Hello everyone,
For the first time I will be working with different enzyme to hydrolyze pea protein. I need to establish which ratio Enzyme: Protein (in %). I have read a lot about enzymes and their activity units but I have still some questions, I hope you could help me.
1. Is this Enzyme Activity specific for the substrate that the companies used for the calculation? In the enzymes I have, there are different units written such as the normal U/mg, and these others AU-A/g, Kilo Novo Proteinase Unit (KNPU)-S/g, Leucine Amino Peptidase Units per gram (LAPU/g), BAEE U/mg
2. If the first is true, can I calculate the enzyme concentration (%)/protein considering the Enzyme Activity (written in the enzyme commercial bottle)? Or is it necessary to calculate the Enzyme Activity in my substrate (pea protein)?
3. The bigger the Enzyme Activity (U/mg) the less amount of enzyme I need?
4. Does it even make sense to consider the Enzyme Activity or can I just establish a concentration for my enzyme (%)?
Thank you everyone in advance
Veronica
Enzyme activity depends on the substrate used, as well as other conditions. If your substrate is different from the substrate used to measure the activity of the commercial preparation and your conditions are also different, it would be better to measure the activity yourself.
The higher the enzyme activity, the less enzyme you need.
The enzyme specific activity will not necessarily tell you how much of the enzyme to use for your application. Specific activity measurements are based on initial rate measurements. Since you are planning to make a hydrolysate, you presumably want the reaction to go nearly to completion. That means the reaction rate will slow down as the substrate gets depleted, so you can't predict based on initial rate measurements how long it will take or how much enzyme it will take to run the reaction to completion. The sensible thing to do is to try some small-scale experiments to figure out how much enzyme to use, then scale up.
Thank you Adam, I might then try in small scale to be sure. And thank you Pia, I will look them up :)
Commercial enzyme preparations mentions on the label about specific activity of enzyme determined by using substrate of their choice. It may not be suitable for your needs. Hence any researcher has to determine specific activity of enzyme at his/her experimental conditions. Higher the specific activity lower specific substrate requirements. Generally enzyme activity is determined before substrate is depleted. Since you wish to measure hydrolysis of substrate and keeping in mind that enzyme activity is not a first order reaction better to start with less enzyme for better result.
For the application of commercial enzyme preparations, the enzyme activity should always be determined with the substrate you use. While you would like to use legume proteins, a determination of the enzyme activity with this protein is highly recommended. For the initial peptidase activity determination you could determine the release of alpha-aminogroups, for example with o-phythalaldehyde (OPA). The application of your commercial enzyme should then be standardized on the initial peptidase activity. A standardization of the enzyme solution in %, is highly risky. Commercial enzyme preparations can differ in their activity, from batch to batch. Also the storage conditions and time can result in major activity losses. Therefore, an enzyme application based on % is most likely not reproducible.
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