Salt dependent resistance against chemical denaturation of alkaline protease from a newly isolated haloalkaliphilic Bacillus sp.

Only few enzymes from haloalkaliphiles are biochemically characterized for their kinetic behaviour and stability. In view of this realization, an alkaline protease from Bacillus sp. AH-6, displaying salt-dependent resistance against chemical denaturation by Urea and Guanidium hydrochloride was investigated for denaturation and in vitro protein folding. The crude enzyme was highly resistant against urea (8 M) denaturation up to 72 h; however, on purification, it turned sensitive and got denatured within 2 h. Interestingly, the purified enzyme regained the resistance in the presence of NaCl. Effective refolding of the purified enzyme was achieved with glycerol; however, other approaches such as lower protein concentrations, rapid dilution and slow removal of the denaturant did not further add to refolding. The results are important from the viewpoint that only few enzymes from haloalkaliphilic bacteria are characterized. Since the resistance against chemical denaturation is a rare phenomenon, the findings would enrich the knowledge on protein stability and denaturation. Besides, such biocatalysts would definitely have novel applications under harsh chemical environments.

Purification and stability characteristics of an alkaline serine protease from a newly isolated Haloalkaliphilic bacterium sp. AH-6.

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.