Several variants derived from the thermostable subtilisin 8397 were made in order to create an enzyme that is more stable toward organic solvents or has a broader specificity for the P-1’ residue in amidation or is more effective for peptide segment ligation in aqueous solution. To improve the stability in organic solvents, one of three surface charges was removed each time from 8397 to create the variants : Lys43 --> Asn (K43N), Lys256 --> Tyr (K256Y), and Asp181 --> Asn (D181N). Although the stabilities of these variants in high concentrations of hydrophilic organic solvents were higher than that of the wild-type enzyme, the D181N variant was less stable than the 8397 variant. It appears that removal of isolated surface charges does not necessarily improve the enzyme stability in polar organic solvents. A dramatic change of the enzyme stability in dimethylformamide (DMF) was, however, observed in the presence of different counterions. Subtilisin BPN’ lyophilized from Tris-HCl buffer (50 mM, pH 8.4) and suspended in DMF (solid partially soluble), for example, was completely inactivated in 30 min at 25 degrees C, while the enzyme still retained about 70% of the original activity in a week if lyophilized from sodium phosphate buffer (50 mM, pH 8.4) (solid completely insoluble in DMF). In general, the enzyme lyophilized from organic buffers deactivates in DMF much faster than that from inorganic buffers. A similar counterion effect was observed with other variants. These studies suggest that subtilisins are very unstable when exposed directly to DMF; the stability is, however, markedly improved if the enzyme is protected by water or salts from contact with the solvent. To use subtilisins and variants in transesterification or aminolysis in organic solvents, water (3-30%) is usually present in order to have significant reactivity, and for transesterifications, it was found that a good rate and yield could be achieved in ethanol containing 30% water. For use in peptide segment ligation in aqueous solution, the active-site serine of subtilisin 8397/C206Q was converted chemically to cysteine, forming thiosubtilisin 8397/C206Q, and the aminolysis:hydrolysis ratio was found to be several orders of magnitude higher than that for subtilisin BPN’ and comparable to that for thiosubtilisin BPN’. The 8397 variant was also modified at the S-1’ site via M222A/Y217W mutations to broaden the P-1’ specificity.