Hi there,

For measuring enzymatic activity you always have to respect these:

concentration of enzyme should be far less than substrate concentration (ideally substrate concentration should be at least 10Km)

measurement should be done when reaction velocity is constant (dP/dt=constante)

consumption of substrate should be negligible towards total substrate present in the assay at  t=0.

In your particular situation, the assay is colorimetric (pNP released from hydrolysis of pNPG) but, according to sigma website, it is run at pH4 but pNP yellow color needs basic pH to be revealed. So the assay can't be monitored continuously in a spectrophotometer. You will have to withdraw aliquots of the reaction mix at given time and mix it with a base (0.1M NaOH or Na2CO3 for instance) to get the color developed and then measure intensity at 405nm.

Reconstitute lyophilized enzyme in a buffer or water as per supplier's suggested method at fairly high concentration of protein and keep it in ice.

Purchase or synthesize (easily done)  p-Nitrophenol. You will need it for quantitation of your enzyme. The product of your reaction (pNP) will allow you to construct a standard curve (line) against which you will read the product generated by your enzyme.  

The supplier of enzyme normally provides with either a number or at least a range of specific activity in units/mg. Use this number as your starting point and empirically find the appropriate dilution of enzyme in your reaction mixture that gives you an amount of product which is in the linear range of the reaction. Since it is an end-point measurement, you will need to test multiple dilutions.

If your spectrophotometer is not equipped with a way to control the temperature of the cuvette, use room temp as your reaction temp. rather than 37C. This means that all your buffers and substrate solutions should be brought to room temp well in advance. Prepare the enzyme solution at fairly high concentration (according to the supplier's suggested solution - usually a buffer or water, if the enzyme powder includes buffer in it already), keep it in ice and use a small aliquot for dilution. 

Adjust the amount of enzyme such that the reaction is not over within seconds. Ideally, you want enzyme amount to be such that under saturating substrate conditions, the reaction takes 10-30min so accurately timing the reaction does not pose a problem. Once you have found the appropriate enzyme dilution, the reaction should be linear beyond the time point you use for routine measurements. This can be determined by stopping the reaction at various time intervals, such as 5, 10, 15, 20, 30, 45, and 60min. The background (blank) will have everything except the enzyme.

Thanks @Dominique Liger and @Tausif Alam. I will start with your suggestions.