An organic solvent-tolerant bacterium producing organic solvent-stable protease was isolated from fishing industry wastewater and identified as B. cereus. The enzyme retained more than 95% of its initial activity after pre-incubation 24 h at 30 degreesC in the presence of 25% methanol, DMSO, acetonitrile and DMF. Above 37 degreesC, Ca2+ was required for enzyme activity. The optimum temperature for the protease activity was at 60 degreesC in the presence of 2 mM Ca2+ and 50 degreesC in the absence of Ca2+. At 60 degreesC, Ca2+ (2 mM) stimulated the protease activity by 500%. Other bivalent metal ions such as Mg2+ and Mn2+ also increased activity by 285 and 157%, respectively, while Zn2+ and Cu2+ had a strong inhibitory effect. Thermostability of the enzyme was enhanced by Ca2+ at temperature values above 40 degreesC. In the presence of 10 mM Ca2+ the enzyme retained 100, 93 and 26% of its initial activity after heating for 15 min at 55, 60 and 70 degreesC, respectively. However, the enzyme was completely inactivated when incubated at 55 degreesC for 15 min in the absence of calcium. The pH optimum was 8.0 and the enzyme was quite stable in various pH buffers between pH 6.0 and 9.0 when incubated at 50 degreesC for 1 and 3 h. Enzyme activity was inhibited by EDTA, suggesting that the preparation contains metalloprotease(s). (C) 2003 Elsevier Science Inc. All rights reserved.