Isolation and screening of a novel extracellular organic solvent-stable protease producer

Using enrichment procedures, 68 organic solvent tolerant strains were screened from sea mud samples. Twelve of these strains demonstrated high protease activity on skim-milk agar. Among them, the DS11 isolate was selected based on the stability of its proteolytic enzyme in the presence of various organic solvents and later identified as Bacillus sphaericus by morphological, physiological, biochemical tests and 16S rDNA gene sequence analysis. Strain DS11 was able to sustain and grow in a wide range of organic solvents. The crude protease also exhibited remarkable solvent stability and retained most of the activity at least up to 14 days at 37 °C and 200 rpm in the presence of various organic solvents at 25% (v/v) concentration. More than 80% activity was recovered when the log P value of organic solvents were from 1.8 to 3.5, whereas in methanol ethanol, and 1-butanol the residual activity was 35%, 36%, and 42%, respectively. However, when n-decane, octane, isooctane, and heptane, of which the log P values are equal to or more than 4.0 were added, the activities of the proteases was enhanced. This organic solvent-stable protease could be used as a biocatalyst for peptide synthesis in organic media.