Structure and Function of Subtilisin BPN‘ Solubilized in Organic Solvents

Abstract

Enzyme structure and function have been studied for subtilisin BPN‘ solubilized in organic solvents by ion pairing with low concentrations of an anionic surfactant (Aerosol OT) in the absence of reversed micelles. Soluble subtilisin shows strikingly different behavior in octane and tetrahydrofuran (THF). In octane, the k cat/K m for the transesterification of N-acetyl-l-phenylalanine ethyl ester (APEE) is 370 M-1 s-1, within one order of magnitude of the enzyme's hydrolytic activity in water. Moreover, the observed half-life of the soluble enzyme in octane is nearly three orders of magnitude greater than in water, presumably because of the absence of autolysis in the organic solvent. In contrast, the catalytic efficiency of the enzyme dissolved in the polar solvent THF is 0.04 M-1 s-1, and the enzyme loses 99% of its activity within 10 min. Comparable enzyme inactivation could also be observed in octane, but only at elevated temperatures such as 70 °C. Therefore, the mechanisms of deactivation of the soluble enzyme were investigated in both octane and THF. Kinetic and spectroscopic (CD and EPR) studies support the existence of multiple inactive forms of the soluble enzyme in THF at 25 °C and in octane at 70 °C. Notably, in both cases a denatured form can be renatured in anhydrous octane at 25 °C, the first demonstration of enzyme renaturation in a bulk organic solvent. A model explaining the THF- and thermally-induced inactivation processes of soluble subtilisin BPN‘ is proposed, and the apparent reasons for the exceptionally high activity and stability of the soluble enzyme in octane are discussed.

Enzyme Stability and Activity in Non-Aqueous Reaction Systems: A Mini Review

“…Although these re-orientations of side chains do not necessarily alter the active site of the enzyme, they affect the enzyme activity by changing the substrate affinity and specificity, as well as the hydration of enzyme. For example, Pramod et al [28] reported that although octane almost had no influence on the secondary and tertiary structures of subtilisin BPN', the catalytic efficiency k cat /K m of the enzyme in octane was only 10.6% of that in aqueous solution. Nevertheless, the stability of subtilisin BPN' in octane was 645 fold of that in aqueous solution, owing to the absence of autolysis in octane.…”

Effect of crown ethers on structure, stability, activity, and enantioselectivity of subtilisin Carlsberg in organic solvents

“…To counter this limitation, many methods have been introduced leading to improved enzyme activity in organic solvents. For example, control of the pH value (Yang et al, 1993), colyophilization with lyoprotectants (Dabulis and Klibanov, 1993) and salts (Khmelnitsky et al, 1994;Ru et al, 1999), addition of water-mimicking agents Kitaguchi et al, 1990), imprinting with substrates and substrate analogs (Rich and Dordick, 1997;Russell and Klibanov, 1988), immobilization (Orsat et al, 1994;Petro et al, 1996;Ruiz et al, 2000), solubilization Okahata et al, 1995a,b;Paradkar and Dordick, 1994;Wangikar et al, 1997;Xu et al, 1997), mutagenesis (Chen and Arnold, 1993), and solvent precipitation (Dai and Klibanov, 1999) represent methods that have been successful for improving the catalytic activity of enzymes. One of the most successful groups of activating additives identified thus far are macrocyclic compounds, which includes cyclodextrins Ooe et al, 1999;Santos et al, 1999) and crown ethers (Broos et al, 1995a;Engbersen et al, 1996;Itoh et al, 1996;Reinhoudt et al, 1989;van Unen, 2000;van Unen et al, 1998van Unen et al, , 2001.…”

Testing for diffusion limitations in salt-activated enzyme catalysts operating in organic solvents

“…A great deal of effort has therefore been devoted to the engineering of biocatalysts and biocatalytic processes for increased activity in organic solvents. Techniques for improving nonaqueous enzyme activity include: protein engineering, both site-directed (Wangikar et al, 1993Xu et al, 1994) and random (Economou et al, 1992); effective control of catalyst water activity (Bell et al, 1995); immobilization (Adlercreutz, 1996;Clark, 1994;Suzawa et al, 1995); solvent engineering (Parida and Dordick, 1993;Wescott and Klibanov, 1994;Yang and Russell, 1996); ultrasound irradiation (Lin and Liu, 1995;Vulfson et al, 1991); enzyme solubilization (Meyer et al, 1995;Paradkar and Dordick, 1994;Wangikar et al, 1997); and lyophilization in the presence of various excipients. Among the various approaches used, lyophilization in the presence of excipients may be the simplest, and several variations have been employed including the addition of lyoprotectants (Dabulis and Klibanov, 1993) and substrates (Rich et al, 1995;Rich and Dordick, 1997).…”