DETERMINATION OF THE EFFECT OF TEMPERATURE ON THE ACTIVITY OF ENZYME INVERTASE

Tan, J. G.; Tiongson, I.; Tomagan, J. M.; Torres, P. M.; Torres, S. M. Group 9 2BMT Biochemistry Laboratory

Abstract
In this particular activity, Invertase, the enzyme found in Bakers yeast, was extracted and the activity of this particular enzyme is the main point of study. The aim of this experiment is to determine several factors that affect enzyme reaction rates with the enzyme Invertase. Temperature is one of those factors. Invertase is a hydrolase which reacts with sucrose to form glucose and fructose. Several test tubes with equal amounts of sucrose and invertase were subjected to varying temperatures, and then the DNS reagent was added. Each test tube was subjected to heat, and was then analyzed with the spectrophotometer with the absorbance value of 540 nm.

Introduction
Of all the functions of proteins, catalysis is probably the most important. In its absence, most reactions in biological systems would take place far too slowly to provide products at an adequate pace for a metabolizing organism. The catalysts that serve this function in organisms are called enzymes [1]. An enzyme is a substance-usually a protein-that acts as a catalyst for a biological reaction. Like all catalysts, enzymes dont affect the equilibrium constant of a reaction and cant bring about a chemical change that is otherwise unfavorable. It acts only to lower the activation energy of a reaction, thereby making the reaction take place more rapidly. Sometimes, the rate acceleration brought about by enzymes is extraordinarily high. Unlike many of the catalysts that chemists use in the laboratory, enzymes are usually specific in their action. Often, it will catalyze only a single reaction of a single compound, called the enzymes substrate [2]. The objective of the experiment is to extract Invertase form Bakers yeast and afterwards determine the effects of changes in pH as well as temperature on the reaction rates of an enzyme catalyzed reaction. Although both pH and temperature affect enzyme activity, this report is limited only to the effect of temperature on enzyme activity. Invertase, the object of study, was extracted from Bakers yeast. Baker's yeast is the common name for the strains of yeast commonly used as a leavening agent in baking bread and bakery products, where it converts the fermentable sugars present in the dough into carbon dioxide and ethanol. Baker's yeast is of the species Saccharomyces cerevisiae. saccharo- being the combining form "sugar", myces being "fungus" and cerevisiae comes from Latin and means "of beer" [3]. Invertase is an enzyme that catalyzes the hydrolysis (breakdown) of sucrose into fructose and glucose. Sucrose + H2O -> Glucose + Fructose.

Glucose is needed by the human body to function. The human body does not directly absorb sucrose, a complex carbohydrate. Rather, it is only interested in one of the simple sugars that compose it which is glucose. To keep functioning, the body must break down these carbohydrates into glucose, also known as blood sugar. Complex carbohydrate sucrose, found in many foods, is converted to glucose which is to be absorbed by the body. The other sucrose product fructose is also converted into glucose for even more energy. Sugar creates a quick energy boost because it is easily and quickly absorbed into the blood stream [4]. Fructose, another product from sucrose is also much preferred that its source for commercial use. Fructose is often used commercially in foods and beverages not only because of its low cost but also its high relative sweetness. It is the sweetest of all naturally occurring carbohydrates and is generally regarded as being 1.73 times as sweet as sucrose . DNS or 3,5-Dinitrosalicylic acid is an aromatic compound that reacts with reducing sugars and other reducing molecules to form 3-amino-5-nitrosalicylic acid, which absorbs light strongly at 540 nm. It was first introduced as a method to detect reducing substances in urine and has since been widely used, for example, for quantificating carbohydrates levels in blood [5]. This study aims to determine the effect of temperature on enzyme activity in order to get the optimum temperature, the temperature at which the enzyme is most active, and use the information to either speed up or slow down enzyme activity depending on the objective of action.

Methodology
A. Compounds tested In the experiment, two compounds were tested: sucrose and the enzyme Invertase. Sucrose is a dissacharide sugar and is commonly called the common table sugar.

Figure 1. Chemical formula of Sucrose

Invertase is an enzyme that is vital to the digestion of carbohydrates. As an enzyme it is classified as a hydrolase as it hydrolyzes the disaccharide sugar, sucrose into simple sugars, glucose, and fructose.

