Deep-sea fungi as a source of alkaline and cold-tolerant proteases

Fungi from coastal environments have been widely studied with respect to the production of secondary metabolites and biotechnologically useful lignocellulolytic enzymes. A few studies on mycology of deep-sea sediments, however, have been carried out. This paper reports a study on alkaline, cold-tolerant proteases from deep-sea fungi. A total of 221 deep-sea isolates of fungi from 5000 m in the Central Indian Basin were screened for the enzyme. Many of these grew and produced alkaline protease at 5 and 30 °C and 1 bar pressure. Aspergillus ustus (NIOCC #20) producing the highest amounts of the enzyme was selected for further studies. The growth yield was substantial at 30 and 5 °C at 1 bar and elevated hydrostatic pressures. The fungus produced alkaline, cold-tolerant protease when grown at 30 °C and 1 bar pressure. The enzyme was active at combinations of 30, 5 °C and 50 and 300 bar pressure. However, protease production was negligible when the fungus was grown at 5 °C, under 1 bar or elevated hydrostatic pressures. The enzyme produced at 30 °C and 1 bar pressure was further characterized. The fungus produced a maximum of 1639 ACU mL−1 of protease by day 7. The enzyme, with molecular mass of 32 kDa and pI values of 6.6 and 6.9 showed several interesting properties. It had a broad pH range of 6–10, with an optimum at pH 9. The optimum temperature for protease activity was 45 °C and approximately 10% of the activity was retained at 2 °C. The enzyme was totally inhibited in the presence of 2 mM PMSF suggesting it to be a serine protease. It was active in the presence of several commercial detergents at 2 g L−1 concentration and in the presence of 0.5 M NaCl, equivalent to 29 parts per thousand salinity. In the presence of stabilizing agents such as glycerol, CaCl2 its thermostability at 60 °C was enhanced. Heavy metal ions Cu, Hg, Fe, Ni and Zn did not inhibit the enzyme activity considerably. This study indicates that fungi from deep-sea sediments could be a useful source of proteases.