The Isolation of Invertase from Yeast and the Effect of pH on Enzyme Activity

J.D. Vergara, J.M. Villena, R.Z.M.L. Walde, R.L. Zamora, K.M. Zhu Group 10 2A-Biochemistry Biochemistry1 Laboratory

ABSTRACT
Proteins that function as biological catalysts are called Enzymes. . The objective of this experiment is to extract invertase from Yeast and to be able to determine the effects of pH on the reaction rates of an enzyme-catalyzed reaction. The Enzymes used in the experiment was derived from Yeast. A portion of the enzyme extracted was subjected denaturation. Sucrose Assay was done using the Dinitrosalicylic Colorimetric Method. The invertase and the denatured invertase were subjected to tests. Test tubes containing certain amounts of sucrose solution and H2O added with different pH of a certain values were prepared and are to be used for the effect of pH on enzyme activity. Another set of test tubes were prepared with the same procedure as the first batch this time with the use of denatured enzyme as blank reagents. Both batches of test tubes were tested using a spectrophotometer. The results show that there are certain pH in which absorbance is at its highest.

INTRODUCTION
Enzymes are protein molecules that act as catalysts by speeding up chemical reactions. Most biological life forms need enzymes, because with it, reactions work faster. Materials at the start of an enzyme reaction are called substrates while the end materials after the reaction are called products. Enzymes work by reducing or lowering the amount of activation energy for reaction, thus increasing the rate of the reaction. Due to the increased rate of the reaction, products are formed much quicker and reach their equilibrium state faster. Although enzymes speed up chemical reactions, they dont usually get used up in the reaction. Examples of biological enzymes are Amylase, which comes from our salivary glands and pancreas digest starch to maltose, Lipase which comes from the pancreas digest lipids to fatty acids and glycerol in the small intestine, another one is Pepsin, a protease that breaks proteins to peptides and amino acids, also Trypsin, a protease from the pancreas secreted in the small intestine which also break down proteins. Enzyme activity can be affected by things called inhibitors. Inhibitors are molecules that decrease enzyme activity while molecules that increase enzyme activity are called activators. Enzyme activity can also be affected by temperature, pH and substrate concentration.

Sucrose, commonly known as table sugar, is a disaccharide composed of an alpha-D-glucose molecule and a beta-D-fructose molecule linked by an alpha-1,4-glycosidic bond. When this bond is cleaved in a hydrolysis reaction, an equimolar mixture of glucose and fructose is generated. This mixture of monosaccharides is called invert sugar, which is derived from the fact that sucrose rotates plane polarized light to the right i.e., dextrorotatory, +66.5, whereas the hydrolysis products rotates plane polarized light to the left i.e., levorotatory, -20 for the mixture (+52.5 for D(+)-glucose and -92 for D(-)-fructose). Other common disaccharides are maltose and lactose.

Figure 2 Structure of Glucose Sucrose can be hydrolyzed in the presence of an enzyme called invertase or sucrose. Sucrose + H2O ---> glucose + fructose

Figure 3 Structure of Fructose The official name for invertase is betafructofuranosidase (EC3.2.1.26), which implies that the reaction catalyzed by this enzyme is the hydrolysis of the terminal nonreducing betafructofuranoside residues in betafructofuranosides. Note that alpha-D-glucosidase,

Figure 1 Structure of Sucrose

which splits off a terminal glucose unit, can also catalyze this reaction. Sucrose can be hydrolyzed relatively easily, the reaction proceeds in an acidic environment without the aid of invertase. Invertase is mainly used in the food industry where fructose is preferred over sucrose because it is sweeter and does not crystallize as easily. However, the use of invertase is rather limited because another enzyme, glucose isomerase, can be used to convert glucose to fructose more inexpensively. For health and taste reasons, its use in food industry requires that invertase be highly purified. A wide range of microorganisms produce invertase and can, thus, utilize sucrose as a nutrient. Commercially, invertase is biosynthesized chiefly by Yeast strains of Saccharomyces cerevisiae or Saccharomyces carlsbergensis. Even within the same Yeast culture, invertase exists in more than one form.

filled with 1.5mL of the sucrose solution and with no added water. After filling the test tubes with sucrose solutions, 3 drops of concentrated HCL was placed on each tube, they were then mixed and placed in a water bath of 90 oC for 5 minutes. 0.15mL of 0.50M KOH, 2.80mL of 0.10M Buffer solution at pH5 and 3mL of Dinitrosalicylic acid was added to each tube and then mixed. The test tubes were then placed in a water bath of 95oC for 10 minutes; the solutions were cooled and measured for absorbance at 540nm. D. Effect of pH on Invertase Activity Two sets comprising of 6 test tubes were used for this test. The first set was used as the sample reagents. Six test tubes were prepared and labeled pH 2, 3, 5, 7, 8, and 11. Approximately 2.90mL of 1M buffer solution at different pH (2, 3, 5, 7, 8, and 11) was added to each of their respective labeled test tubes. 0.10mL of Enzyme solution from procedure A was added to each test tube, mixed thoroughly, and placed in a water bath of 60oC for 5 minutes. After that, 1.5mL of 10mg/L sucrose solution was added, mixed and placed in the same water bath of 60oC for 5 minutes. Then, 3mL DNS reagent was placed on the tubes, mixed and placed in a water bath of 95oC for 10 minutes. The solutions were then measured for absorbance at 540nm.

