The activity of enzymes is highly affected by changes in pH and temperature and as such each enzyme works best at a given pH and temperature (Jencks, 1987). Changes in pH alter the state of ionization of charged amino acids that may play a crucial role in substrate binding and/or the catalytic action itself. Similarly, hydrogen bonds are easily disrupted by increasing temperature which disrupts the shape of the enzyme such that its affinity for its substrate diminishes (Groves, 1997).
In this experiment, each group of four students was provided with a tube of concentrated -amylase that was labeled A, B or C. The tube with enzyme was kept on ice. Each group performed part 1 and 2 of the experiment.
First, -amylase preparation C was recorded and then one test tube was labeled "blank" and five others as 40C, 230C, 370C, 650C and 1000C. 1ml of 1% starch solution at pH 7 was added to each test tube, whereby the starch was the substrate for the reaction. Each tube was placed in a water bath that was set as one of the indicated temperatures. The blank and the 230C were placed at room temperatures while the 40C on ice. All the tubes were allowed to equilibrate to the desired temperatures for ten minutes.
A fresh dilution of the unknown -amylase was made by mixing 100l of the concentrated enzyme stock with 9.9 of dH20 shaking to mix. The stock and the diluted solutions were kept on ice. All the tubes were retrieved after 10 minutes of pre- incubation step. 1ml of dH20 was added to the blank tube only. To the other five tubes, a timer