The mixtures were incubated at 37o
C for 2 minutes.
After that, 50 µl of 4 mM pNPG was added in the well
finally. The mixture was required to incubate again for 5
minutes at same condition. The pNP was performed and
monitored at 405 nm every 30 seconds for 5 minutes by
micro plate reader. The velocity was determined by linear
relationship equation between absorbance and time as
following equation (1).