Assay of enzyme from thermophile

From: Kayashree M
Date: 18 October 2011 11:18


Dear users,

Pardon me for the non-crystallography related question,
Can anyone provide some papers regarding the assay of
enzymes from hypertherphilic /thermophilic organisms? ie,
assays at high temperatures..

Thanking you
With Regards
M. Kavyashree

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From: Partha Chakrabarti

You can do it without having to do an assay at high temperature, because the substrate stability would also change at higher temperature. What you could do is the following:

 

1. Use a PCR machine to heat the enzyme, and then cool it down.

2. Keep one fraction at 4C / RT separately.

3. Do the assays and take the ratio (residual activity).

4. Make sure you normalize against protein concentration if you are checking different mutations.

5. Do it at different pH and duration of heating.

 

HTH,

Partha