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

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 overall coherence was evident between the patterns reflected in phenogram and phylogram. Four isolates of different geographical locations belonging to Geomicrobium halophilum were scattered in the phenogram, displaying significant variations in their phenotypes. Further, stability and catalysis of the extracellular alkaline proteases of these isolates were studied. The bacteria were able to grow in the range of pH 7-10 and temperatures between 37-55 °C. The proteases were active and stable at pH 7-13 with the optima between 9.5 and 10.5, although a rapid loss of activity was observed at pH 6.5 and below. Most of the 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 of incubation at 60-90 °C. As a unique feature, the enzymes were resistant to urea denaturation (2-8 M) for a period of 2-24 h. The addition of NaCl had profound effect on the resistance against chemical denaturation.