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

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. <br> Data The following data sets are included in this collection <b>Data from purification table</b> 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 <b></b><br> <b>Kinetic Data</b> 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 <br> <b>Derived slope data</b> Mean &amp; SD Acetaldehyde 100mM &amp; 10mM combined.txt Mean &amp; SD Acetaldehyde 100mM stock.txt Mean &amp; SD Acetaldehyde 10mM stock.txt Mean &amp; SD Butyraldehyde 100mM &amp; 10mM stock combined.txt Mean &amp; SD Butyraldehyde 100mM stock.txt Mean &amp; SD Butyraldehyde 10mM stock.txt Mean &amp; SD Hexanal 2 10mM stock.txt Mean &amp; SD Propionaldehyde 100mM &amp; 10mM stock combined.txt Mean &amp; SD hexanal 100mM &amp; 10mM stock combined.txt Mean &amp; SD hexanal 100mM stock.txt Mean &amp; SD pentanal 100mM &amp; 10mM stock combined.txt Mean &amp; SD pentanal 100mM stock.txt Mean &amp; SD pentanal 10mM stock.txt Mean &amp; SD propionaldehyde 100mM stock.txt Mean &amp; SD propionaldehyde 10mM stock.txt <br> 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, <br> -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 &amp; SD values -Analyze, Nonlinear regression (curve fit), Michaelis-Menten or substrate inhibition -Data points that were outliers were excluded from the analysis<br>