I have purchased a solid cellulase from Aspergillus niger (CAS Number 9012-54-8). I want to use it in solution as a positive control for CMC breakdown using agarose plate assays (incubate and use Congo red to visualize clearings).
The bottle states that it contains 29000 unit/gram, and defines one unit as liberating 10µg glucose from CMC per minute at 40°C at pH 4.5.
What do I mix it with? Do I need to sterilize the solution, or just add 0.02% sodium azide? How many units do I want in my final product?
Hi Matan Shelomi,
Usually, when you buy powdered enzymes, you can dissolve them in the same buffer you want to perform the enzyme assay or, in your situation, the same buffer/pH you use for agarose plate assay. I suggest you suspend the powdered cellulase in an ice-cooled buffer and keep it on ice or 4 °C till the complete dissolution (the time depends on the chemical nature of the enzyme preparation). Avoid mix the enzyme solution on vortex!
Cheers
Hi Matan,
Besides the point suggested by Dyoni, I think it is better to add sodium azide to the solution because it would be a partially purified enzyme, which may contain several contaminants. After the complete dissolution, I suggest you to make serial dilutions of the enzyme solution and determine the detection limit of the CMCase activity under your experimental conditions.
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