Profile of BAPNA hydrolysis by the purified enzyme. (A) 50 μl of purified enzyme was assayed at pH 8.5 for BAPNA hydrolysis for different time periods up to 5 hrs using 50 mM BAPNA substrate (B) BAPNA hydrolysis was carried out for 1 hour with increasing volume of purified enzyme. (C) pH optimum of BAPNA hydrolysis by the purified CESP was studied in 100 mM buffers of varying pH. The buffers used were: Na Ac (5.1), MES (5.5, 6.0), PIPES (6.5, 7), Tris HCl (7.5 to 9) and CAPS (9.7, 10). (D) Measurement of Michaelis-Menten constant (Km): p-nitroaniline liberated was measured by varying substrate concentrations. Conditions of reactions are same as mentioned earlier. (E) SDS/PAGE zymogram analysis of the purified CESP, 2 μg (1st lane) & 3 μg (2nd lane) of the purified CESP after storage for one week.