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questions about enzymatic acitivity assay


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#1 califoria

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Posted 24 September 2009 - 08:29 PM

hi,

I am going to do some experiments to measure the extracellular enzymatic activity in bacteria.
These are Pectate Lyase, Protease, Polygalacturonase, and Cellulase secreted by Pectobacterium carovotorum.

I read some related articles, and briefly know how to do these measurements.
But I am confused that every article leaves no calculation formulas but one definition like "one unit of Pectate Lyase is defined as the amount enzyme that produces 1 umol of unsaturated products per minute. Activity is expressed as micromoles of unsaturated products liberated per minute per OD580 unit" !

How can I connect the OD value to the amount of ** products in the reaction solution ? :P

Thanks in advance.

#2 Gerard

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Posted 25 September 2009 - 01:21 AM

How can I connect the OD value to the amount of ** products in the reaction solution ? :P


As usesual in a quantitative assay:
You have to establisch a calibrationline with the ** products
Ockham's razor
Pluralitas non est ponenda sine necessitate
-- "You must assume no plural without necessity".

#3 califoria

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Posted 25 September 2009 - 02:32 AM

How can I connect the OD value to the amount of ** products in the reaction solution ? :P


As usesual in a quantitative assay:
You have to establisch a calibrationline with the ** products


Thank you, Gerard.
I knew what you said was a good way.
But consider this: these reaction products are in umol. These solutions would be very hard to prepare, I think.

But thank you very much for reply, anyway.

#4 DRT

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Posted 26 September 2009 - 07:22 PM

But I am confused that every article leaves no calculation formulas but one definition like "one unit of Pectate Lyase is defined as the amount enzyme that produces 1 umol of unsaturated products per minute. Activity is expressed as micromoles of unsaturated products liberated per minute per OD580 unit" !

How can I connect the OD value to the amount of ** products in the reaction solution ? :D



It looks as if the OD580 is correcting the enzyme activity for the amount of bacteria present ie activity/cell (or have I misread your confusion?).

#5 califoria

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Posted 27 September 2009 - 07:57 AM

But I am confused that every article leaves no calculation formulas but one definition like "one unit of Pectate Lyase is defined as the amount enzyme that produces 1 umol of unsaturated products per minute. Activity is expressed as micromoles of unsaturated products liberated per minute per OD580 unit" !

How can I connect the OD value to the amount of ** products in the reaction solution ? :D



It looks as if the OD580 is correcting the enzyme activity for the amount of bacteria present ie activity/cell (or have I misread your confusion?).



No, OD580 is used for detecting the amount of the unsaturated products in the solution, not for the amount of bacteria cells here.
Thanks, DRT.




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