Effect of pH on Invertase Activity

Kristel Van Cuarentas, Jesspeed Marvel De Los Santos, Kathrina Antheia Dimaano, Gia Directo, Clare Ducut, Toni Encinares Group 3, 2 G Pharmacy, Pharmaceutical Biochemistry Laboratory
Abstract Invertase is a type of enzyme, a natural catalytic agent for biochemical reactions, can be obtained in Bakers Yeast. Determination of the effect of PH on invertase activity is the primary objective of the experiment. Dinitrosalicyclic acid (DNS) Assay method is utilized to monitor the enzymatic activity of invertase. Invertase was subjected to different pH (3, 5,7 and 10) of buffer solution and was observed under 540 nm absorbance using spectrophotometer. After observation and analysis, a peak (optimum pH) was observed by plotting absorbance versus pH.

Introduction Enzymes are proteinaceous catalysts, which speed up the rate of a biochemical reaction. They reduce the activation energy that is essential for starting any type of chemical reaction. With a low energy requirement for activation, the reaction takes place faster. The overall performance of an enzyme depends on various factors, such as temperature, pH, cofactors, activators and inhibitors. Invertase, which was used in the experiment, is a yeast derived enzyme which is classified as a hydrolase. Having the official name, fructofuranosidase (EC 3.2.1.26), it is classified as a hydrolase. Generally, invertase can break peptide bonds and specifically hydrolyzes sucrose to glucose and fructose. Dinitrosalicyclic acid (DNS) Assay is the method utilized to monitor its enzymatic activity. Furthermore, pH

is discussed in the experiment and specifically tackled on the effect of pH on invertase activity. pH, also called the potential of hydrogen, is defined as the measure of the activity of hydrogen ions in a solution. Spectrophotometer was used to determine the absorbance of the solution. The objectives of the experiment are: (1) to extract invertase from Bakers yeast, and (2) to determine the effects of changes in pH on reaction rates of an enzyme-catalyzed reaction.

Methodology Extraction of Invertase from Yeast Bakers yeast weighing 0.25 g was dissolved in distilled water to make a 250 ml. solution. The solution was allowed to stand for 20 minutes at room temperature. Supernatant was collected as few sedimentation occurred. The supernatant served as

the enzyme stock solution that was used for the succeeding experiments.

characteristic red-brown color. solutions were allowed to cool.

The

Preparation of Stock Solution

Denatured

Invertase

Enzyme stock solution amounting to 100 ml was incubated in a boiling water bath for 10 minutes. The solution was allowed to cool. The supernatant was collected as frothing occurred. This served as the denatured enzyme stock solution which was used for the succeeding experiments.

Blank solutions were prepared by following the steps previously described. Denatured enzyme was added instead of enzyme stock solution. The absorbance was measured at 540 nm. The amount of sucrose hydrolyzed was determined using hydrolyzed-sucrose standard curve constructed in the dinitrosalicylic colorimetic method.

Results and Discussion Effect of pH on Invertase Activity Six-numbered test tubes were prepared. Afterwards, 2.90 ml appropriate 0.1 M buffer solution was added as described below. The test tubes were labeled accordingly. Invertase was extracted from Brewers Yeast. Since invertase is classified as a hydrolase, they are able to break peptide bonds and can split sucrose to glucose and fructose (Figure 1).

Table 1: The pH of Buffer Solution per Test Tube Tube No. 1 2 3 4 pH of Buffer solution 0.3 0.5 0.7 1.11 Figure 1: Reaction of Sucrose and Invertase to Yield Glucose and Fructose

Enzyme stock solution measuring 0.10 ml was added to each test tube. The solution was mixed thoroughly and incubated in 60 degrees C water bath for 5 minutes. A mixture of 1.50 mL of sucrose solution was added. Reaction mixture was then incubated in 60 degrees C water bath for 5 minutes. DNS reagent amounting to 3 mL was added. The test tubes were immersed in 95 degrees C water bath for 10 minutes to develop the

Two Stock Solution was prepared in the experiment: the enzyme stock solution and the denatured invertase stock solution (which served as the blank). The result of test tubes that contains a pH of 3, 5, 7, and 10 was tested in the experiment.

Table 2: pH and Absorbance of Invertase pH 3 5 7 10 Absorbance 0.145 0.987 0.632 0.081

Figure 3 shows that the optimum pH of the extracted invertase was 5.

Enzymes are affected by changes in pH. The most favorable pH value - the point where the enzyme is most active - is known as the optimum pH. This is graphically illustrated in Figure 2.

Figure 3: Activity.

Graph

of

the

Invertase

Figure 2: Graph Showing the Optimum pH of an Activity.

The rate of a chemical reaction and/or the enzyme activity is greatly influenced by the structure of the enzyme. Or in other words, a change in the structure of the enzyme affects the rate of reaction. When pH of a particular medium changes, it leads to alteration in the shape of the enzyme. Not only on enzymes, the pH level may also affect the charge properties and shape of the substrate. Within a narrow pH range, changes in the structural shapes of the enzymes and substrates may be reversible. But for a significant change in pH levels, the enzyme and the substrate may undergo denaturation. In such cases, they cannot identify each other. Consequently, there will be no reaction as such. This the reason why, pH affect enzyme activity.

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