At first, the optimal pH and temperature of enzyme activity must be determined. For pH stability, enzyme solution was pre-incubated at different pH in the range of 3.5–9.0 for 30 min at optimum temperature for enzyme. The residual enzyme activity was determined.

Read the attached papers

Article Reduction of raffinose oligosaccharides in chickpea (Cicer a...

Article Immobilization of invertase on Celite and on polyacrylamide ...

Article Characteristics and stability of free and immobilized an Alp...

I don't understand how you were able to achieve a pH range between 3 and 9 with the same buffer or why you think the pH was the same in each case when the assay was started. Do you mean that you made up a variety of different buffers to cover the pH range? Did you measure the pH in each sample before and after starting the reaction to make sure it was correct?

Also, why did you store the enzyme at 4C during the incubation at various pHs? Why not at the assay temperature? The stability will differ depending on the temperature.

Do you really need to examine the stability at such acidic pH? Is it an enzyme found in lysosomes or in the digestive tract?

Sounds like you're going about this in the right way overall, but maybe missing a step. The idea of an assay like this is to incubate your enzyme at each pH, then take small samples out at various time points and measure their activity at the optimal pH. These incubations should be at a relatively high concentration so that when you take a sample out and add new buffer and substrate it is at a 'normal' concentration for measuring activity. From your question, it seems like the part you are missing is the change in buffer. The easiest way to do this is simply by dilution. If you have the enzyme at a relatively high concentration at each pH (and provided it is stable at these high concentrations), when you take an aliquot out and put it in a substrate/buffer mix at the optimal pH (possibly with a relatively high concentration of the optimal pH buffer, assuming this does not inhibit) the original varied pH buffers will be diluted to negligible concentrations. You can double check the validity of this dilution at the two extreme pH values to make sure that the dilution doesn't give any detectable pH change in the substrate buffer mix. Essentially this experiment involves two reactions - one is the incubation of your enzyme at each pH, the other is the catalytic reaction of your enzyme to measure how much of it is still active. As a guide, I would do this experiment something like this: A small reaction at each pH, maybe 30 µL, 20 mM buffer, 4 °C. At a few time points (say, 10, 30, 60, 120 min), take 5 µL of this and dilute with 195 µL substrate/buffer mix (try to use one large stock for all timepoints and pH values) with 50 mM buffer and substrate at 5-7 times Km, so that activity is directly proportional to enzyme conc and not influenced by substrate conc. Measure activity for each timepoint and each pH, then plot residual rate as a measure of active enzyme concentration. Hope this helps, and feel free to ask if there are details that don't make sense.

Stability of the enzyme depends on both pH and temperature. If 4C was optimal for the reaction, then dilution can help to achieve the optimal pH after incubation. 

However, dilution requires standardisation (enzyme concentration varies with dilution).

At first, the optimal pH and temperature of enzyme activity must be determined. For pH stability, enzyme solution was pre-incubated at different pH in the range of 3.5–9.0 for 30 min at optimum temperature for enzyme. The residual enzyme activity was determined.

Read the attached papers

Article Reduction of raffinose oligosaccharides in chickpea (Cicer a...

Article Immobilization of invertase on Celite and on polyacrylamide ...

Article Characteristics and stability of free and immobilized an Alp...

You can add about 5% glycerol (V/V) to your buffer and then assay enzyme activity at 25-36 °C in different pH ranges 4-9 and look for highest enzyme activity in different pH

good luck

hello dear Adam

I prepare 100 ml Mix buffer containing chemical I said then  I adjust different PHs for each 5 ml of that. my question is how can I reach the same PH when I want to assay enzyme in each PH.

i incubate in 4 C according to literature about my enzyme. my enzyme is cellulase deleted small domain from them.

i assay stability of my enzyme in different temperature. I found my truncated enzyme aggregate in acidic PH with comparing with wildtype. so I'm interested in determining PH stability

You should first determine the optimum pH and the ionic strength for your enzyme. A mixture of "Good" buffers is best with NaCl or KCl added to maintain a (low) constant ionic strength. Then determine the optimum inoic strength at optimal pH. To ensure that the extremes of pH are not inactivating the enzyme, incubate a concentrated sample of enzyme at the desired pH and then assay an aliquot at optimal conditions as suggested above. A time course can also be measured in this way at any chosen temperature.

Good luck!

hello dear seino

thanks a lot for your really good and complete explanation.

this is the logical way for which I've looked

I understand your guide. but I have a problem! unfortunately I don't have a lot of enzyme, so I can't prepare different PH buffer with high concentration enzyme!

Could you incubate your enzyme in a low capacity buffer and then adjust the pH with high capacity pH buffer at the optimum pH? You could check if this would work by mixing buffers in the absence of your precious enzyme and measure the pH change.

The 'high' concentration is only relative. As long as you can accurately measure the residual activity after dilution it should work. Doing a smaller reaction for each pH, and only one time point as you mentioned in your original question, will help conserve enzyme. Extending the time for the residual activity assay should also let you use less enzyme, as it will give you a bigger signal to detect. Finally, using different buffer capacities in the pH stability incubations and the residual activity assays might help, as suggested by Dr Fairlamb. In the end, if you plan the assays carefully, you should only end up using the same amount of enzyme that you would use for around 12 'normal' activity assays (for pH 3-9 at 0.5 intervals).

thanks a lot a dear Alan and Seino. I try it!

First note  the optimum temp and pH for your enzyme.The enzyme may be incubated at various pH for 30 min,1h,3h and estimate the activity of the enzyme.

I think you have jumbled your experiment. There should be only one variable in a single set of experiment. You please follow the guidelines suggested by Esam H. Mansour and analyze the results systematically and methodically. you are sure to get what you want. Experimentation is learning, not following.

In order to determine the pH stability of the enzyme can you test its activity in a wide range of pH around proper isoelectric point. Enzyme assays are common laboratory methods for measuring enzymatic activity.

Article Nylon 6 film and nanofiber carriers: Preparation and laccase...

Article Immobilization of fungal (Trametes versicolor) laccase onto ...

For the determination of enzyme stability you have to determine the residual activity by pre-incubation of enzyme in various pH solutionfor different interval. After defined time period take the defined amount of enzyme and perform assay under assay optimum pH. 

Just incubated your enzyme at various pH in 1 or two hours, then check the residual activities.

1. Determine the optimum PH and Temperature.

2. Establish a PH range e.g. 3.0,3.5,4.0-9.0.

3. Incubate the enzyme at various PH for 30mins and determine the residual enzyme activity.

Good luck 😊

Question: There is need to make different pH standard curve of product when we are finding enzyme activity on different pH.

Please explain

Whether or not it is necessary to prepare separate standard curves at each pH depends on the assay method. If the slope of the standard curve does not vary with pH, then one standard curve is sufficient. If, on the other hand, the slope does change with pH, then you will need a different standard curve for each pH.