Effects of organic solvents on the enzyme activity of Subtilisin Carlsberg proteas

Proteases are one of the most important groups of industrial enzymes. . Subtilisin (EC 3.4.21.62) is important serine protease Extensive research has been conducted on the stability of proteases in organic solvents, because they have numerous applications in organic media. Organic solvents are advantageous in enzyme-catalyzed peptide synthesis, both to solubilize substrates and product. In this study, the activity and stability of the subtilisin were investigated at different concentrations of organic solvent of ethanol , DMSO and n-hexane. For study of activity and stability of the subtilisin, it was treated with different concentrations of ethanol , n-hexane and DMSO (0-60%) and their combinations(0-10%), then was assayed at 0/65% of casein as substrate, after that Folin Ciocalteau reagent absorbance was measured at 660 nm.The results showed that subtilisin retained more than 82% of its initial activity after 1 h incubation with 10% organic solvent at 40 ºC. Relative activity of the enzyme monotonically decreased with increasing concentration of this organic solvent . The reducing effect of DMSO on enzymatic activity was lower than ethanol and n-hexane.
Organic solvents have effect on electrostatic interactions of proteins because their dielectric constant differs from water. In general reduction in solvent polarity and dielectric constant, lead to loosening the protein structure. Inhibitory effect of solvent on enzyme activity increases with logP elevation, since hexane and ethanol has more inhibitory effect on subtilisin activity than DMSO