Control of Subtilisin Substrate Specificity by Solvent Engineering in Organic Solvents and Supercritical Fluoroform

Abstract

We have investigated the effect of physical properties of 30 nonaqueous solvents on the specificity of Subtilisin Carlsberg for nucleophiles in the transesterification of N-acetyl-l-phenylalanine ethyl ester by methanol, 1-propanol, and 1-butanol at fixed thermodynamic water activity. In organic solvents, enzyme activity and nucleophile specificity are solvent-dependent, while in supercritical fluoroform, the activity and specificity are pressure-dependent. Losses in catalytic efficiency and substrate specificity are observed when subtilisin is exposed to hydrophilic organic solvents such as dioxane, tetrahydrofuran, and acetonitrile as compared to hydrophobic solvents (hexane and heptane). Log P is an important descriptor for correlating both the rate and the specificity of deacylation with solvent properties. A linear model of log initial rate against both log P and nonpolar unsaturated area provides the best two-variable fit to the data for solvents of high log P. A nonlinear model of specificity against log P provides the best fit for the complete data set. Correcting the activity for partitioning of nucleophilic substrates shows a similar trend for the intrinsic activity dependence of nucleophiles as a function of log P. In propane, under subcritical conditions, there is no significant effect of pressure on either the activity or the nucleophilic specificity of subtilisin. In fluoroform, however, where the physical properties of the solvent are pressure-dependent, the specificity of the enzyme is solvent density-dependent.

Effect of Treatment with Compressed Propane on Lipases Hydrolytic Activity

“…In fact, enzyme stability and activity may depend on the enzyme species, the characteristics of the compressed fluid, the water content of the enzyme/support/reaction mixture, and the process variables involved, which means that very distinct effects can be achieved depending on the characteristics of the system under investigation (Chaudhary et al 1996;Kamat et al 1995;Kamat et al 1993;Kamat et al 1992;Taniguchi et al 1987;Tedjo et al 2000;Chen et al 1992;Ishikawa et al 1996;Oliveira et al 2006a;Oliveira et al 2006b;Gieβauf and Gamse 2000;Fricks et al 2006).…”

Peptide synthesis: chemical or enzymatic

“…Medium engineering refers to the rational manipulation of the reaction medium to positively influence the properties of the enzyme with respect to the reaction of synthesis (Clapés et al 1990a;Clapés et al 1990b;Khmelnitsky et al 1991;Ryu and Dordick, 1992;Wescott and Klibanov, 1994;Carrea et al 1995, Chaudhary et al 1996. This frequently implies the substitution of the usual aqueous medium for a non conventional medium in which water has been replaced partially or almost totally by another solvent (Hari Krishna, 2002).…”

Enzymatic catalysis in cosolvent modified pressurized organic solvents

“…The minimum amount of Na 4 P 2 O 7 .10H 2 O required to buffer the a w of the system was inferred from the measurement of enzyme activity as a function of salt concentration (Chaudhary et al, 1996;Kamat et al, 1995a). Hexane had a very low initial water content and hence would absorb enough water to form the anhydrous species of the salt requiring the addition of Na 4 P 2 O 7 .10H 2 O only (Halling, 1994).…”

“…The rate and selectivity of enzymatic reactions in organic solvents may be directly affected by solvent strength (Chaudhary et al, 1996) or influenced by indirect measures of solvent strength including thermodynamic water activity (a w ) and the solubility of the substrate (e.g., Kamat et al, 1993;Martins et al, 1992;Marty et al, 1990;Rantakylä and Aaltonen, 1994;Valivety et al, 1991). Enzymatic catalysis in SCFs exploits the ability to dramatically alter solvent strength with small changes in temperature and pressure in the near-critical region.…”