Detergentless microemulsions as media for enzymatic reactions: spectroscopic and ultracentrifugation studies
The Journal of Physical Chemistry
Analytical ultracentrifugation and time-resolved fluorescence techniques were used to investigate structure and microenvironment of microemulsion droplets existing in ternary systems composed of n-hexane, isopropyl alcohol, and water. In fluorescence measurements, rhodamine B was used as a molecular probe sensitive to the environment. Well-defined microdroplets were found only inside a limited area in the triangle phase diagram, their molecular weight and radius being in the range of 3000-5000
... altons and 11-13 A, respectively. Density and microviscosity of the droplets correspond to those of a water-isopropyl alcohol binary mixture containing approximately 20 vol % of isopropyl alcohol, whereas the micropolarity of droplets interior was close to the polarity of 60-70 vol % solution of isopropyl alcohol in water. The area of existence of well-defined microemulsion droplets in the phase diagram was found to overlap with previously determined locations of maxima of catalytic activity of several enzymes dissolved in n-hexane-isopropyl alcohol-water ternary systems. From this observation, the conclusion is drawn that the presence of well-defined microdroplets is a prerequisite for achieving high catalytic activity. Fluorescence anisotropy decay measurements of tryptophan in trypsin dissolved in detergentless microemulsions were used to estimate the size of protein-containing microemulsion droplets. The radius was found to be in the range of 27-37 A, depending on the composition of the system. The results suggest that at least 10 empty microdroplets are required to produce a droplet large enough to accommodate a protein molecule. On the basis of obtained results, a structural model of protein-containing microemulsion droplets is proposed. According to this model, an enzyme molecule is surrounded by a water-rich layer of 7-10-A thickness, which serves to protect the enzyme against the denaturing contact with the outer organic phase, thus ensuring the retention of catalytic activity. of the equilibrium of enzymatic reactions towards desired products, etc. (for reviews see ref 1). One of the most promising approaches to conduct biocatalytical processes in nonaqueous media has been developed in the framework of micellar enzymology,2 which deals (1) (a) Martinek, K.; Semenov, A. N. Khmelnitsky, Yu. L.; Levashov, A. V.; Klyachko, N. L.; Martinek, K. Enzyme Microb. Technol., in press.