I am following a procedure for a beta-Glucosidase assay using pNPG. The reaciton mixture of 250 µL of pNPG (1mM), with 50 µL of crude enzyme and 700 µL of sodium acetate buffer (PH 4.8, 50mM) is kept at 50 celcius (tried both incubator and water bath) for 30 minutes, followed by the addition of Sodium carbonate (2M, 2ml) and measurement of Absorbance at 410 nm.
Now the problem is, the reaction mixture doesn´t show the development of any kind of colour, even the slightest change, after the addition of Sodium Carbonate, where the protocol I am following says "add Na2CO3 and measure the Absorbance after the development of colour". What shall i do?
I have a completly inverse problem. I use exactly your protocol to measure B-glucosidase activity, but my problem is a very high absorbance (A>2) The problen with the p-nitrophenol is the need of a very basic pH to set the color yellow. You can try with NaOH 1mM . it's the method I use. Don't forget to do a "Enzyme Blank" of reaction only with buffer and Enzyme. Check the pH after addition of Sodium Carbonate it be over 10-11.
I have a relevant question.
I have been trying to express glucosidase in bacillus system and look for extracellular activity in LB Broth. I am using standard procedures for pNPG assay. But the issue is I do get positive result for the clone using MUG assay, but was not able to find any extracellular activity through pNPG for the same clone ( there was no distinct yellow coloration after the reaction was allowed) what could be the reason behind the failure in pNPG assay but being positive in MUG assay (Fluorescent substrate from SIGMA)
I wish to know one more thing, when we over lay the agar plate containing clone with these fluorescent substrates/congo red staining/ trypan blue staining. Will there be any chances that the clear zones are observed even if the enzyme is produced intracellularly.? As I am working on bacillus systems I wonder if I get a clear zone does it mean the enzyme is secreted extracellular or intracellular.
See:
Tabatabai MA (1982). Soil Enzymes. In: Page, A.L. (Editor), Methods of soil Analysis, Part 2. Am. Soc. Agron. pp. 903-948.
ISBN-10: 0891180729
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