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

Abstract

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 4 h) 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 were characterized by several physicochemical properties, and multiple linear regression analysis (MLRA) together with non-linear regression 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.

Isolation, Purification, and Optimization of Thermophilic and Alkaliphilc Protease Originating From Hot Water Spring Bacteria

“…N-hexane and toluene caused greater inhibitory effect showing 25% and 29% residual activity for the enzyme. Organic solvents alter the catalytic mechanism by interfering the hydrogen bonding and hydrophobic interactions among active protein leading to unfavorable changes in dynamics and conformation [31]. Previous studies have reported organic solvent-tolerant strains from the genus Pseudomonas and Bacillus producing solvent stable enzymes (Fig.…”

A Highly Stable Biocatalyst Obtained from Covalent Immobilization of a Non-Commercial Cysteine Phytoprotease

“…The general conditions for the immobilization of araujiain on glyoxyl-agarose were selected in function of i) the immobilization of other enzymes on glyoxyl-agarose; ii) previous knowledge on the preparation and characterization of the proteolytic extract from A. hortorum (Asclepiadaceae) fruit latex, and iii) the non-deactivating conditions for the proteases present in the enzymatic extract: the use of high pH values does not affect the performance of araujiain [13,26,28,36]. Since biocatalysts are designed to perform their activities in aqueous medium and in organic media, the pH value is important, since it is well established that enzymes "remember" the pH of the last aqueous solution in which they were dissolved and, after water removal, their ionization state remains unchanged (i.e., the optimum to display its maximum activity) [37].…”

“…Nevertheless, organic solvents may affect the conformational stability of biocatalysts because these may interact with the hydration layer essential for activity and proper folding, or may alter the protein structure by direct interactions with protein solvation sites [51]. It has been previously demonstrated that free araujiain is not inactivated in aqueousimmiscible organic systems and that it has higher activity in these systems than in buffer [36,52,53]. The partition of the organic solvents into the aqueous phase reduces the autolysis degree and produces a considerable activation of araujiain [52].…”

Applied Biocatalysis: An Overview

“…The limitations of such a predictive model may be ascribed to the mechanisms involving biocatalyst/solvent interaction, which are far too complex for solvent biocompatibility to be predicted by a single physical parameter [102,103] . In the design of the bioconversion system, adequate hydrodynamic conditions and phase volume ratio values have to be selected, in order to enhance mass transfer and thus increase the overall reaction rate.…”