Study of phytoproteases stability in aqueous-organic biphasic systems using linear free energy relationships

In this paper we study the effect of different water-immiscible organic solvents (benzene, toluene, 1-butanol, 1-octanol, dichloroethane, dichloromethane, diethyl ether, hexane, chlorobenzene, acetophenone, n-dodecane, trichloroethylene, ethyl acetate) on the stability (residual caseinolytic activity after 4h) of soluble phytoproteases, such as araujiain, funastrain and papain in aqueous-organic biphasic systems. Besides, the effect of organic solvents on enzymatic catalysis was quantitatively studied by means of linear free energy relationships (LFERs). The organic solvents werecharacterized byseveral physicochemicalproperties, and multiple linear regression analysis (MLRA)togetherwithnon-linearregression were the methods used to search the relationships between the residual caseinolytic activity data and several physicochemical parameters. Those enzymes show much greater activity and stability in some biphasic media than in water. On the other hand, all developed correlations represented highly significant LFERs models and showed that non-specific polar and hydrophobic factors are of prime and approximately equal importance for the biocatalytic activity of araujiain, funastrain and papain in the studied biphasic systems, while the specific polar interactions are of little importance for activity. The results suggested that araujiain, funastrain and papain do not suffer unfolding in the studied biphasic media and they are able to retain their native or native-like configurations, though with altered characteristics or properties. This fact was demonstrated by means of a comparative FTIR spectroscopy study in both, buffer and biphasic media, for each studied enzyme.Fil: Barberis, Sonia Esther. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis; Argentina. Universidad Nacional de San Luis. Facultad de Química, Bioquímica y Farmacia. Departamento de Farmacia. Laboratorio de Bromatología; ArgentinaFil: Quiroga, Evelina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis; Argentina. Universidad Nacional de San Luis. Facultad de Química, Bioquímica y Farmacia. Departamento de Farmacia. Laboratorio de Bromatología; ArgentinaFil: Morcelle del Valle, Susana Raquel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Laboratorio de Investigación de Proteínas Vegetales; ArgentinaFil: Priolo, Nora Silvia. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Laboratorio de Investigación de Proteínas Vegetales; ArgentinaFil: Luco, Juan Maria. Universidad Nacional de San Luis. Facultad de Química, Bioquímica y Farmacia. Departamento de Química; Argentin