EXPERIMENTAL
A. Extraction of Invertase from Yeast Approximately 0.25 grams of Bakers Yeast was weighed, dissolved in 250mL of distilled water and let the solution stand for 20 minutes. After letting the solution stand for 20 minutes, it is then filtered and the supernatant is collected. B. Preparation of Denatured Enzyme solution. 100mL is taken from the supernatant portion filtered from the 250mL yeast solution. This is then subjected to a water bath at boiling temperature of 100 oC. Allow the solution to cool then collect the supernatant. C. Sucrose Colorimetric Method. Assay using DNS

RESULTS AND DISCUSSIONS

A. Sucrose Assay using DNS Colorimetric Method Table 1 Results obtained from Sucrose Assay using DNS Colorimetric Method Test Tube Blank 1 Sucrose Concentration 0 0.675 -4.388 -1.875 -7.764 0.0675 -8.186 Absorbance 540nm 0 0.025 0.013 0.019 0.005 0.025 0.004

Seven test tubes were prepared for the assay. 1.50mL of water was placed on the first test tube. 1.25mL of water and 0.25mL of the 100mg/L sucrose solution was placed on the second test tube. On the third one, 1.00mL of water and 0.50mL of sucrose solution was placed. The fourth test tube is composed of 0.75mL of water and 0.75mL sucrose solution. The fifth test tube is a mixture of 0.50mL water and 1.00mL sucrose solution. In the sixth test tube, 0.25mL of water and 1.25mLsucrose solution was placed. The last test tube is only

2 3 4 5 6

Table 1 shows the results obtained from the sucrose assay done. It showed the absorbance value of the test tube with the sucrose solutions at 540nm. In the Sucrose DNS assay, enzyme activity is monitored by measuring the amount of reaction products that reacted with DNS reagent. Absorbance is equivalent to the amount of DNS reduced and the amount of substrate consumed which is also equal to the rate of the reaction. 0.03 Absorbance 540nm 0.02

Table 2 shows that the enzyme activity is clearly affected by the different pH levels present in the set of test tubes. Enzymes have a unique characteristic pH in which their activity is at its highest or peak; this peak is called optimal pH. The pH-activity relationship of an enzyme can be affected by the acid-base behavior of an enzyme and its substrate. Rate of reactions involving acid-base catalysis depends upon the concentration of both basic and acidic forms of groups in both the substrate and its enzyme. Equilibrium between these acidic and basic forms depends on the level of pH.

0.06 0.01 0 Sucrose Concentration 0.04 0.02 0 -0.02 -0.04 0 5 10 15

-10

-8

-6 -4 -2 0 2 Sucrose Concentration y = 0.0016x + 0.0177 R = 0.35

Figure 4 Sucrose Assay Std Curve Computations on the Sucrose concentration for the sucrose assay graph were done using the formula:

pH

y = 0.003x - 0.0071 R = 0.1443

Figure 5 pH effect on Enzyme Activity From table showed that Enzyme Activity differs at certain pH levels, this is due to the fact that pH levels alter the rate of enzyme activity. The graph expressed is a linear one due to errors committed that affected the obtained results plotted in the graph. Human Error is again a big factor why such big discrepancy is made in the experiment. Human factor comes down to the way the volume of the solutions were pipette out, the time it takes to place reagents in the test tubes and even the usage of the spectrophotometer. Another key element in the errors obtained from the experiment is the water baths that were used in the procedures. The temperatures in the water baths were not maintained perfectly at a constant temperature causing errors on the reading this is probably because the fact that temperature can also affect the pH of substances, just like water, an increase in temperature will lower the pH value of water but in a very slight change negligible in some instances.

From table 4 we can see that a linear graph was formed from the concentration of Sucrose versus the absorbance reading. Although the graph formed is a linear one which is the ideal theoretical graph, there are negative values of concentrations obtained from the computations. These negative values are possible errors. Human error might have dealt the biggest influence on the negative results obtained. B. The Effect of pH on Enzyme Activity Table 2 Results obtained from the Effect of pH on Enzyme Activity Test Tube 1 2 3 4 5 6 Sucrose Concentration 7.6 -2.14 1.3 2.43 -5 15.91 pH 0.025 -0.009 0.003 0.010 -0.019 0.054

References
Books: McGilvery, Robert. Biochemical Philadelphia. (1975). Pages 95-96 Concepts.

Lehninger, Albert. Biochemistry. New Worth Publishers. (1970). Pages 195-196 Internet:

York:

http://terpconnect.umd.edu/~nsw/ench485/lab1 4.htm http://www.eng.umd.edu/~nsw/ench485/lab9d.h tm http://en.wikipedia.org/wiki/Invertase http://www.123helpme.com/view.asp?id=12070 9 http://www.rpi.edu/dept/chem-eng/BiotechEnviron/IMMOB/enzymeac.htm http://www.buzzle.com/articles/ph-effect-onenzymes.html http://www.worthingtonbiochem.com/introBiochem/effectspH.html http://www.ehow.com/about_6837207_effectstemperature-ph-water.html