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Edward
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 Posted: Sun Aug 24, 2003 1:00 am Post subject: Unusual enzyme behaviour |
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I have been doing experiments in which I hydrolyse a particular
hemicellulose in wood pulp fibers using a hemicellulase. I have used two
concentrations of enzyme, on a 100 times higher than the other. At the
end of the treatment I separated the liquid from the fibers, boiled it
to denaturate the enzyme, centrifuged it to remove any fiber fragments
and rehydrolysed the filtrate using a mixture of enzymes. I used an HPLC
and a calibration curve to determine the amount of the monosacharide of
the hemicellulose that was removed from the fibers.
This is my problem: at high enzyme concentrations I detect _less_
product than at low enzyme concentrations. At the low enzyme
concentration the amount of liberated product is 15%-40% less than at
the high concentrations. I repeated a couple of experiments to measure
the weight loss of the pulp, which gave similar results.
I can think of several causes for a lower activity of the enzyme at
higher concentrations. However, I can>t think of anything that can lower
the total hydrolysis rate at a high enzyme concentration to below that
of the lower enzyme concentration. For example, if substrate limitation
would play a role I would expect that the hydrolysis rate would be more
of less equal to that of the low concentration, but not much lower.
Any ideas?
Edward |
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Bob
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| Posted: Sun Aug 24, 2003 1:17 am Post subject: Re: Unusual enzyme behaviour |
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On Sat, 23 Aug 2003 22:00:18 +0200, Edward <nospam@student.utwente.nl>
wrote:
[quote]I have been doing experiments in which I hydrolyse a particular
hemicellulose in wood pulp fibers using a hemicellulase. I have used two
concentrations of enzyme, on a 100 times higher than the other. At the
end of the treatment I separated the liquid from the fibers, boiled it
to denaturate the enzyme, centrifuged it to remove any fiber fragments
and rehydrolysed the filtrate using a mixture of enzymes. I used an HPLC
and a calibration curve to determine the amount of the monosacharide of
the hemicellulose that was removed from the fibers.
This is my problem: at high enzyme concentrations I detect _less_
product than at low enzyme concentrations. At the low enzyme
concentration the amount of liberated product is 15%-40% less than at
the high concentrations. I repeated a couple of experiments to measure
the weight loss of the pulp, which gave similar results.
I can think of several causes for a lower activity of the enzyme at
higher concentrations. However, I can>t think of anything that can lower
the total hydrolysis rate at a high enzyme concentration to below that
of the lower enzyme concentration. For example, if substrate limitation
would play a role I would expect that the hydrolysis rate would be more
of less equal to that of the low concentration, but not much lower.
Any ideas?
[/quote]
Formally, you could get your result if the enzyme prep contained a
non-competitive inhibitor. The conc of the inhibitor is continuing to
increase, even though you have added saturating levels of enzyme.
How about if your enzyme stuck to the fibers, and bound product, so
that your assay is not truly measuring the hydrolysis?? Or maybe some
fraction of your enzyme is dead, and binds irreversibly to the fibers?
Running only two E levels, 100-fold apart is very limited info. I
appreciate the difficulty of assaying this enzyme, but 100-fold extra
enzyme is a huge jump.
bob |
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Bob
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| Posted: Sun Aug 24, 2003 9:51 pm Post subject: Re: Unusual enzyme behaviour |
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On Sun, 24 Aug 2003 10:27:43 +0200, Edward <nospam@student.utwente.nl>
wrote:
[quote]Bob wrote:
On Sat, 23 Aug 2003 22:00:18 +0200, Edward <nospam@student.utwente.nl
wrote:
This is my problem: at high enzyme concentrations I detect _less_
product than at low enzyme concentrations. At the low enzyme
concentration the amount of liberated product is 15%-40% less than at
the high concentrations. I repeated a couple of experiments to measure
the weight loss of the pulp, which gave similar results.
I can think of several causes for a lower activity of the enzyme at
higher concentrations. However, I can>t think of anything that can lower
the total hydrolysis rate at a high enzyme concentration to below that
of the lower enzyme concentration. For example, if substrate limitation
would play a role I would expect that the hydrolysis rate would be more
of less equal to that of the low concentration, but not much lower.
Formally, you could get your result if the enzyme prep contained a
non-competitive inhibitor. The conc of the inhibitor is continuing to
increase, even though you have added saturating levels of enzyme.
How about if your enzyme stuck to the fibers, and bound product, so
that your assay is not truly measuring the hydrolysis?? Or maybe some
fraction of your enzyme is dead, and binds irreversibly to the fibers?
Ah, I think I see what you mean. You say that the 'dead' fraction binds
to the fibers, making the substrate inaccessible to the 'living'
fraction. Using the high concentration might block so much of the
substrate compared to the low concentration, that the hydrolysis rate
actually is lower.
[/quote]
yes, as an example.
Remember, as with chemical kinetics, kinetics per se cannot tell you
the mechanism. It can only be consistent or inconsistent with a
proposed mechanism. So as you start thinking about formal
possibilities that could lead to the observed kinetics, it is up to
you to test them -- assuming you want to resolve that issue.
[quote]I have to think about that...
A possibility that I just thought of:
1. The enzyme is likely to have a higher activity on insoluble substrate
than on the soluble substrate.
2. At high concentrations the DP of the liberated oligosacharides might
be lower, at low concentrations the DP will be higher (and since the
enzyme is active on insoluble substrate, these will not be hydrolysed)
3. Small oligosacharides might inhibited the enzyme
[/quote]
which at least is testable. This might also predict some complex
kinetics -- and points to why one tries to measure _initial_ rates for
enzymes.
[quote]
So, the enzyme at high concentration produces inhibitors, while the
enzyme at low concentration does not.
Running only two E levels, 100-fold apart is very limited info. I
appreciate the difficulty of assaying this enzyme, but 100-fold extra
enzyme is a huge jump.
You>re right. I also did experiments using a 10 fold increase, but I>m
not able to do more HPLC experiments at the moment.
[/quote]
How about just measuring reducing sugars as a simple assay? You
probably like the additional info that the HPLC provides. But if as a
practical matter HPLC is limiting your assay capacity, doing old
fashioned simple reducing sugar measurements could be a way to let you
get more info more quickly about basic kinetics. Since you mentioned
that you are measuring mainly mono-saccharides at the final step,
total reducing sugars should be a useful number. Of course, you can
save the samples and run selected ones thru the HPLC when time
permits.
You might consider whether you can also measure the oligos from the
initial treatment. Assuming they are soluble, you can probably get a
useful number from the reducing sugar assay; this would count number
of ends = number of oligos. This might help you with the model you
proposed above.
bob |
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