Papain in organic solvents: determination of conditions suitable for biocatalysis and the effect on substrate specificity and inhibition.

Synthesis of N-CBZ-(N-Carbobenzoxy)-1-amino-acid methyl esters from N-CBZ-amino acids and methanol has been used as an assay to examine the properties of papain in organic solvents containing small amounts of water. Papain is active in solvents ranging in polarity from acetonitrile to tetrachloromethane. The optimal activity in each solvent varied only about three to four fold, but the amount of added water required to achieve it varied from 4% (v/v) in acetonitrile to 0.05% (v/v) in tetrachloromethane. The enzyme was generally more stable in hydrophobic solvents and at lower water contents. The apparent K(m) value of CBZ-glycine was 26 times higher in acetonitrile than in toluene due to differential partitioning of the substrate between aqueous and organic phases. The substrate specificity of the enzyme was qualitatively little different from that in aqueous solution, with amino acid derivatives still the best substrates. Nitrile analogs of substrates inhibited the enzyme, as they do in aqueous solution, and inhibition by a variety of substituted aromatic hydrocarbons showed that the main specificity of papain for hydrophobic side chains at its S(2) subsite, was little affected. The results show that papain can catalyze reactions under a variety of conditions in organic solvents but its substrate specificity is little changed from that in aqueous media.