THE EXTRACTION OF INVERTASE FROM YEAST AND ITS EFFECTS ON PH AND TEMPERATURE

Kathleen S. Corrales, Lorenz Rael D. Cruz, Jezreel Yanah A. De Leon, Deanne Louise E. Dela Cruz, Micaella Anne A. Duquil Group III, 2E Pharmacy, Faculty of Pharmacy, University of Santo Tomas

ABSTRACT
Invertase is extracted from yeast to test the activity of an enzyme. It can be affected by different factors like concentration, pH, and temperature. This experiment was conducted to understand the effects of pH and temperature on the invertase activity. The extract from Bakers yeast was subjected to dinitrosalicylic colorimetric method, and varying pH levels and temperatures to discern the effects of physical and chemical factors to the activity of sucrose. The results were shown by using a graph in which the best-fit line and graph curve are indicated.

INTRODUCTION
Enzymes are biomolecules with catalytic activity. An enzyme increases the rate of reaction by lowering the activation energy of a certain reaction. The process is achieved through the binding at the active site of the enzyme to a specific reactant called substrate forming an enzyme-substrate complex. The substrate from said complex is then converted into the product at a faster rate thus releasing the enzyme. The net behavior of the enzyme depends on distinct factors like enzyme concentration, substrate concentration, coenzyme concentration, pH, and temperature. Invertase is a substance obtained from yeast (Saccharomyces cerevisiae) by means of extraction with water. It belongs to the IUBMB Class 3 enzymes (hydrolases) and it is also referred to as -fructofuranoside fructohydrolase. It is considered a simple globular protein and a non-allosteric enzyme. Its function is to split sucrose into its main parts: glucose, and fructose. It plays an important role in the human body through conversion of complex sugars into blood sugar that is used by the body for energy. Dinitrosalicylic acid (DNS) Colorimetric Method is used to test the presence of a free carbonyl group (C=O) referred to as the reducing sugars. The reaction is dependent on the nature of the reducing sugars which yield different color intensities. The availability of 3,5-dinitrosalicylic acid is competed for the decomposition of sugar thus affecting the calibration curve through the increase in color intensity. pH and temperature are two of the main factors in determining the enzymatic activity. pH is a quantitative measure of the acidity or basicity of aqueous or other liquid solutions. It shows the concentration of Hydrogen ions. The most favorable pH value is known as the optimum pH where the enzyme is most active. Temperature affects enzymatic activity through direct influence on the reaction rate constant, and in thermal denaturation of the enzyme at elevated temperature.

The experiment aims to extract invertase from Bakers yeast and to determine the

effects of changes in pH and temperature on reaction rates of an enzyme-catalyzed reaction.

MATERIALS AND METHODS

Materials Dinitrosalicylic acid (DNS) was used as a reagent for the sucrose assay in the experiment. It is also referred to as 3, 5dinitrosalicylic acid. A spectrophotometer was used to measure the absorbance or the amount of light absorbed by the invertase. It operates by passing a beam of light through a sample and measuring the intensity of light reaching a detector.

100 milliliter of enzyme stock solution was incubated through a water bath for ten minutes and allowed to cool. The supernatant was collect only if frothing occurred. This served as the denatured enzyme stock solution that will be used for the following experiments.

Sucrose Assay Using Colorimetric Method

Dinitrosalicylic

Six test tubes were prepared according to the table below and covered with marbles to prevent evaporation of solvent.

No. mL sucrose std. soln. mL dH2O

Blank 0

1 0.25

2 0.50

1.50

1.25

1.00

Figure 1. A spectrophotometer

Methods

Extraction of Invertase from Yeast 0.25 grams of Bakers yeast was dissolved in distilled water to make a 250 milliliter solution. After it was allowed to stand for twenty minutes, the supernatant was collected. It served as the enzyme stock solution that will be used for the following experiments.

3 4 5 6 0.75 1.00 1.25 1.50 0.75 0.50 0.25 0 Three drops of concentrated HCl was mixed and added to each test tube and incubated by a water bath at 90C for five minutes. 0.15 milliliters of 0.5M KOH was then added to neutralize the solution. The group added 2.80 milliliters of 0.1M buffer solution, pH = 5 and mixed it well. 3 milliliters of the DNS reagent was then added right before immersing the test tubes in 95C water bath for ten minutes to develop the red-brown color. It was allowed to cool and the absorbance was measured at 540 nanometer. The hydrolyzed-sucrose standard curve was constructed by plotting A540 against the concentration.

Preparation of Denatured Invertase Stock Solution Effect of pH on Invertase Activity

Six test tubes were labeled and prepared according to the table below.

No. pH of buffer soln. mL of buffer soln.

