Effects of Change in pH and Temperature on the Reaction Rates of Enzyme-Catalyzed Reaction

Yanquiling, Nicole Anne P.; Yonzon, Ma. Julianne Denise U.; Yu, Kenzo Donovan B.; Yu, Kevyn Sikorsky D.; Zara, Nerissa Jeanne M. Group 8 2GPH Faculty of Pharmacy University of Santo Tomas
Abstract: Enzymes are biological catalysts, mainly proteins, generated by an organism to speed up chemical reactions. The objectives of the experiment are to extract invertase that breaks existing bond in sucrose to form glucose and fructose from Bakers yeast and to determine the effects of the change in pH on the reaction rates of an enzyme-catalyzed reaction. To accomplish its objective, In this experiment, the sucrose assay using a DNS colorimetric method measured and showed the maximum capacity of invertase activity in increasing concentration of the standard through a graphical representation, the best fit straight line. Factors that affect the invertase is also investigated such as pH and temperature .The results obtained were represented by bell-shaped graphs that shows the optimum amount of pH and temperature.

INTRODUCTION
Enzymes are catalysts that make biochemical reactions happen faster than they would, sometimes the essential reactions would not happen at all without the help of enzymes. Being catalysts also means that enzymes are not part of the final product. They make things happen. When the job is done, enzymes are ready to catalyze a new biochemical reaction. The activity of enzymes depends on various factors, such as the effect of enzyme concentration, pH, and temperature. This experiment focuses on the effect of pH on enzymatic activity. The objectives of the experiment are to extract invertase that breaks existing bond in sucrose to form glucose and fructose from Bakers yeast and to determine the effects of the change in pH on the reaction rates of an enzymecatalyzed reaction.

METHODOLOGY
A. Extraction of Invertase from Yeast 0.25g of Bakers Yeast was dissolved in distilled water to make a 250mL solution. The solution was left to stand for 20-minutes at room temperature. The supernatant liquid was collected which has served as the enzyme stock solution for the succeeding experiments. B. Preparation of Denaturated Invertase Stock Solution 100mL of the 250mL stock solution prepared beforehand was incubated in a boiling water bath for 10-minutes. After the incubation it was left to cool down. The supernatant liquid was collected which has served as the denaturated enzyme stock solution for the succeeding experiments. C. Sucrose Assay Using Dinitrosalicylic Colorimetric Method

7 test tubes were prepared containing the assigned amount of sucrose standard solution and distilled water. 3 drops of concentrated HCl was added to each test tube, well mixed and incubated at 90-degrees Celcius water bath for 5 minutes. After incubating, 0.15mL of 0.5M KOH was added to to each text tube to neutralize the solution. Litmus paper was used to check their pH. 2.80mL of 0.1M Buffer solution with the pH of 5 was added to each test tube and mixed thoroughly in the solution. 3mL of DNS reagent was added to each test tube, and were immersed in 95-degrees Celcius water bath for 10 minutesto develop the characteristic color of red-brown. After cooling, the absorbance at 540nm was measured D. Effect of pH on Invertase Activity 2.50mL of assigned buffer solution, 0.50mL of enzyme stock solution and 1.50mL of sucrose solution are mixed together in a test tube. The solution then was incubated for 15 minutes in a 60-degrees Celsius water bath. 3mL of Dinitrosalicylic (DNS) reagent was added then the solution was incubated for 10 minutes in a 95-degrees Celsius water bath. The solution was cooled after, A540 was measured E. Effect of Temperature on Invertase Activity

4 0.050 - (0.170) 5 0.067 0.170 6 0.250 0.183 7 0.100 0.226 The table shows the results of Acid-Hydrolyzed Sucrose
Table 2: Results of Effects of pH on Invertase Activity

pH

AMOUNT OF ACIDHYDROLYZED SUCROSE (mg/mL)

ABSORBANCE540

0.075 2 0.026 0.161 3 0.146 0.147 5 0.126 0.192 7 0.189 0.279 7.5 0.310 0.022 8 -0.048 0.040 9 -0.023 0.023 11 -0.047 The Table shows the results of the measured absorbance at 540 nm in regards with the pH used in the solution. Table 1: Results of Temperature on Invertase Acitivity

TEMPERATURE

2.50 mL of acetate buffer solution, 0.50mL RESULTS & DISCUSSION

Table 1: Results of Sucrose Assay using Dinitrosalicylic Colorimetric Method

TEST TUBE NO. BLANK 2 3

AMOUNT OF ACIDHYDROLYZED SUCROSE (mg/mL) 0 0.017 0.033

ABSORBANCE540 0.00 0.015 - (0.226)

AMOUNT OF ACIDABSORBANCE540 HYDROLYZED SUCROSE (mg/mL) 0.186 20 0.180 0.142 30 0.119 0.209 50 0.212 0.259 60 0.282 0 70 -0.079 0 90 -0.079 The Table shows the results of the measured absorbance at 540 nm in regards with the Temperature used to incubate the solution.

SUCROSE ASSAY CURVE

1 0.8 0.6 0.4 0.2 A540 0 -0.2 -0.4 -0.6 -0.8 -1 Acid-Hydrolyzed Sucrose (mg/mL) 0 0.017 0.033 0.05 0.067 0.25 0.1 0 0.017 0.033 0.05 0.067 0.25 0.1

http://nationalenzyme.com/education/whatare-enzymes/ [3] Novozymes. (2013). what are enzymes? Retrieved January 12, 2014, from Novozymes: http://www.novozymes.com/en/about-us/ourbusiness/what-are-enzymes/pages/default.aspx [4] Welcome Trust, Co. (2011).Who am I?. Retrieved January 12, 2014, from sciencemuseum: http://www.sciencemuseum.org.uk/whoami/fin doutmore/yourbody/whatdoyourcellsdo/whatis acellmadeof/whyareenzymesimportant.aspx [5] Whitford, D. (2005). Proteins: Structure and Function. Wiley, John & Sons, Inc. New Jersey, USA [6] Whitford, D. (2005). Proteins: Structure and Function. Wiley, John & Sons, Inc. New Jersey, USA

Figure 1: Sucrose Assay Curve using Dinitrosalicylic Colorimetric Method

The Figure shows a Sucrose Assay Curve using Dinitrosalicylic Colorimetric Method. The two red dots represent data that have been measured but are wrong.

CONCLUSION REFERENCES
[1] Crisostomo, A. C., Daya, M. L., Guia, R. M., Farrow, F. L., Gabona, M. G., Liu, M. I., et al. (2010). Laboratory Manual in General Biochemistry. Quezon City: C&E Publishing, Inc. [2] Nemiroff, P. (2012). WHAT ARE ENZYMES? Retrieved January 12, 2014, from National Enzyme Company: