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Determining the Effect of Temperature on Invertase Activity

Jiana Tresmaria, Francesca Valdes, Melanie Velasco, Marielle Villarino, and Faye
Wong
Group 8 2A Pharmacy Biochemistry Laboratory

ABSTRACT

The reaction rate of enzymes were affected by factors like temperature. Equal amounts of
sucrose and invertase inside test tubes were placed in different temperatures.
Dinitrosalicyclic Acid (DNS) reagent was also placed inside the test tubes. After which, the
test tubes were put to 95 degrees water bath in order to form a red-brown color. The result
was subjected to analyzation with spectrophotometer at 540 nm. The idea for this is that it
will be able to determine the effect of changes in temperature with the reaction rates of
enzyme-catalyzed reaction with the use of the enzyme invertase. Invertase was a yeast
from enzyme that separates sucrose into glucose and fructose.
INTRODUCTION EXPERIMENTAL
Enzyme is a macromolecule that A.Test Compounds used
catalyzes a biochemical reaction. The In 5 test tubes:
starting molecules in this kind of chemical ● 1 mL of 0.03M sucrose per
reaction are called substrates. Enzyme test tube
interacts with a substrate to form a new
● 1.4 mL of distilled water per
product forming an Enzyme Substrate
Complex. It is a globular protein which is test tube
affected by several factors particularly ● 0.5 mL of 0.05 M acetate
changes in pH and temperature. An buffer (pH 4.7) per test
extremely high or low temperature will tube
take effect by lessening the enzymatic ● 0.1 mL of invertase solution
activity.
● 2 mL of DNS reagent
In test tube labeled blank:
● 1.4 mL of distilled water per
test tube
● 0.5 mL of 0.05 M acetate
buffer (pH 4.7) per test
tube
● 0.1 mL of invertase solution
Figure 1. Invertase Activity ● 2 mL of DNS reagent

Invertase, a digestive enzyme B. Procedure
derived from Saccharomyces cerevisiae, Prepare water baths with
separates sucrose into its main temperatures 20°C, 30°C, 50°C, 60°C,
components, fructose and glucose. It acts 70°C and 90°C. In six test tubes, place 1
as a catalyst for the hydrolysis of sucrose. mL of 0.03 sucrose, 1.4 mL distilled water
The objective of this experiment is to and 0.50 mL of 0.05 M acetate buffer
observe the optimum temperature of solution with pH 4.7 and incubate them
invertase and its reaction with sucrose by separately in each of the water baths for
using a spectrophotometer. five minutes (one of these test tubes will
serve as the blank reagent). Next, add 0.1

plot the temperature against the Figure 2. Guevarra.D.I.T. 3 50 0. Lastly.. thus resulting into a drastic drop of the rate of reaction. G. [2] invertase. and graphed (Figure 2.L.07 (2017). & Farrell.W.. Santiago. 2 30 0. M. C.I. Test Temperature Absorbance [2] Tube (°C) (A540) REFERENCES Blank 60 0. Revised Edition. [3] Since enzymes.) newly extracted proteins.C. Laboratory Manual in General Biochemistry. R. L.L.. Voet. A. 5 70 0..A. 1 20 0. G. C. Principles of Biochemistry Fourth Edition In the graph shown at Figure 2..C.. J. Ysrael. D. Cruz. L. Then add 2 mL of the DNS reagent and immerse in 95°C water bath for ten minutes until it develops a red-brown color. Crisostomo. Connecticut. It also increases just like in Temperature affects the invertase inorganic reactions. Effect of Temperature on [2] Voet.. S. Cool the test tubes. [1] with the Change of Temperature It was shown that as the RESULTS AND DISCUSSION temperature increases the rate of reaction. The Change of Absorbance amount of inverted sugar. they can be denatured when spectrophotometer.G. were proteins with tertiary After observation under the structure. add 5 mL of distilled water to each one and mix well.L. (2013).08 Manila.). Peña. P. A very low temperature will not there would be a sudden drop at the rate allow the enzyme to react while a very of reaction thus resulting into a bell- high temperature will denature the shaped curve.mL of the enzyme solution to the test tubes and incubate again for another five minutes without removing them from their respective water baths. Farrow.S.M. was low. Test tubes labeled 1 to 5 had an Biochemistry (8th ed. Daya. Denatured proteins do not react as much as the down (Table 1.G...058 F.. Santiago. Liu.T. M. and Torres. the data were noted exposed to high temperatures.. Vargas. Gabona. Table 1.. Peña.A.. A.G.B. Metro 4 60 0.C.. But once the rate of reaction reaches its peak temperature activity..A. Quezon City: C & E Publishing.)..014 [1]Bathan.123 Inc. M.. the absorbance for the blank labeled test tube [3]Campbell. M. Pratt. M. Invertase Activity (2013). Cengage Learning . USA: increasing absorbance. A. Sarile. M.. De Guia.063 L. like protein. Measure the absorbance at 540 nm and determine the amount of invert sugar produced in each test tube using the invert sugar standard curve.