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Effect of Temperature on Invertase Activity Jan Carlo Marcus T. Doria, Camille Joy V. Galapon, Innamari Justinne Q.

Go, Anjelica V. Gonzales, Elizar N. Gonzales Group 5 2G Pharmacy Biochemistry Laboratory ABSTACT
Enzyme reaction rates were affected by some factors, an example of which was Temperature. A set of test tubes with equal amounts of sucrose and invertase were subjected to varying temperatures, and then Dinitrosalicylic acid (DNS) reagent was added to the said test tubes. Each test tube were subjected to 95C water bath to form a red-brown color characteristic, then the result was analyzed with the spectrophotometer at 540 nm. The aim of the activity was to determine the effects of changes in temperature on reaction rates of enzyme-catalyzed reaction, using the enzyme Invertase. Invertase was a yeast derived enzyme that splits sucrose into glucose and fructose.

INTRODUCTION
Enzymes were protein molecules that act as a biological catalyst. Its basic function was to increase a rate of reaction. Most enzymes act specifically with only one reactant, called a substrate, to produce products. And its most remarkable characteristic was that enzymes were regulated from a state of low activity to high activity and vice versa. The efficiency of enzymes was affected by some factors just like pH and temperature. Invertase was the enzyme used and can be extracted from bakers yeast which will also be done in this activity. 100mg/L Sucrose standard solution, Concentrated Hydroalcoholic acid, 0.5M Potassium hydroxide, Dinitrosalicylic acid (DNS) reagent, 10g/L sucrose solution, 0.1M buffer solutions (pH 5), test tubes, pipettes, beakers, volumetric flasks, paraffin film, hot plate, UV-Vis spectrophotometer.

B. Procedure a. Extraction Yeast of Invertase from

Figure1. Invertase Activity


The aim of this experiment was to observe and mark the optimum temperature in which the invertase enzyme react with sucrose. This activity also allows the students to use spectrophotometer to study and take note the results of the experiment in different temperatures.

In order to acquire invertase, 0.25g bakers yeast was weighed and dissolved in distilled water to make a 250-mL solution. The solution was then allowed to stand for 20 minutes at room temperature. If sedimentation occurs, collect the supernatant in order to serve as the enzyme stock solution that would be used for future experiments.

b. Preparation of Denatured Invertase Stock Solution


For 10 minutes, 100mL of enzyme stock solution was incubated in a boiling water bath. The solution was then allowed to cool and if frothing occurred, the supernatant would be collected and would act as the denatured enzyme stock solution for future purposes.

EXPERIMENTAL A. Materials and Compounds Used a. Extraction Yeast: of Invertase from

c. Determining Temperature Activity

the on

Effect of Invertase

0.25g Bakers yeast and Distilled water

b. Effect of Temperature Invertase Activity:

on

Water baths with 20, 30, 50, 60, 70, 90C were set and 6 test tubes containing 1.5mL sucrose solution were prepared. The test tubes were

then incubated separately for 5 minutes in each water bath. In another test tube, 0.80mL enzyme stock solution and 19.20mL 0.1M buffer solution with a pH of 5 were mixed. Next was 3mL of dilute enzyme solution was added to all test tubes and was again incubated for 5 minutes. Afterwards, 3mL of DNS reagent was also added to all test tubes. The test tubes were then immersed in a 95C water bath for 10 minutes so that it could develop the characteristic red-brown color, then the solution was allowed to cool. The said procedures were also used to make solutions for denatured enzymes. Absorbance at 540nm was measured. Lastly, the amount of sucrose hydrolyzed using the sucrose standard curve was determined. Sucrose standard curve was made in the Dinitrosalicylic colometric method.

Figure 2. Hydrolyzed-Sucrose Standard Curve REFERENCE From Internet:


1. http://www.elmhurst.edu/~chm/vchembo ok/570enzymes.html(01/06/13) 2. http://ehis.ebscohost.com/ehost/detail?vi d=10&hid=4&sid=ecd180dc-d949-4f1fbc7c4d7706d2779e%40sessionmgr10&bdata= JnNpdGU9ZWhvc3QtbGl2ZQ%3d%3d#db =bmh&AN=14387865 (01/06/13) 3. http://www.ncbi.nlm.nih.gov/books/NBK2 1166/(01/06/13) 4. http://media.collegeboard.com/digitalServ ices/pdf/ap/biomanual/CB_Bio_Lab_13_WEB_1_24_12.p df(01/06/13) 5. http://ehis.ebscohost.com/ehost/detail?vi d=11&sid=ebebc610-ce21-488e-9355354b06c52a0b%40sessionmgr4&hid=2&b data=JnNpdGU9ZWhvc3QtbGl2ZQ%3d%3 d#db=mnh&AN=7784506 (01/06/13) 6. http://ehis.ebscohost.com/ehost/detail?vi d=5&hid=3&sid=41618542-1239-40e5bb1c435b34fec565%40sessionmgr10&bdata=J nNpdGU9ZWhvc3QtbGl2ZQ%3d%3d#db= mnh&AN=10028694 (01/06/13)

RESULTS AND DISCUSSION


After observation under the spectrophotometer, the data were noted down (Table 1.), and graphed (Figure 2.)

Test Temperature Absorbance Tube (C) 1 20 0.513 2 30 0.441 3 50 0.687 4 60 0.432 5 70 0.421 6 90 0.377 Table 1. Effects of Temperature on Invertase Activity
In the graph shown at Figure 2, it was obvious that there was a peak absorbance at 50C. This was the optimum temperature where in the enzyme was most effective. It was shown that as the temperature increases the rate of reaction also increases just like in inorganic reactions. But once the rate of reaction reaches its peak temperature there would be a sudden drop at the rate of reaction thus resulting into a bell-shaped curve. Since enzymes, like invertase, were proteins with tertiary structure, they can be denatured when exposed to high temperatures. Denatured proteins do not react as much as the newly extracted proteins, thus resulting into a drastic drop of the rate of reaction.

7. http://ehis.ebscohost.com/ehost/detail?vi d=5&hid=3&sid=41618542-1239-40e5bb1c435b34fec565%40sessionmgr10&bdata=J nNpdGU9ZWhvc3QtbGl2ZQ%3d%3d#db= mnh&AN=16656885 (01/06/13)

From Books:
1. Campbell M., Farrell S. (2012). Biochemistry (7th Ed.). China: China Translation & Printing Services Limited

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