(City, State)
Abstract
Reactions taking place in a biological system are usually catalyzed by enzymes. One of these enzymes is the cellobiase, which is works to degrade cellobiose into glucose monosaccharides. Just like any other enzyme, cellobiase works under very specific conditions and alteration of these factors may result in a reduction, in its activity. The experiment was done with an aim of studying the cellobiase in terms of its reaction rate and how factors such as temperature, pH concentration of enzyme and the substrate affect the rate of cellobiase activity. It was determined that cellobiase has an initial rate of a reaction of 11.25nmole/minute. The enzyme is also affected by low as well as high temperatures. From the experiment the optimum temperature of the cellobiaose enzyme was 37OC. Very high and low pH levels reduced its activity and the enzyme was able to work optimally at pH5. Low substrate content also reduced the activity of the enzyme.
Introduction
Reactions that occur in a biological system are usually catalyzed by enzymes. One of these enzymes is the cellobiase, which is involved in breaking down cellobiose into glucose monosaccharides (Reis, et al., 2014). The activity of cellulose enzyme is usually found in organism such as the fungi, bacteria and termites (Brune & Moriya, 2011). The enzyme enables the organisms to breakdown cellulose as a source of food. Those organisms that lack the enzyme lack the ability to use cellulose as a source of food. Breakdown of cellulose to glucose may be done chemically or using enzymes (Wang, 2009).
In enzyme catalyzed reactions, the activation energy that is needed in a reaction is usually low compared to a reaction that is not catalyzed (Cooper, 2000). Since enzymes are proteins in nature, their activity is usually affected by those factors that may alter the 3-D configuration of the protein structure. Some of these factors include temperature, where high temperatures denature the enzyme and low temperatures inactivate the enzyme, pH levels where extreme pH levels, the concentration of the substrate, as well as the concentration of the enzyme (Eed, 2012). This experiment aimed to study the cellobiase in terms of its rate of reaction and how factors such as temperature, pH concentration of enzyme and the substrate affect the rate of cellobiase activity.
Methods
The procedures for this experiment were followed according to the laboratory manual provided.
Results
Measurement of Absorbance and Recording of Standard Curve
The absorbance of the standard samples were measured and recorded in Table 1 below.
Using the data that was gathered a standard curve was plotted that showed the correlation between absorbance at 410 nm and the quantity of the p-nitrophenol (Figure 1).
Figure 1: A correlation between absorbance of p-nitrophenol and its concentration
Experiment 1: Determine the Reaction Rate in the Presence or Absence of an Enzyme
In the determination of the reaction rate of cellobiose, in the presence, as well as in the absence of an enzyme, the absorbance of the different cuvettes that were prepared was done and their values recorded as shown in Table 2 below. Using the standard curve that was developed above, the quantity of p-nitrophenol produced in the reaction cuvettes was determined and the values recorded in Table 2 below.
A graph of p-nitrophenol against time in minutes was plotted as shown in Figure 2 below.
Figure 2: Rate curve for the cellubiase enzyme reaction
Gradient= Change in yChange in x
=57.398-23.6484-1
=33.753
=11.25 nmol/min
Experiment 2: Determine the Effect of Temperature on the Reaction Rate
The absorbance values that were obtained in this experiment for the 4 cuvettes were recorded in Table 3. Using the standard curve that was prepared earlier, the absorbance obtained was changed into the concentration of the product in nmol and the obtained results were recorded in Table 3 below.
Using the above data, the initial rates of the product were calculated as follows:
At 0 OC,
Initial rate=12.568-02-0
=12.5682
=6.284 nmol/min
At 22OC,
Initial rate=57.136-02-0
=57.1362
=28.568 nmol/min
At 37OC,
Initial rate=99.898-02-0
=99.8982
=49.949 nmol/min
At 80OC,
Initial rate=2.057-02-0
=2.0572
=1.029 nmol/min
The initial rates of product formation for the different temperatures were used to plot an initial rate against temperature (Figure 3).
Figure 3: Initial reaction rate against temperature for the cellubiase enzyme
The graph indicates that the enzyme has an optimum temperature of 37OC.
