It is a fact that enzymes affect by temperature. I assayed trypsin, papain and alpha amylase enzyme on insect crude extraction on different temperature I found that the increase of temperature (80 and 100 Celsius) make increase on the amount of these enzyme then repeat it again I obtained the same result how can I explain the results?
At times the environment of the organism where the enzyme was extracted can affect the factors of its optimum activity e.g optimum temperature, pH, or even mineral content. So the environment of the insects might need to be considered in your case.
It is very unlikely that these enzymes would show increased activity (or any activity at all) at such high temperatures, at which they would most likely be denatured. If you would provide the details of the experiment, perhaps it will be apparent where the problem is.
Some enzymes have been extracted from microbes from hot springs - water at temperatures around 80 to 95 degrees. There are a number of publications on these topics.
Quick question: what type of assay are you using to assay the activity?
Any controlls?
best
Firstly I heated the sample at each temperature for 1 hour exactly at 100 the sample denatured .
For alpha amylase I used starch as a substrate and Dinitro salyslic acid to stop the reaction .
For trypsin and chymotrypsin use BAPNA and BATPNA as a substrate and glacial acetic acid for stop the reaction.
after I had the result I repeated it again and shown the same result .
@Adam B Shapiro @Adam Lesner
@Isabella Nyambayo
What about isoenzymes if is it possible the results come back to isoenzymes?
Have you subtracted the absorbance signal from a blank reaction treated the same way, but without the enzyme, to correct for instability of the substrate at high temperature?
no, the absorbance of blank is zero
i heated the sample only then i assayed the enzyme with the same protocol to assay this enzyme at any sample
for instance with details the trypsin enzyme
i heated the sample at 100 c and kept it then at the second day i put the sample with the substrate then put this mixture at water bath for 20 minutes at 30 c after this i put 200 microliter glacial acetic acid then the absorbance was read at 410 nm Adam B Shapiro
Since the crude enzyme sample was heated, then cooled and set aside for a day prior to testing the activity, a few possibilities come to mind.
1. The heating process inactivated an inhibitor, then the enzyme renatured subsequently.
2. The heating process released enzymes from a compartment that formerly prevented them from interacting with the substrate. As in (1), renaturation of the heat-denatured enzymes must have occurred.
OK, is there are a confirmatory test that must I do to confirm this result? Or now what should I do
In the first case, you could examine the activity of the heated preparation as a function of time after heating to see whether the activity increases with time.
In the second case, you could use an ultracentrifuge to pellet any subcellular organelles and see whether that reduces the activity of the lysate supernatant. Or, if it is only cells that are responsible, a regular centrifuge would be sufficient.
OK thank you very much.. but I have last 2 question, Firstly should I put the sample after heating at the centrifuge? I didn't put it at the centrifuge
Secondly to calculate the specific activity I estimate the total protein of each sample after heating at each temperature degree or I estimate the total protein on the sample before heating?
I would recommend centrifuging the sample after heating and cooling it back down, to remove precipitated protein. Then, measure the total protein concentration of the supernatant for the specific activity calculation.
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