Partial characterization of a crude alkaline protease from Bacillus circulans and its detergent compatibility
R Jaswal, G Kocher
alkaline protease, and detergent compatibility, ph, temperature
R Jaswal, G Kocher. Partial characterization of a crude alkaline protease from Bacillus circulans and its detergent compatibility. The Internet Journal of Microbiology. 2006 Volume 4 Number 1.
An extracellular alkaline protease of Bacillus circulans MTCC7906 was produced using cotton deoiled meal replacing nitrogen source in the production medium at a pH of 10.5, temperature of 25C in 96 hours of incubation. The enzyme purified by (NH4)2SO4 precipitation and dialysis showed optimum activity at 60C and pH 9.0 with an enzyme concentration of 3.33% of reaction mixture at a casein concentration of 14mg ml-1. SDS PAGE of the crude enzyme revealed it as 24.5 kDa protein. The enzyme was stable at room temperature for 20 days and its activity was enhanced by Ca2+, Mn2+, Zn2+, Co2+ and Ba2+ while inhibited by Hg2+, Cu2+, NH4OH and EDTA suggesting it as a metal dependent serine protease. The alkaline protease was compatible with six local powdered detergent brands retaining more than 90% activity.
Alkaline proteases alone account for 20% of the world enzyme market with their predominant use in detergent and leather processing industries (Kumar and Takagi, 1999; Oberoi et al., 2001). The application of such proteases requires their improved properties like usage at optimum to high temperatures, higher alkaline pH values, stability over a range of time etc. which make them detergent compatible (Gupta et al., 2002; Adinarayana et al., 2004). Furthermore, bacterial alkaline protease activity has been reported to be enhanced by various metal ions, surfactants etc. that also help to know their biochemical nature. In general, alkaline proteases have been reported to be serine centered or metallo centered (Kumar et al., 1999).
In the present study,
Material and Methods
Results and Discussion
The alkaline protease of
The use of different casein concentration (1-15 mg ml -1 ) in a reaction mixture revealing a typical hyperbolic curve with 14mg ml -1 as the best concentration suggesting that enzyme follows Michael Menten's equation. A double reciprocal plot was prepared that showed an apparent Km of 5mg ml -1 and a Vmax of 1000 mol tyrosine min -1 ml -1 (Fig 1d). Similarly, Kaur et al (1998) reported Km of 3.7 mg ml -1 in
The two metal ions Hg 2+ and Cu 2+ inhibited the enzyme activity by 95.4% and 23.8% respectively. The liquor ammonia used at the rate of 10 mM in the incubated reaction mixture resulted in 23% reduction in alkaline protease activity, which suggested that the enzyme may be carrying serine residue at its active site also. The results thus suggest that alkaline protease of
Among the six detergents for detergents, four detergents were already containing the enzyme as quoted by their manufacturers. Therefore, their suitable controls were also run and their activities were found to be very low compared to those obtained by supplementation with enzyme of
The present study thus reveals a metal dependent serine protease of