Experiment 3: Quantitative Analysis of the Amount of Product Formed at Different pH Levels
The data on absorbance and the concentration of the product obtained from the standard were recorded in Table 4 below.
Using the p-nitrophenol concentration values obtained, the initial rate of product formation was calculated as follows
At pH 3.0,
Initial rate=67.625-02-0
=67.6252
=33.813 nmol/min
At pH 5.0,
Initial rate=67.739-02-0
=67.7392
=33.869 nmol/min
At pH 6.3,
Initial rate=53.591-02-0
=53.5912
=26.786 nmol/min
At pH 8.6,
51.375-02-0
=51.3752
=25.687 nmol/min
A graph of the initial rate of p-nitrophenol formation against pH was plotted as in Figure 4 below.
Figure 4: Initial rate of product formation at different temperatures
The graph indicates that the optimum pH for the enzyme is pH 5 since it is the pH that gave the highest initial rate of reaction.
Experiment 4: Determine the Effect of Substrate Concentration on the Reaction Rate
The values for the absorbance and the concentration of p-nitrophenol that were obtained using the standard curve were recorded in Table 6 below.
The concentrations of p-nitrophenol for the two levels of substrate concentration were plotted against substrate concentration in a graph (Figure 5).
Figure 5: Amount of the p-nitrophenol against substrate concentration
Calculations for the initial rates of reaction for the two levels of substrate concentration were calculated as follows
At high substrate concentration,
Initial rate=107.74-02-0
=107.742
=53.87 nmol/min
At low substrate concentration,
Initial rate=47.91-02-0
=47.912
=23.955 nmol/min
Discussion
The initial rate of product formation in an enzymatic reaction indicates the extent at which an enzyme catalyzes a given reaction. The cellobiase enzyme recorded a rate of 11.25nmol/min. This rate shows that cellobiase is an excellent enzyme in catalyzing the breakdown of cellobiose to glucose molecules. The current study has also demonstrated that various factors have differing ways in which they affect the activity of an enzyme. Increase or decrease in temperature above the optimum temperature has been shown to reduce the activity of an enzyme drastically. For the current enzyme, cellobiase, the optimum temperature may be said to be 37OC. Increasing the amount of the substrate in a reaction has been shown to cause an increase in the rate of enzyme activity. This is mainly due to the increased interaction between the substrate and the enzyme (Adam-Day, 2012). Increase or decrease in pH above the optimum pH has been shown to reduce the activity of an enzyme drastically. For the current enzyme, cellobiase, the optimum pH may be said to be pH 5. Changes in pH levels have been associated with alteration of the 3-D configuration of the active site of an enzyme (Adam-Day, 2012). This is the site where enzyme interacts with the substrate.
Conclusion
This experiment aimed to study the cellobiase in terms of its rate of reaction and how factors such as temperature, pH concentration of enzyme and the substrate affect the rate of cellobiase activity. It can be concluded that the initial rate of a reaction of cellobiase enzyme is 11.25nmol/min and has an optimum temperature of 37OC and optimum pH of 5. High substrate concentration increased the activity of cellobiase enzyme.
Reference List
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Brune, A. & Moriya, O., 2011. Role of the termite gut microbiota in symbiotic. In: D. E. Bignell, Y. Roisin & N. Lo, eds. Biology of Termites: a Modern Synthesis. Netherlands: Springer, pp. 439-475.
Cooper, G. M., 2000. The Cell. 2nd ed. Sunderland (MA): Sinauer Associates.
Eed, J., 2012. Factors Affecting Enzyme Activity. ESSAI, 10(1), pp. 47-51.
Reis, A. et al., 2014. Oxygen-limited cellobiose fermentation and the characterization of the cellobiase of an industrial Dekkera/Brettanomyces bruxellensis strain. Springer Plus, 3(1), pp. 1-9.
Wang, N. S., 2009. Cellulose Degradation. [Online] Available at: http://www.eng.umd.edu/~nsw/ench485/lab4.htm[Accessed 26 February 2014].
