Determining The Effects Of PH, Temperature, Enzyme Concentration, And Substrate Concentration On The Activity Of Catacholase In Solanum Tuberosum

By varying temperature, pH, and enzyme and substrate concentration in this experiment, the effects that these factors have on catecholase activity were determined by measuring the amount of benzoquinone produced.

As shown in Figure 2, increasing the concentration of 0.1 catechol increases the rate of benzoquinone formation, as shown by the increasing levels of absorbance. This trend is observable in all four substrate concentrations until the 4 minute mark. After 4 minutes graphs 3 and 4 begin to decrease in slope, which describes a leveling off of benzoquinone production. Furthermore, at 8 minutes graphs 3 and 4 decline slightly. Graphs 1 and 2 maintain a slightly more linear shape, which implies that their enzymatic reactions continue at a relatively steady pace throughout the experiment.

As was expected, increasing the concentration of catecholase increased the rate of the enzymatic activity (ie. more benzoquinone was formed in the same amount of time). According to Dixon (1958), with an excess of substrate, two enzyme molecules can convert substrate to product at twice the rate of one enzyme molecule. This boils down to the fact that the greater the enzyme concentration, the greater the rate of product formation. Our results are fairly consistent with this statement however graphs 3 and 4 do show some discrepancies. At the 4 minute mark, graph 3 starts to decline. This is most likely due to insufficient mixing of the ingredients or perhaps from improper use of the spectrophotometer. Also, at 6 minutes graph 4 keeps increasing past graph 3 and by 8 minutes has reached a maximum point. Nevertheless, up until the 4 minute mark all graphs show an increase in benzoquinone formation as enzyme concentration is increased.

The enzymatic activity of catecholase is responsible for tissue browning in fruits and vegetables. This enzymatic activity causes the formation of unstable and highly reactive o-quinones, which subsequently react with themselves, proteins, or amino acids, to form brown, black, or red heterogeneous polymers (Paul and Gowda, 2000). The substrates of catecholase in this experiment are catechol and oxygen. Catechol is oxidized to produce benzoquinone, and oxygen is reduced to form water, according to the following equation (Albisu, King and Kozlov, 1989):

Experiment 3: Effect of temperature on Enzyme Activity:

As shown in Figure 3, increasing the temperature has little effect on the reaction until the 30 minute mark. At this time, the reaction takes on a negative slope, which tells us that benzoquinone is being lost. The highest absorbance recorded is at 30C, which could be considered the optimal temperature. The lowest absorbance recorded is at 50C.