Phenotypic characteristics, phylogenetic analysis and characterization of alkaline proteases of marine bacteria Geomicrobium halophilum, Oceanobacillus oncorhynchi, and Oceanobacillus khimchii

Abstract

In this report, protease-producing haloalkaliphilic bacteria from sea water have been investigated. Seven bacterial strains belonging to Geomicrobium halophilum, Oceanobacillus oncorhynchi, and Oceanobacillus khimchii were isolated and characterized based on their colony characteristics, cell morphology, biochemical properties, antibiotic sensitivity, and 16S rRNA gene sequence analysis. The analysis based on 53 phenotypic characters (cluster analysis) generated a phenogram establishing correlation with the phylogenetic placement. A maximal level of phenotypic similarity among the protease producing bacteria was evident at only 46% in the phenogram. An overallcoherence was evident between the patternsreflected in phenogram and phylogram. Fourisolates of different geographical locationsbelonging to Geomicrobium halophilum werescattered in the phenogram, displaying significantvariations in their phenotypes. Further, stabilityand catalysis of the extracellular alkaline proteasesof these isolates were studied. The bacteria wereable to grow in the range of pH 7-10 andtemperatures between 37-55 °C. The proteases wereactive and stable at pH 7-13 with the optimabetween 9.5 and 10.5, although a rapid loss ofactivity was observed at pH 6.5 and below. Most ofthe proteases had temperature optima at around 60°C, while they were stable in the range of 37-60 °C,with the retention of 100-80% activities after 1h ofincubation at 60-90 °C. As a unique feature, theenzymes were resistant to urea denaturation (2-8M) for a period of 2-24 h. The addition of NaCl hadprofound effect on the resistance against chemicaldenaturation