B. Procedure I. Sucrose Assay Using Dinitrosalicylic Colorimetric Method Seven test tubes were following the amount given below: used

Table 1. Test tube preparation for Sucrose Assay Tube No. Blank 1 2 3 4 5 mL Sucrose 0 0.25 0.5 0.75 1 1.25 standard solution mL distilled 1.5 1.25 1 0.75 0.5 0.25 water For each test tube, 0.5 mL conc. HCl was added and mixed thoroughly. It was incubated at 90C water bath for 5 minutes.

6 1.5

To neutralize the solution, 0.5 mL 0.5 M KOH was then added. After that, 2.80 mL 0.1 M buffer solution was added. Finally, 3 mL of DNS reagent was added. The test tubes were then immersed in a 95C water bath for 10 minutes until the characteristic red-brown color appeared. After cooling, the absorbance at 540 nm was measured and a hydrolyzed-sucrose standard curve was worked upon. II. Effect of Temperature on Invertase Activity Series of water baths were prepared with varied temperatures; 20, 30, 50, 60, 70, and 90C. First, seven test tubes were prepared including a blank test tube. Then, 1.5 mL of sucrose solution, 0.1 mL of enzyme and 1.4 mL buffer pH 5 were added. After that, we incubated the test tubes in a water bath with the mentioned temperatures for 10 minutes. After incubating, 3 mL of DNS (3,5dinitrosalicylic acid) reagent was added to the test tubes, followed by immersion of test tubes in water bath for 5 minutes in order to develop the red brown color characteristic. Meanwhile, in the blank test tube, 3mL distilled water was added with the 3.0 mL DNS. After the cooling process, all solutions were transferred to separate cuvettes. The absorbance was measured by using a spectrophotometer with 540 nm wavelength. The absorbance of the blank solution was put to zero. The resulting absorbances were tabulated and the concentration of each hydrolyzed sucrose was determined using the standard curve constructed in the dinitrosalicylic colometric method. All the data collected were tabulated and interpreted using figures and tables.

Results and Discussion

A. Sucrose Assay using Dinitrosalicylic Colorimetric Method The observable result is a distinct orange reddish coloration of the solution after heating. The test tubes that have the most amount of sucrose have produced a darker color. Table 2. Concentration and Absorbance Values Amt of AcidTest Tube Absorbance540 Hydrolyzed No. Sucrose (mg/mL) Blank 0 0.5 1 0.50 1.126 2 0.83 1.647 3 1.33 2.43 4 1.67 2.79 5 2.17 2.88 6 2.50 2.91

As seen from the figures (Table 2), the test tube number 6 has the most distinctive reddish color; this shows that it has the greatest concentration of sucrose, thereby producing more simple sugars that reacted with the DNS reagent. Based on the given data below (Figure 2), the absorbance values and the concentration of the solution are directly proportional. This is because the light transmitted by a solution decreases as the concentration of the solution increases. Therefore, test tube number 6 has the least amount of transmitted light because it has the greatest amount of concentration.

Figure 2. Graph of Sucrose Assay Standard

Figure 3. Temperature vs. Absorbance

References
II. Effect of Temperature on Invertase Activity Enzymes are affected by changes in temperature. The most favorable temperature is known as the optimum temperature. The optimum temperature of Invertase is at 60C.
Table 3. Absorbance obtained

1. Campbell, M.K., Farrell,S.O. (2008), Biochemistry, 6th edition. Belont,California; Brooks/Cole Cengage Learning. 2. McMurry, J. (2010), Foundations of Organic Chemistry, Philippine edition. Cengage Learning Asia Pte. Ltd. 3. http://en.wikipedia.org/wiki/Baker's_ yeast 4. Camacho, F., Gonzalez-Tello, P. Infulence of enzymes, pH and temperature on the kinetics of whey protein hydrolysis. Food Science and Technology International 4(1998) 79-84. 5. http://en.wikipedia.org/wiki/3,5Dinitrosalicy lic_acid

Temperature (C) 20 30 50 60 70 90

Amount of AcidHydrolyzed Sucrose (mg/mL) 0.5 0.83 1.33 1.67 2.17 2.5

Absorbance
540

0.211 0.228 0.232 0.247 0.236 0.217

The table (Table 3) shows that at 60C, maximum absorbance was achieved. This then, indicates that 60C is the optimum temperature for the enzyme Invertase that was extracted from Bakers yeast. At 20C, 30C, and 50C, as the temperature increases, the absorbance also increases. But after the optimum temperature was achieved, the absorbance value decreases as the temperature increases. The relationship between temperature and absorbance can be seen in the next figure (Figure 3.).