Effect of PH On Invertase Activity

Effect of pH on Invertase Activity Subijano, M.G., Silva, J.A., Sunglao, A.J., Supan, E.J., Tan, C.M., Tayag, P.
I Group no. 7, 2G Medical Technology, Faculty of Pharmacy, UST
ABSTRACT
INTRODUCTION Living systems control their activities through enzymes. An enzyme is a protein molecule that is a biological catalyst bearing three essential characteristics. First, the most fundamental of its function is to speed up the rate of reaction. Most cellular reactions take place many times faster than they would in the absence of an enzyme. Second, most enzymes act specifically with only one reactant, called substrate, to produce products. The third and the most notable characteristic an enzyme is that enzymes are regulated from a state of low activity to high activity or vice versa.[1] Enzymes accelerate biochemical reactions by physically interacting to reactants and products to provide a more favorable pathway for the transformation of one to another. They increase the rates of reactions by increasing the probability of the reactant can interact properly. But no matter how reactants increase the rate of a reaction, they cannot promote reactions where G is positive or non-spontaneous reaction.[2] Since enzymes are proteins, they are very sensitive to changes in pH. Each enzyme has its own optimum range where it will be most active. Several factors are influenced directly by the pH in which the reaction takes place: The binding of substrate to the enzyme, the ionization \the catalytic activity of the enzyme, the ionization of the substrate and variation in the protein structure at extreme pH.
Fig. 1 Graph of enzymatic activity against pH
The graph indicated above is described as the bell-shaped curve. It indicates the situation wherein the enzyme works at it best at a particular pH, which is called the optimum pH. Take note that shape of the graph can be altered as changes in pH occur. [3] Temperature can affect an enzyme in two ways. One is a direct influence on the reaction rate constant, and the other is in thermal denaturation of the enzyme at elevated temperatures. To relate the effect of temperature to the reaction rate constant, the Arrhenius equation is used: where k is the rate constant, R is the gas law constant, A is the frequency factor and Ea is the activation energy of the
reaction. The temperature ranges over which enzymes show activity is limited between the melting point (0oC) and boiling point (100oC) of water. If a temperature is too low, there can be no noticeable reaction rate since the enzyme is operating at a temperature far below its optimum. If the temperature at which the enzyme is operating at is well above 100oC, then thermal deactivation can occur.
fructose. Dinitrosalicylic colorimetric method is used to test the presence of free carbonyl group (C=O), the so-called reducing sugars. This involves the oxidation of the aldehyde functional group present in, for example, glucose and the ketone functional group. 3,5-dinitrosalicylic acid is the (yellow) reagent used to determine the sugar content of the given Invertase. However in some instances, glucose can also be used directly in the experiment to save time. [6] The spectrophotometer is an instrument which measures the amount of light of a specified wavelength which passes through a medium. According to Beer's law, the amount of light absorbed by a medium is proportional to the concentration of the absorbing material or solute present. Absorbency is indicated with a capital A. [7] In this experiment, the spectrophotometer was employed to measure the absorbance of Invertase which was plotted against values of the amount of acid-hydrolyzed sucrose to come up with the standard calibration curve which will be the basis for the construction of pH vs. amount of glucose. The objectives include, a.) To extract Invertase from Bakers yeast. b.) To determine the effects of changes in pH and temperature on reaction rates of an enzyme catalyzed reaction. Given the objectives, the experiment was successfully achieved. EXPERIMENTAL A. Samples used Glucose and 3,5-dinitrosalicylic acid. B. Procedure 1. Extraction of Invertase From Yeast Invertase was extracted by dissolving the 0.25g Bakers yeast in distilled water to make a 250 mL solution. Then it was allowed to stand for 20 minutes at a room temperature, allow the solution to settle. If sedimentation occurs, collect the supernatant. The supernatant
Fig 2 Graph of enzymatic activity against Temperature
Sucrose, commonly known as table sugar, is a disaccharide composed of an alpha-D-glucose molecule and a beta-Dfructose molecule linked by an alpha-1,4glycosidic bond. When this bond is cleaved in a hydrolysis reaction, an equimolar mixture of glucose and fructose is generated. This mixture of monosaccharide [4] is called invert sugar. Thereby, dextrorotatory saccharose is transformed to levorotatory variety. [5] Sucrose can be catalyzed in the presence of an enzyme named Invertase or sucrose. Invertase is classified as a hydrolase which basically conducts hydrolysis reactions. The systematic name for the invertase is fructofuranosidase (EC3.2.1.26), which implies that the reaction catalyzed by this enzyme is the hydrolysis of the terminal nonreducing -fructofuranoside residues in -fructofuranosides. [4] In this experiment, the invertase was isolated from the Bakers yeast and is subjected to hydrolysis to form glucose and
serves as the enzyme stock solution that will be used for the succeeding experiments. 2. Preparation of Denatured Invertase Stock Solution Incubate an amount of 100mL enzyme stock solution in a boiling water bath for 10 minutes. Allow the solution to cool the collect only the supernatant if frothing occurs. 3. Preparation of Standard Glucose Prepare a working standard solution by diluting 1.80mL 1% glucose solution with distilled water amounting 1.20mL in a test tube. 4. Effect of pH on Invertase Activity Prepare 3 test tubes having two samples of 0.50mL enzyme stock solution and one 0.50mL denatured stock solution. On the same given test tubes bearing the enzymes, accurately pipette a quantity of 1mL buffer solution (the group used Phosphate buffer with a pH of 8.00) and 1.50mL glucose solution. Mix well the solution then heat in water bath for 15 minutes at 60C. After heating in water bath, add 3.0mL DNS reagent and heat the solution again for 10 minutes at 95C. Allow the test tubes to cool at room temperature. The absorbance of the test tubes was measured at 540nm. Another set of test tubes were prepared using 0.1 mL of distilled water instead of denatured enzyme stock solution. The latter solutions served as blank solution for calibrating the spectrophotometer. 5. Effects of Invertase on Temperature
Prepare 3 test tubes having two samples of 0.50mL enzyme stock solution and one 0.50mL denatured stock solution. On the same given test tubes bearing the enzymes, accurately pipette a quantity of 1mL buffer solution at pH 5.00 and 1.50mL glucose solution. Heat to water bath for 15 minutes at 95C and after which add 3mL DNS reagent then heat to water bath again for 15 minutes and let it cool down at room temperature after the last water bath. RESULTS AND DISCUSSION Effect of pH on Invertase Activity The slope-intercept form obtained from the standard calibration curve was used to calculate the amount of acid-hydrolyzed sucrose using varied pH values. The formula used was where y is the absorbance, b is intercept and m is the slope.

Effect of PH On Invertase Activity

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Effect of pH on Invertase Activity Subijano, M.G., Silva, J.A., Sunglao, A.J., Supan, E.J., Tan, C.M., Tayag, P.

I Group no. 7, 2G Medical Technology, Faculty of Pharmacy, UST

Fig. 1 Graph of enzymatic activity against pH

Fig 2 Graph of enzymatic activity against Temperature

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