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During assaying an enzyme at high temperature, the substrate (Pectin) is degraded by the high temperature rather than by enzyme, so, how can I minimize degradation of the substrate by the temperature?

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  • $\begingroup$ Why do you run the assay at high temperatures? The solution sounds easy, run it at lower temperatures, but is there a reason to not do this? $\endgroup$ – Chris Sep 8 '14 at 16:40
  • $\begingroup$ I wanted to check whether the Pectinase (enzyme) is thermo-stable or not $\endgroup$ – Oli Sep 8 '14 at 16:50
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    $\begingroup$ pretreat the enzyme at high temperature before carrying out the assay at the standard temperature $\endgroup$ – Alan Boyd Sep 8 '14 at 17:07
  • $\begingroup$ Thats what I would test as well. Incubate the enzyme at different temperatures for the same time. How do you perform your experiment? In small tubes? Is it possible to premix the whole enzymatic reaction without the pectin and the pre-incubate it at higher temperatures? $\endgroup$ – Chris Sep 8 '14 at 17:45
  • $\begingroup$ I perform the the experiment in test tubes, I pre-incubate the enzyme and the substrate for about 5 minutes separately, , then I mix the enzyme and substrate then follow by incubating vfor about 10 minutes, I tried to draw the graph enzyme activity vs temp. The rexn is good till 65°c , however, in higher temps.the absorbance values are very high $\endgroup$ – Oli Sep 8 '14 at 18:10
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Pretreat the enzyme at high temperature before carrying out the assay at the standard temperature – Alan Boyd

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