Zymogram Analysis of Alkaline Keratinase Produced by Nitrogen Fixing Bacillus pumilus ZED17 Exhibiting Multiprotease Enzyme Activities

Abstract

Background: One group of significant enzymes produced by Bacillus genus are alkaline proteases with several important applications in the daily life as well as common industries such as food, detergents, leather, alcohol and beer production and medical and sanitary industries, beside wastewater treatment, , biotransformation, hydrolyzed proteins and oil manufacturing. Objectives: In this paper, keratinase activity and zymogram analysis of Bacillus pumilus ZED17 using different conditions and substrates are reported. Materials and Methods: The nitrogen fixing Bacillus, obtained from heated see water, was enriched on feather as the only sources of carbon, nitrogen and energy. It was also determined whether the isolated nitrogen fixing Bacillus exhibited extracellular proteolytic activity on feather, meat, gelatin and casein. Biochemical tests, carbohydrate fermentation patterns and 16srRNA detection were employed for identification of the isolated strain. Furthermore, the extracellular proteolytic activity using different protein substrates was investigated. Results: B. pumilus ZED17 is one of the best strains enriched on feather, of which the extracellular proteolytic activities are exhibited. Activity-pH profiles were resoluted in buffers with different pH levels. Extracellular enzyme activities were assayed using different proteins and feathers. Keratinase activity was observed at neutral and alkaline, but not low pH levels. This enzyme demonstrated to have multi–activity. zymogram test revealed a 50-kD Caseinase produced by this strain. The optimum keratinase activity was at pH 8.0 and 40°C, using keratin as a substrate. Conclusions: Since the isolated strain is halotolerant and nitrogen fixing, it is a good candidate for alkaline protease production, and soil fertilizing, in addition to biofertilizer production out of poultry and fish byproduct.