Isolation of alkaline protease from Bacillus subtilis AKRS3

Abstract

This research study was mainly focused on phenotypic, biochemical characterization, 16s rRNA sequence based species level identification of isolate and determination of the higher production of alkaline protease through optimization study (carbon, nitrogen, incubation period, temperature, pH and sodium chloride concentration), production by submerged fermentation and analytical studies (protease assay, protein and biomass estimation). The produced crude enzyme was been used for dehairing activity on goat and sheep hides. Primary screening was achieved by skim milk casein hydrolysis method. Microbiological, biochemical characterization and 16S rRNA phylogenetic analysis revealed that isolated bacterium was Bacillus subtilis AKRS3 with an optimum alkaline protease producing temperature, 37°C and pH 9.0. The maximum alkaline protease production was achieved at 24 h of incubation period. Among various nitrogen (organic and inorganic) sources, beef extract was found to be the best inducer for alkaline protease in the concentration of 1.5% as was reported for the maximum alkaline protease production. Effect of carbon sources for example xylose, on protease production proved high protease production than the other tested carbon sources and subsequently 2% concentration registered an optimum to enhance the protease production. The halotolerancy of B. subtilis AKRS3 for alkaline protease production indicated that 3% of sodium chloride was optimum to yield maximum protease activity. During production, agitation rate was 250 rpm at air flow rate of 1 VVM. Maximum protease activity of 42.7556 U/ml was observed at the end of 24 h cell free supernatant of fermentation broth. Crude alkaline protease was most active at 55°C, pH 9 with casein as substrate. According to our knowledge, this study demonstrated the first report on alkaline protease producing B. subtilis AKRS3 isolated from fish waste. The produced enzyme could be effectively used to remove hair from goat and sheep hide indicating its potential application in leather processing industry.

Key words: Bacillus subtilis AKRS3, 16S rRNA sequence, alkaline protease, submerged fermentation (SmF), dehairing activity.