I am getting some difficulties in estimating the activity of CELLOBIASE. The result I am getting seems to be much higher then expected.The substrate I am taking is 15 mM Cellobiose solution, still some errors floating around . As per the method, I first tried to know the ABS shown by 1mg/ml glucose diluted in citrate buffer, to check the ABS , i separately run .4mg/ml glucose and confirmed the ABS shown by 1mg/m Now , i m calculating the enzyme conc, by following formula. .0926/ conc. of enzyme required for releasing 1 mg of glucose . ( The conc. of enzyme require to release 1 mg was determined by the graph (attached) and it was .309.The conc. was .0002708 and the CBU as per the conc. is ~ 342, which seems to be higher one . Let me know how to calculate the exact one.. And yet if the glucose conc. multiplied by two the CBU is becoming much higher. Please have a look on the the excel file attached .
Hi Manasa
To be honest, I couldn't understand your graph and the relationship you explained!
What I got is that you first prepared a glucose solution of 1 mg / mL and get the absorbance! Next you prepared some enzyme dilutions and (I think after performing the assay procedure which is missing here) got the absorbances! Then you calculated the glucose concentration based on the division of two absorbances and multiplied that in 2!
Am I right?
I don't know about the protocol you are using, but when we want to calculate the activity of enzymes, we need to have a standard curve for the final product of the desired enzyme (e.g. phosphate for phytase and glucose for amylase). Then we need to follow the procedure of enzyme assay and later we should first determine the concentration of final product of enzyme according to the graph. Later we have to consider the time, dilution, etc to calculate it based on the definition we have for the enzyme activity.
Hope it could help you!
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