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An alkaline protease secreting Haloalkaliphilic bacterium (Gene bank accession number EU118361) was isolated from the Saurashtra Coast in Western India. The alkaline protease was purified by a single step chromatography on phenyl sepharose 6 FF with 28% yield. The molecular mass was 40 kDa as judged by SDS-PAGE. The enzyme displayed catalysis and stability over pH 8-13, optimally at 9-11. It was stable with 0-4 M NaCl and required 150 mM NaCl for optimum catalysis at 37 degrees C; however, the salt requirement for optimal catalysis increased with temperature. While crude enzyme was active at 25-80 degrees C (optimum at 50 degrees C), the purified enzyme had temperature optimum at 37 degrees C, which shifted to 80 degrees C in the presence of 2 M NaCl. The NaCl not only shifted the temperature profile but also enhanced the substrate affinity of the enzyme as reflected by the increase in the catalytic constant (K(cat)). The enzyme was also calcium dependent and with 2 mM Ca(+2), the activity reached to maximum at 50 degrees C. The crude enzyme was highly thermostable (37-90 degrees C); however, the purified enzyme lost its stability above 50 degrees C and its half life was enhanced by 30 and sevenfold at 60 degrees C with 1 M NaCl and 50 mM Ca(+2), respectively. The activity of the enzyme was inhibited by PMSF, indicating its serine type. While the activity was slightly enhanced by Tween-80 (0.2%) and Triton X-100 (0.05%), it marginally decreased with SDS. In addition, the enzyme was highly stable with oxidizing-reducing agents and commercial detergents and was affected by metal ions to varying extent. The study assumes significance due to the enzyme stability under the dual extremities of pH and salt coupled with moderate thermal tolerance. Besides, the facts emerged on the enzyme stability would add to the limited information on this enzyme from Haloalkaliphilic bacteria.

A halotolerant alkaliphilic actinomycete, Kut-8, was isolated from saline desert of Kutch, Western India. It has been identified as Streptomyces aburaviensis based on the chemotaxonomic characteristics, including cell wall constituents. Kut-8 is Gram-positive having a spiral sporophore with dark green and fluffy spore mass. It was able to grow with 15%, w/v NaCl with optimum being in the range of 5-10%. It grew optimally at pH 9 with slow growth at neutral pH. The cell wall contained L-diaminopimelic acid and no diagnostic sugars. It produced an antibiotic that selectively inhibited the growth of Grampositive bacteria, with Bacillus subtilis being the most sensitive. Kut-8 secreted the antibiotic optimally during mid-stationary phase (on day 14 of growth in liquid culture). The crude antibiotic metabolites were separated by various solvent systems with hexane-methanol-water giving the best separation. The results of bioautographs revealed the presence of single active compound in the Kut-8 antibiotic filtrate. Partial purification of antibiotic metabolite by charcoal absorption and methanol extraction resulted in enhanced antimicrobial activity by 4.16-fold. The study holds significance as only few salt-tolerant alkaliphilic actinomycetes from saline deserts have been explored and information on their antimicrobial potential is still scarce.

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Halophiles are excellent sources of enzymes that are not only salt stable but also can withstand and carry out reactions efficiently under extreme conditions. The aim of the study was to isolate and study the diversity There is significant diversity among halophiles from saline habitats of India. Preliminary characterization of crude hydrolases established them to be active and stable under more than one extreme condition of high salt, pH, temperature and presence of organic solvents. It is concluded that these halophilic isolates are not only diverse in phylogeny but also in their enzyme characteristics. Their enzymes may be potentially useful for catalysis under harsh operational conditions encountered in industrial processes. The solvent stability among halophilic enzymes seems a generic novel feature making them potentially useful in non-aqueous enzymology.

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