4 replies to this topic

[califoria]

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 ?  

Thanks in advance.

[Gerard]

califoria, on Sep 25 2009, 08:59 AM, said:

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

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

[califoria]

Gerard, on Sep 25 2009, 01:21 AM, said:

califoria, on Sep 25 2009, 08:59 AM, said:

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

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.

[DRT]

califoria, on Sep 25 2009, 02:29 PM, said:

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 ?  

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?).

[califoria]

DRT, on Sep 26 2009, 07:22 PM, said:

califoria, on Sep 25 2009, 02:29 PM, said:

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 ?  

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.