Validation of a dispenser machine and enzyme kinetics for alcohol dehydrogenase

Abstract: To optimize precision and accuracy of small dispensed volumes, a dispenser was built. A 96-well plate was loaded with four replicates of a linear pyranine dilution series to test the accuracy and precision of the dispenser machine. Three dilution series of pyranine (100-0 percent, 10-0 percent and 1-0 percent) were made. The results showed that the dispenser has very good accuracy and precision. The dispenser was then used in an enzyme kinetic experiment with alcohol dehydrogenase to reveal kinetic properties of this enzyme. A dilution series of the substrate ethanol was loaded onto a new 96-well plate to generate a Michaelis Menten curve. The kinetic parameters for the ethanol concentration 0,2 M and 0,1 M were as follows: KM 10,5 mM and KM 7,48 mM, kcat 1,06 s-1 and kcat 0,39 s-1.