Purification, characterization, and gene cloning of a cold-adapted thermolysin-like protease from Halobacillus sp. SCSIO 20089

Marine sediment is a distinctive habitat of cold enzyme producing bacteria. A protease producing strain Halobacillus sp. SCSIO 20089 was isolated from a marine sediment of South China Sea. Using chromatographic techniques, the extracellular protease was purified to homogeneity from the culture supernatant. The purified protease exhibited maximal activity at 30°C, pH 8.0, and remained more than 20% of its activity at 0°C. Its activation energy was calculated to be 34.4 kJ/mol, suggesting it is a cold-adapted protease. Based on the N-terminal amino acid sequence of the purified enzyme, full gene encoding the enzyme was obtained by combination of degenerate primer PCR and hiTAIL-PCR. The deduced amino acid sequence showed 57% and 52% identity with mesothermal and thermophilic protease in thermolysin family respectively. All these indicate the enzyme is a unique cold-active thermolysin-like protease with potential in both basic research and industrial application areas.