1. High-pressure stabilization of alpha-chymotrypsin entrapped in reversed micelles of aerosol OT in octane against thermal inactivation
R V Rariy, N Bec, J L Saldana, S N Nametkin, V V Mozhaev, N L Klyachko, A V Levashov, C Balny FEBS Lett. 1995 May 1;364(1):98-100. doi: 10.1016/0014-5793(95)00344-9.
alpha-Chymotrypsin (CT) solubilized in reversed micelles of sodium bis-(2-ethylhexyl)-sulfosuccinate (AOT) undergoes thermal inactivation and the enzyme stability decreases significantly when temperature increases (25-40 degrees C). The half-life of CT in micelles shows a bell-shaped dependence on the degree of hydration of AOT (wo) analogous to the previously obtained dependence on wo for the enzyme activity. The optima of catalytic activity and thermal stability have been observed under conditions where the diameter of the inner aqueous cavity of the micelle is close to the size of the enzyme molecule (wo = 10). Application of high hydrostatic pressure in the range of 1-1500 atm (bar) stabilizes CT against thermal inactivation at all hydration degrees (wo) from 7 to 20; the stabilization effect is most pronounced under the experimental conditions being far from the optimum for catalytic activity.
2. Reversed micelles of polymeric surfactants in nonpolar organic solvents. A new microheterogeneous medium for enzymatic reactions
Y L Khmelnitsky, A K Gladilin, V L Roubailo, K Martinek, A V Levashov Eur J Biochem. 1992 Jun 15;206(3):737-45. doi: 10.1111/j.1432-1033.1992.tb16980.x.
A new microheterogeneous non-aqueous medium for enzymatic reactions, based on reversed micelles of a polymeric surfactant, was suggested. The surfactant termed CEPEI, was synthesized by successive alkylation of poly(ethyleneimine) with cetyl bromide and ethyl bromide and was found to be able to solubilize considerable amounts of water in benzene/n-butanol mixtures. The hydrodynamic radius of polymeric-reversed micelles was estimated to be in the range 22-51 nm, depending on the water content of the system, as determined by means of the quasi-elastic laser-light scattering. Polymeric reversed micelles were capable of solubilizing enzymes (alpha-chymotrypsin and laccase) in nonpolar solvents with retention of catalytic activity. Due to the strong buffering properties of CEPEI over a wide pH range, it could maintain any adjusted pH inside hydrated reversed micelles. It was found that catalytic behavior of enzymes entrapped in polymeric reversed micelles was rather insensitive to the pH of the buffer solution introduced into the system as an aqueous component, but determined mostly by acid-base properties of the polymeric surfactant itself. Both catalytic activity and stability of entrapped alpha-chymotrypsin and laccase were found to increase with increasing water content of the system. Under certain conditions, the entrapment of alpha-chymotrypsin into CEPEI reversed micelles resulted in a considerable increase in catalytic activity and stability as compared to aqueous solution. CEPEI reversed micelles were demonstrated to be promising enzyme carriers for use in membrane reactors. Owing to the large dimensions of CEPEI reversed micelles, they are effectively kept back by a semipermeable membrane, thus allowing an easy separation of the reaction product and convenient recovery of the enzyme.
3. [Thermodynamics changes in catalytic properties of immobilized enzymes]
L V Kozlov, L Ia Bessmertnaia Biokhimiia. 1982 Feb;47(2):179-83.
Study of the temperature dependence of hydrolysis of the ethyl ester N-acetyl-L-tyrosine and p-nitroanilide N-succinyl-L-phenylalanine by chymotrypsin and polymaleic acid-modified chymotrypsin showed that the enthalpy and enthropy of dissociation of the enzyme-substrate complex and those of activation of the enzyme acylation in the course of catalysis increase during the native enzyme transition to its modified form. The data obtained can be explained terms of changes in the native conformation of the enzyme during its modification, which is confirmed by the previously obtained data on thermodenaturation. Some part of the energy of substrate binding is consumed for changing the conformation of the modified protein, rendering it close to the native one and thus providing a further enzymatic catalysis which is similar to that by the native enzyme.
