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

Abstract

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.

Immobilization of α-amylase from Exiguobacterium sp. DAU5 on Chitosan and Chitosan-carbon Bead: Its Properties

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Extreme Environments: Goldmine of Industrially Valuable Alkali-stable Proteases

“…The first haloalkaliphilic bacterium was isolated by Tindall in 1984 [164]. Enzymes derived from such bacteria are potential biocatalysts due to their optimal activities under the conditions of high salt, surfactants, high temperature and alkaline pH [74], whereas the mesophilic counterparts fail to act under similar conditions [165].…”

“…The enzyme was tolerant to toluene, xylene, n-decane, n-dodecane and n-undecane. Dodia et al [165] isolated an alkaline protease secreting haloalkaliphilic bacterium (Gene bank accession number EU118361) from the Saurashtra Coast in Western India. The enzyme displayed catalysis and stability over pH 8.0-13.0, optimally at 9.0-11.0.…”

“…The enzyme displayed catalysis and stability over pH 8.0-13.0, optimally at 9.0-11.0. It was stable in 0-4 M NaCl and required 150 mM NaCl for optimum catalysis at 37 o C [165]. A haloalkaliphilic Bacillus pseudofirmus Mn6 isolate was found to produce an alkaline protease with a high potency (500 U/ml/min) at 60 o C, pH 10.0 and salinity 0.5% [183].…”