An organic solvent-tolerant bacterium that produced an organic solvent-tolerant protease was isolated from soil. The protease secreted from the organic solvent-tolerant bacteria had very high tolerance in the presence of various organic solvents. The organic solvent-tolerant protease was characterized, and its conformational transition in the presence and absence of an organic solvent was monitored. By using site-directed mutagenesis and random mutagenesis, amino acid residues that influenced organic solvent tolerance were investigated. And the organic solvent-tolerant protease was used as a catalyst of peptide synthesis in the presence of various organic solvents. Furthermore, the organic solvent-tolerant protease, which had high peptide synthetic activity, was developed by protein engineering.