i want to test the alpha amylase inhibitory activity of various pharmacuticals. is it possible to use alpha amylse from Aspergillus oryzae alpha (800EAU/Gram) to analyse in vitro alpha-amylase inhibitory activity using DNS Method. and what is the amount to enzyme (in ml) should i use?
May 9, 2024
Dear Mukunda,
Yes, you should be able to use the A. oryzae enzyme to test for a-amylase inhibitory activity of your pharmaceutical agents. I would also suggest you try amylase from several other sources, e.g., porcine a-amylase, Bacillus, sp., a-amylase, plant amylases, etc. The protein structure of amylase from widely selparated biological sources will probably be different. The pharmaceutical agent may not inhibit A. oryzae amylase, but it might inhibit amylase from another biological source. That in itself would be an interesting observation, and perhaps give a clue about the mechanism of inhibition.
The last part of your question is confusing. Are you asking how much enzyme to use in the assays or what dosage of agent to use to inhibit the amylase? If you are asking how much enzyme to use in the assay, this you should be able to calculate from the potency of the enzyme, specifically the # of activity units per gram (or per ml for liquid enzyme). For the A. oryzae amylase this is specified (800 EAU/gram). You need to check to identify how the manufacturer defines a unit. DNS measures reducing sugar, so you want to use enough amylase to release an amount of reducing sugar that you can conveniently measure after a suitable reaction period (e.g., 10-15 min). You may have to determine this experimentally.
As far as how much of the pharmaceutical agent to use to inhibit the enzyme, this can be determined by adding increasing amounts of the agent to a fixed amount of the amylase. Again, some experimentation (trial and error) may be required.
I hope this information helps you.
Bill Colonna Dept. Food Science & Human Nutrition, Iowa State University, Ames, Iowa, USA [email protected]
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