Effect of pH on Invertase Activity

EFFECT OF pH LEVEL ON THE ENZYME ACTIVITY OF INVERTASE
John Dale Cruz, Michael Ray Dela Cruz, Karen Angelica Domalaon, Abegail Dulay, Shirley Ann Felipe
Group 5 2C Medical Technology Biochemistry Laboratory

ABSTRACT
Invertase is a type of enzyme, a natural catalytic agent for biochemical reactions, can be obtained in Baker’s 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 (3.87, 4.0, 5.5, 7.3 and 10.55) 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 is an enzyme which is usually found in plants. It acts as a catalyst for the hydrolysis of sucrose. Sucrose is a disaccharide composed of glucose and fructose linked by a glycosidic bond. When this bond is cleaved in a hydrolysis reaction, an equal amount of glucose and fructose. Invertase is a significant enzyme because glucose is an important product of photosynthesis. Invertase is also used in the confectionery industry where fructose is preferred over sucrose because it is sweeter and does not crystallize easily. Enzymes are affected by changes in pH. Extreme pH values generally result in loss of activity for most enzymes. Furthermore, there is a most favorable pH for enzyme – the point where the enzyme is most active. This point is known as the optimal pH. The aim of this experiment is to find out the range of pH which invertase is effective.
The objectives of this experiment are: to extract invertase from Baker’s Yeast and to determine the effects of changes in pH on reaction rates of an enzyme-catalyzed reaction.
MATERIALS
The materials used in this experiment are:
Baker’s Yeast, Sucrose Standard Solution (100 mg/L), Concentrated HCl, 0.5 M KOH, DNS reagent, 0.1 M buffer solutions (pH 1, 3, 5, 7, 9, 11), ucrose solution (10 g/L), test tubes, pipets, beakers, volumetric flasks, paraffin film, hot plate and UV-Vis Spectrophotometer.
METHODOLOGY
Extraction of invertase from yeast
To extract the invertase from Baker’s Yeast, 0.25 g of it was dissolved in distilled water to make a 250-mL solution. When the solution is prepared (complete dissolvation of Baker’s Yeast) it is then allowed to stand for 20 minutes at room temperature. Provided that the sediments form, the supernatant must be collected as it will be used as the enzyme stock solution that will be used in the succeeding experiment.
Sucrose Assay Using Dinitrosalicylic Colorimetric Method
In preparation of this part of the experiment, a series of test tubes were prepared as follows:
Tube No.
Blank
1
2
3
4
5
6
mL sucrose std. solution
0
0.25
0.50
0.75
1.00
1.25
1.50
mL distilled water
1.50
1.50
1.25
1.00
0.75
0.50
0.25

After, 3 drops of concentrated HCl (0.05mL) were introduced to each test tube. Noted that the tubes were mixed well and then incubated after at a 90 degrees Celsius water bath for 5 minutes. After the incubation, 0.15 mL of 0.5 M KOH was added to neutralize the solution. Another 2.80 mL of 0.1 M buffer solution at pH 5 were added, then the solution was mixed well again. Then, 3 mL of DNS reagent was added before the test tubes were immersed in a water bath at 95 degree Celsius for 10 minutes to develop the characteristics of a red-brown colour solution. After cooling, the solution were subjected into spectrophotometry to measure the absorbance at 540 nm.
Effect of pH on Invertase Activity
In finding the effect of pH on invertase activity, six numbered test tubes were prepared with 2.90 mL appropriate 0.1 M buffer solution as shown below:
Tube No.
1
2
3
4
5
6 pH buffer solution
0.1
0.3
0.5
1.7
1.9
1.11

Then, 0.10 mL enzyme stock solution was added to each test tube. After mixing thoroughly, all test tubes were incubated in 60 degrees Celsius water bath for 5 minutes. When the time was right, another 1.50 mL of sucrose was added. The solution was then incubated again and treated to the same water bath for the same amount of time, 5 minutes. Then, 3 mL of DNS reagent was added before immersing the solution in a water bath (95 degrees Celsius) for 10 minutes until the solution turns into a red-brown colour solution. After cooling the first test tube, blank solutions were prepared by following steps 1-4 again, but instead of using the enzyme stock solution, denatured enzyme was added. All the test tubes containing the solution were then subjected to spectrophotometry to measure the absorbance at 540 nm.
EXPERIMENTAL
Sucrose Assay Using Dinitrosalicylic Colorimetric Method
A. Materials used
Sucrose Standard Solution, Distilled Water, Concentrated HCl, 0.5 M KOH, 0.1 M Buffer Solution, DNS Reagent, and UV-Vis Spectrophotometry.
B. Procedure
After collecting the supernatant from the enzyme stock solution, each test tube were introduced to 3 drops of conc. HCl before incubating at 90oC water bath for 5 minutes. 0.5 M KOH was then added to neutralize. Then, 2.80 mL of 0.1 M buffer solution was added before the solution was introduced to DNS reagent. The solution was in water bath at 950C for 10 minutes (until it is a red-brown solution). After cooling, it is subjected to spectrophotometry to measure absorbance at 540 nm.
Effect of pH on Invertase Activity
A. Materials used
Buffer Solution, Enzyme Stock Solution, 1.50 Sucrose Solution, 3 mL DNS Reagent, Test Tubes, UV-Vis Spectrophotometry.
B. Procedure
After preparing the required test tubes, they were introduced with 0.10 mL enzyme stock solution before being incubated for 5 minutes in a water bath at 600C. Then, 1.50 mL sucrose solution was added before the solution was incubated again for 5 minutes in a water bath with the same temperature. After cooling, 3 mL DNS reagent was added before immersing the test tubes again in a water bath at 950C until the red-brown color appears. Repeat steps 1-4 but this time, instead of adding the enzyme stock solution, add the denatured enzyme. After all the test tubes were prepared, they were sunjected to UV-Vis Spectrophotometry to measure absorbance at 540 nm.

Image 1. The red-brown coloration after water bath
RESULTS
Sucrose Assay Using Dinitrosalicylic Colorimetric Method
The following table shows the results from the UV-Vis Spectrophotometer of Sucrose Assay using DNS Colorimetric Method:

Test Tube No.
Amount of Acid-Hydrolized Sucrose
Absorbance
Blank
0.0
0.000 A
1
0.56
0.335 A
2
1.11
-0.456 A
3
1.67
1.248 A
4
2.22
1.800 A
5
2.78
-0.238 A
6
3.33
-0.319 A
Table 1. Results of Sucrose Assay using DNS Colorimetric Method
The students were also asked to plot the hydrolized-sucrose standard curve by plotting Absorbance against Concentration (mg/mL)

Chart 1. Standard Curve of Absorbance against Concentration.

Effect of pH on Invertase Activity
The following table shows the results from the UV-Vis Spectrophotometer in respect to the Effect of pH on Invertase Activity:

pH
Amount of Acid-Hydrolized Sucrose
Absorbance
Blank
0.0
0.000 A
3.87
2.02
0.162 A
4.0
9.12
0.78 A
5.5
12.6
0.975 A
7.3
1.883
0.151 A
10.55
9.33
0.748 A
Table 2. Results of the Effect of pH using Colorimetric Method.

We were also asked to plot the graph of the amount of sucrose-hydrolyzed using hydrolized-sucrose standard curve in the Dinitrosalicylic Colorimetric Method:

Chart 2. Hydrolyzed-Sucrose Standard Curve.

CONCLUSION