The Effects of low pH value on the Activity of an Enzyme

Enzymes are basically biological catalysts. Their structural components include properties that make biological processes unique. They require a particular optimum temperature and pH value to reach their highest reactive potential. They are also proteins and can become denatured at temperatures above 45 degrees. In other cases, their denaturing occurs as a result of extreme pH values. Enzymes are basically catalysts that can be involved in reactions but can never be used completely. According to Bolz, (2013), they are regenerable since they can be utilized completely in any reaction. The experiment used the barley amylase enzyme to spur the breakdown of starch into simple sugars. The reason why barley amylase was used in the experiment was due to its ubiquitous nature in the environment (Bubalo, et al., 2014). The main objective of the experiment was to examine the effects of three parameters of the enzyme reaction, pH, and temperature and enzyme concentration. The experiment aimed at examining the how these parameters react on the activity of amylase enzyme. Spectrophotometric techniques were utilized in the experiment to make it successful and achieve the required results.

The following formulated hypothesis was proved during the experiment; low pH lowers the activities of barley amylase enzyme. Many biological catalysts performs best at optimum pH. An optimum pH condition is possible with the presence of both H+ and OH- that have a significant influence on the active sites of the enzymes. The impact results in either a decrease or increase once the pH of the solution is changed.

Materials and Methods

The experiment required 28mls of barley enzyme, 21mls of distilled water. The 28mls of barley enzyme were poured in a flask labeled 1 while distilled water was added into two flask 2 and 3. Out of the 28mls of the enzyme in the experiment, 7mls was poured into flask 2, with a dilution of 1:4. 7mls of the contents in flask 2 were poured into flask 3 with a dilution ration of 1:16. After the reaction, 7mls of the contents in flask 3 were extracted and the rest disregarded. The disregarded content was referred to as the undiluted flask. For each of the flask, a blank was prepared through pipetting 1.5mls of the solution from every single flask, 1.5mls of distilled water in a test tube and addition of 3 drops of Lugol’s iodine. The contents of the mixture were later mixed in vortexes.

The spectrophotometer used in the experiment was hit it off with 540nm of heat and later zeroed using the blank. Starch solution of 20mls was poured into flask 3 and swirled. The time taken for the contents of flask 3 to mix was noted. Another 3mls of the solution was poured in a cuvette holding the Lugol's iodine and vortex while recording the absorbance. The procedure was repeated at intervals of 30 seconds for a period of 240 seconds equivalent to 4 minutes. The results of the repeated procedure were recorded at the specific time intervals and the results are presented in table 1a below. At the end of the 4 minutes, the spectrophotometer was later blanked and the process repeated using a starch solution. The results of the second procedure were recorded are presented in table 1b below. The values obtained from the experiment were then used to plot a graph.

The preparation of the blank used in the experiment was prepared by mixing 3mls and 3 drops of iodine in a cuvette at 540nm. All the flasks used were clearly labeled to avoid any confusion. 22mls of buffer was transferred into four different flasks each containing different concentration of pH, 4, 5, 6, and 7. A mixture of the enzyme and substrate in ratios of 1mls and 2mls respectively were mixed into the cuvette and later transferred to a cuvette having droplets of iodine vortex. The table 1a below represents the recorded time of absorbance. The procedure was repeated for 7 minutes at intervals of 1 minute each. The other three flasks containing different concentrations of pH were also processed following same the same procedure. All the results obtained from these procedures were recorded and presented in table 1b.

Results

Table 1 (a): pH against Absorbance

Table 1 (b): pH against Percentage Starch

Discussion

From the presented results in tables 1a and 1b, it is evident that the hypothesis of the study and research objective were achieved. The results give rise to three significant conclusions. The first being, an increase in temperature results in a corresponding increase in the rate of reaction of the enzyme amylase (Vasudevan, 2013). The second conclusion is that the concentration rate of amylase enzyme is directly proportional the reaction of the same enzyme. This implies that an increase in the concentration levels of the amylase enzyme would result in an increase in the reaction rate of the enzyme (Bergmeyer, 2012). The third conclusion drawn from the experiment is that amylase reacts best at optimum pH concentration. In case the concentration of the pH is altered it will affect the action of amylase enzyme.

Conclusion

The experiment was successful since it achieved the objectives and met satisfied the formulated hypothesis. The reason why it was a success is that the optimum pH concentration for amylase enzyme was found to be pH4. Enzymes have divergent features, therefore, a general standardization of enzyme amylase is not possible. One significant recommendation would be stabilize the pH through the use of buffers and make sure that its pKa corresponds to the pH optimum of the enzyme. Amylase is an enzyme that is picked from germinating barley seeds. The enzyme hydrolyzes starch particles into glucose that the seedling could use. Starch with the help of amylase enzyme has the ability to hydrolyze starch at temperatures in a situation where the reaction fails to take place under normal conditions. Varying the pH of solution helps to identify the optimal condition for the starch hydrolysis. The optimal ranges of temperature and pH are dependent on the substrate concentration, and the amount of enzyme used during the experiment.

References

Bergmeyer, H. U. (Ed.). (2012). Methods of enzymatic analysis. Elsevier.

Bolz, F. (2013). Advanced materials in catalysis. Elsevier.

Bubalo, M. C., Hanousek, K., Radošević, K., Srček, V. G., Jakovljević, T., & Redovniković, I. R. (2014). Imidiazolium based ionic liquids: effects of different anions and alkyl chains lengths on the barley seedlings. Ecotoxicology and environmental safety, 101, 116-123.

Vasudevan, D M (2013). Textbook of biochemistry for medical students. London: Jaypee Brothers Medical Publishers