0.6 ml of sulfanilamide(1% w/v) was added to it followed by 0.6 ml NED (0.1% w/v). The test tubes were kept in dark for 15 min for pink color development. Then absorbance was measured at 540 nm with the help of spectrophotometer using distilled
water as blank. The amount of nitrite produced was calculated from the standard curve plotted using known concentrations of nitrite. Enzyme activity was defined as nanomoles of nitrite consumed per ml extract per hour. Specific activity was defined as enzyme activity per mg protein.