2 3

3 5

4 7

2.90mL

2.90mL

2.90mL

5 6 7 8 7.5 8 9 11 2.90mL 2.90mL 2.90mL 2.90mL 0.10 milliliters of enzyme stock solution was then added to each test tube and thoroughly mixed. The tubes were incubated in a 60C water bath for five minutes. 3 milliliters of the DNS reagent was added right before immersing the test tubes in 95C water bath for ten minutes to develop the red-brown color. It was allowed to cool. Blank solutions were prepared same as the enzyme stock solution but denatured enzyme was added instead of enzyme stock solution. The absorbance was measured at 540 nanometer. The amount of sucrose hydrolyzed using hydrolyzed-sucrose standard curve was determined.

respective water baths. Three milliliters of DNS reagent was added right before immersing the test tubes in 95C water bath for ten minutes to develop the red-brown color. It was allowed to cool. The test tubes were covered with marbles to prevent evaporation of the solvent. Blank solutions were prepared same as the enzyme stock solution but denatured enzyme was added instead of enzyme stock solution. The absorbance was measured at 540 nanometer. The amount of sucrose hydrolyzed using hydrolyzed-sucrose standard curve was determined.

RESULTS AND DISCUSSION

Hydrolases are able to break peptide bonds. Invertase was obtained from the extraction from Bakers yeast and since invertase is classified as a hydrolase, it breaks the peptide bonds. This makes sucrose undergo hydrolysis, thus splitting into glucose and fructose as shown in the figure below.

Figure 2 Hydrolysis of sucrose: reaction of sucrose and invertase yielding glucose and fructose

Effect of Temperature on Invertase Activity Each group prepared varying water baths with the following temperatures respectively: 20, 30, 50, 60, 70, and 90C. Six test tubes containing 1.5 milliliters of sucrose solution were prepared and incubated separately for five minutes in each water bath. 0.80 milliliters of enzyme stock solution was mixed with 19.20 milliliters of 0.1M buffer solution, pH = 5 in another test tube. Three milliliters of dilute enzyme solution was added to all the test tubes and incubated for another five minutes. The test tubes were not to be removed from their Test Tube No. Amt. of AcidHydrolyzed Glucose (mg/mL) 0.000 0.003 0.007 0.010 0.013 0.017 0.021 Absorbance540

Blank 1 2 3 4 5 6

0.000 0.208 0.435 0.839 2.176 2.435 2.670

Table 1. Amount of Acid-Hydrolyzed Glucose and Absorbance540.

0.025 0.02

3 2.5 2

0.015 0.01 0.005

1.5 1 0.5 0 0 0.005 0.01 0.015 0.02 0.025 0 0 2 4 6 8 10 12

Figure 4 Effect of pH on Invertase activity

Figure 3 Hydrolyzed glucose standard curve

Since there was no sucrose available, the class was advised to use glucose instead. With the use of dinitrosalicylic colorimetric method, reducing sugars indicating free carbonyl groups and other reducing molecules used to form 3-amino-5nitrosalicylic acid subject to basic conditions that highly absorbed light at 540 nanometers. A red-brown color shows a positive result. See Table 1 and Fig. 3 for the results. Amt. of Acid- Absorbance540 Hydrolyzed Glucose (mg/mL) 3 0.000 0.000 5 0.021 2.940 7 0.021 2.750 7.5 0.021 2.640 8 0.003 0.029 9 0.017 1.550 11 0.003 0.021 Table 2. pH and Absorbance of Invertase pH

One of the factors of affecting enzymes is pH. The most appropriate pH value where the enzyme is most active is called the optimum pH. As seen in Fig. 4, the optimum pH for the extracted invertase was 5. pH affects the enzymes through alteration of its structure which further affects the rate of reaction. If there is a narrow pH range, the modification in the structure of enzymes is reversible. For cases wherein the pH range is remarkable, the enzyme and substrate may undergo denaturation. No reaction would occur since the enzyme and substrate would not be able to recognize each other. Temperature Absorbance540 (C) Blank 0.000 20 -1.009 30 -0.125 50 -0.210 60 -2.780 70 -0.813 90 -0.043 Table 3. Temperature and Absorbance of Invertase A bell shaped curve should have been plotted illustrating why temperature affects the absorbance of invertase activity. As the temperature increases, enzyme activity

would also increase. It would only stop until 60C where the enzyme activity would start to decrease and eventually become inactive. This concept represents denaturation. The absorbance of the invertase showed negative results due to human and mechanical errors (i.e. lack of experience in using spectrophotometer, unmonitored temperature of the water, contaminated curvettes).

http://www.eng.umd.edu/~nsw/ench 485/lab4a.htm Worthington Biochemical Corporation. (n.d.). Introduction to Enzymes. Introduction to Biochemistry. Retrieved January 19, 2014, from http://www.worthingtonbiochem.com/introbiochem/Enzymes .pdf

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