutions , 2000 .

[8]  J. Cabral,et al.  Reverse micelles as reaction media for lipases. , 2000, Biochimie.

[9]  Das,et al.  Activity and Conformation of Yeast Alcohol Dehydrogenase (YADH) Entrapped in Reverse Micelles. , 2000, Journal of colloid and interface science.

[10]  G. R. Castro.  Properties of soluble α-chymotrypsin in neat glycerol and water , 2000 .

[11]  N. M. Correa,et al.  FTIR and 1H NMR Studies of the Solubilization of Pure and Aqueous 1,2-Ethanediol in the Reverse Aggregates of Aerosol-OT , 2000 .

[12]  N. M. Correa,et al.  Properties of AOT Aqueous and Nonaqueous Microemulsions Sensed by Optical Molecular Probes , 2000 .

[13]  J. Cabral,et al.  An Integrated Model for Enzymatic Reactions in Reverse Micellar Systems: Nominal and Effective Substrate Concentrations , 2000 .

[14]  E. Lissi,et al.  INTERACTIONS OF SMALL MOLECULES WITH REVERSE MICELLES , 1999 .

[15]  S. P. Moulik,et al.  Structure, dynamics and transport properties of microemulsions , 1998 .

[16]  N. Bec,et al.  Thermobarostability of alpha-chymotrypsin in reversed micelles of aerosol OT in octane solvated by water-glycerol mixtures. , 1998, Biotechnology and bioengineering.

[17]  Silber,et al.  Micropolarity of Reversed Micelles: Comparison between Anionic, Cationic, and Nonionic Reversed Micelles , 1996, Journal of colloid and interface science.

[18]  D. Birch,et al.  The fluorescence and circular dichroism of proteins in reverse micelles: application to the photophysics of human serum albumin and N-acetyl-L-tryptophanamide. , 1996, Biophysical chemistry.

[19]  Yoshikazu Miyake.  Enzymatic reaction in water-in-oil microemulsions , 1996 .

[20]  L. Setti,et al.  Superactivity of peroxidase solubilized in reversed micellar systems , 1995, Applied biochemistry and biotechnology.

[21]  A. Maitra,et al.  Solution behaviour of Aerosol OT in non-polar solvents , 1995 .

[22]  N. M. Correa,et al.  Micropolarity of Reverse Micelles of Aerosol-OT in n-Hexane , 1995 .

[23]  A. Maitra,et al.  The effect of droplet dynamics on the enzyme kinetic reactions by yeast alcohol dehydrogenase in reverse micelles , 1994 .

[24]  S. P. Moulik,et al.  Dynamics and thermodynamics of aerosol OT-aided nonaqueous microemulsions , 1994 .

[25]  M. Teramoto,et al.  Enzymatic reaction in water-in-oil microemulsions. Part 2.—Rate of hydrolysis of a hydrophobic substrate, 2-naphthyl acetate , 1994 .

[26]  B. Binks,et al.  Adsorption and aggregation of sodium bis(2-ethylhexyl) sulfosuccinate in systems containing toluene plus water, 1,2-ethanediol, or 1,2,3-propanetriol , 1993 .

[27]  A. Maitra,et al.  Activity and stability of yeast alcohol dehydrogenase (YADH) entrapped in aerosol OT reverse micelles. , 1992, Biotechnology and bioengineering.

[28]  E. Lissi,et al.  Incorporation of n-alkanols in reverse micelles in the AOT/n-heptane/water system , 1992 .

[29]  E. Ruckenstein,et al.  On the enzymatic superactivity in ionic reverse micelles , 1990 .

[30]  M. Vermuë,et al.  Description of enzyme kinetics in reversed micelles. 1. Theory. , 1990, European journal of biochemistry.

[31]  P. Luisi,et al.  Reverse micelles as hosts for proteins and small molecules. , 1988, Biochimica et biophysica acta.

[32]  R. Freedman,et al.  Kinetic properties of α-chymotrypsin in water-in-oil microemulsions: studies with a variety of substrates and microemulsion systems , 1985 .

[33]  R. Freedman,et al.  Reactivity of α-chymotrypsin in water-in-oil microemulsions , 1984 .

[34]  K. Martínek,et al.  Ultracentrifugation of reversed micelles in organic solvent: new approach to determination of molecular weight and effective size of proteins. , 1981, Analytical biochemistry.

[35]  K. Gekko,et al.  Mechanism of protein stabilization by glycerol: preferential hydration in glycerol-water mixtures. , 1981, Biochemistry.

[36]  P. Luisi,et al.  Micellar solubilization of biopolymers in organic solvents. 5. Activity and conformation of .alpha.-chymotrypsin in isooctane-AOT reverse micelles , 1981 .

[37]  K. Martínek,et al.  The principles of enzyme stabilization. VI. Catalysis by water-soluble enzymes entrapped into reversed micelles of surfactants in organic solvents. , 1981, Biochimica et biophysica acta.

[38]  P. Luisi,et al.  Micellar Solubilization of Biopolymers in Hydrocarbon Solvents III. Empirical Definition of an Acidity Scale in Reverse Micelles , 1980 .

[39]  F. Menger,et al.  Enzyme catalysis in water pools , 1979 .