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Enzyme activity of Clostridium phytofermentans acylating aldehyde dehydrogenase Cphy_1178 against various aldehyde substrates

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posted on 04.02.2016, 20:43 by Jon Marles-WrightJon Marles-Wright, Laura TuckLaura Tuck

Method

Kinetic assays were performed using a multimode plate reader (Molecular Devices, M5), in flat bottomed 96 well plates (Corning) at 21°C. Each 300 μl reaction contained 100 mM Tris.HCl, pH 8.0; 0.66 mM NAD+; 100 mM KCl; 10 mM 2-mercaptoethanol; 100 nM protein; and various concentrations of aldehyde substrates, using both 100 mM and 10 mM stock solutions to increase assay accuracy. The enzyme activity was monitored by measuring the formation of NADH at 340 nm using an absorption coefficient of 6.22 mM-1 cm-1. Enzyme activity is expressed as mM NADH produced per second. Steady state kinetic data, obtained with five technical repeats, were analysed by non-linear regression to the Michaelis-Menten equation using GraphPad Prism. 


Data

The following data sets are included in this collection

Data from purification table

1178 his cell extract.txt

1178 his cell lysate.txt

1178 his gel filtration.txt

1178 his his-trap .txt

1178_20 Q-sepharose.txt

1178_20 cell extract.txt

1178_20 cell lysate.txt

1178_20 gel filtration.txt


Kinetic Data

Acetaldehyde 100mM stock.txt

Acetaldehyde 10mM stock.txt

Butyraldehyde 100mM stock.txt

Butyraldehyde 10mM stock.txt

Hexanal 100mM stock.txt

Hexanal 10mM stock.txt

Pentanal 100mM stock.txt

Pentanal 10mM stock.txt

Propionaldehyde 100mM stock.txt

Propionaldehyde 10mM stock.txt


Derived slope data

Mean & SD Acetaldehyde 100mM & 10mM combined.txt

Mean & SD Acetaldehyde 100mM stock.txt

Mean & SD Acetaldehyde 10mM stock.txt

Mean & SD Butyraldehyde 100mM & 10mM stock combined.txt

Mean & SD Butyraldehyde 100mM stock.txt

Mean & SD Butyraldehyde 10mM stock.txt

Mean & SD Hexanal 2 10mM stock.txt

Mean & SD Propionaldehyde 100mM & 10mM stock combined.txt

Mean & SD hexanal 100mM & 10mM stock combined.txt

Mean & SD hexanal 100mM stock.txt

Mean & SD pentanal 100mM & 10mM stock combined.txt

Mean & SD pentanal 100mM stock.txt

Mean & SD pentanal 10mM stock.txt

Mean & SD propionaldehyde 100mM stock.txt

Mean & SD propionaldehyde 10mM stock.txt


Data processing method

The following method was used to process the data above

-Export data from plate reader (time format)

-Subtract data  (5 repeats) from blank (no protein)

-Correct for NADH extinction coefficient (6220 M cm-1), correct delta NADH for protein from nM to mM (100nM*10 to give µM*1000 to give mM) correct for pathlength [8mm] (1.25)

-Beer-Lambert law A=ecl, c=A/el, c=A/(6.22)*(correction factor), c=(A/6.22)*12500

-Gives [NADH] mM/mM protein, 


-Export processed data to GraphPad (File, New, data table and graph, Enter 5 replicate values in side-by-side subcolumns)

-Analysis, linear regression of data

-Take best fit values of slope, transpose data 

-New, data table and graph, Enter and plot error values already calculated elsewhere [Enter: Mean, SD, N]

-Take 10mM and 100mM data and combine Mean & SD values

-Analyze, Nonlinear regression (curve fit), Michaelis-Menten or substrate inhibition

-Data points that were outliers were excluded from the analysis


Funding

BBSRC EastBio DTP (BB/M010996/1)

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