Purification and Biochemical Characterization of Phytase from newly isolated Bacillus subtilis C43

Bacillus subtilis C43, an isolate from cattle shed soil sample, produces phytase, which catalyzes the hydrolysis of phytic acid into myo-inositol and inorganic phosphates. The phytase was purified using ammonium sulphate precipitation and dialysis followed by anion and cation exchange chromatography. Molecular weight of the purified phytase was 55 kDa. The optimum temperature for phytase activity was found to be 50oC .The enzyme retained over 80% of its activity over a temperature range of 37 to 55° C. An abrupt decrease in enzymatic activity was observed above 55°C. This enzyme was almost completely inactive at 70°C. The enzyme was stable at temperatures below 60°C and partial and complete loss of activity was recorded at 70°C (32% activity remained) and 80°C respectively. The highest phytase activity was observed at pH 5 and a decline of enzyme activity was observed on both sides of pH optimum. The enzyme was stable over the pH range of 2 to 5. The enzyme activity was stimulated by the addition of Ca+2, Na+, Cu+2, K+, Co+2 and Ni+2; inhibited by Mg+2, Fe+2 and Mn+2 and unaffected by Zn+2. Of all those tested, EDTAwas the agent that greatly inhibited the enzyme activity as it caused about 55% reduction in activity.