Enzymes [Isolation of Enzyme and Determination of Enzyme Activity] Pineda, M., Puno, J., Quan, J., Ramil, J.

, Recabo, K., Sevilla. A. Group 6, 2-G Medical Technology, Faculty of Pharmacy, UST

Abstract Enzymes play an important role in Biochemistry and our everyday life. Enzymes are complex proteins that cause a specific chemical change in the human body. In this experiment, the invertase enzyme was extracted from Bakers yeast. Furthermore, colorimetric tests were done and the effect of pH and temperature was observed. In the Glucose assay using DNS colorimetric method, it can be observed that the absorbance is directly proportional to the amount of hydrolyzed glucose. Moreover, enzyme activity increases as the temperature increases but within a certain limitation. In addition to that, pH also affects the activity of the enzyme. The optimum temperature and pH at which the enzyme has maximum potency and efficiency to catalyze the reaction in the experiment is at 60 degrees Celsius and pH 5.0, respectively. Therefore it is a must to understand how an enzyme works along with its effects on variables (temperature and pH).

Introduction An organism or a living system controls its activity through the use of enzymes. Enzymes are complex proteins that cause a specific chemical change in all parts of the body. For example, they can help break down the foods we eat so the body can use them. Blood clotting is another example of enzymes at work. [1] An enzyme is a protein molecule that is a biological catalyst with three characteristics. First, the basic function of an enzyme is to increase the rate of a reaction. Most cellular reactions occur about a million times faster than they would in the absence of an enzyme. Second, most enzymes act specifically with only one reactant (called a substrate) to produce products. The third and most remarkable characteristic is that

enzymes are regulated from a state of low activity to high activity and vice versa. The individuality of a living cell is due in large part to the unique set of some 3,000 enzymes that it is genetically programmed to produce. If even one enzyme is missing or defective, the results can be disastrous. [2] . Much of the information about enzymes has been made possible because they can be isolated from cells and made to work in a test tube environment. Extensive work has also been done with X-Ray diffraction techniques to elucidate the three-dimensional structure of some enzymes.

Transferases Hydrolases Lyases Isomerases Ligases or Synthetases

Transfer of functional groups Hydrolysis reactions Addition to double bonds or its reverse Isomerization reactions Formation of bonds with ATP cleavage

Figure 1: 3D structure of a Carboxypeptidase. [2] The ribbon and backbone form of carboxypeptidase is shown on the left. The substrate is shown in magenta. [2] A special region on the enzyme, called the active site, has a shape that fits with specific substrate molecules. An enzyme works by binding to one or more specific molecules called reactants or substrates. Binding occurs at the active site. The enzyme and substrates form an enzyme-substrate complex. The interaction between the substrates and the enzymes stresses or weakens some of the chemical bonds in the substrates. These stresses encourage a link between the two substrates leading to the formation of a different molecule. As a result of the chemical interactions within the active site, a new product is formed. The product is released from the active site, the enzymes assumes its original shape and is free to work again. Although this reaction has specifically illustrated the formation of a single product from two substrate molecules, other enzymes catalyse the formation of two products from a single substrate. [3] The table that follows show the classification of enzymes. Table 1: IEC Classification of Enzymes [2] Group Name Oxidases Dehydrogenases Type of Reaction Catalyzed or Oxidation-reduction reactions

The objectives of the experiment were to extract invertase from Bakers yeast and to determine the effects of changes in pH and temperature on reaction rates of an enzymecatalysed reaction. An invertase is an enzyme that catalyzes the hydrolysis of sucrose into glucose and fructose. It is also called saccharase, sucrase. [4] Invertase is mainly used in the food (confectionery) 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. [7] The resulting mixture of fructose and glucose is called inverted sugar syrup. Invertases cleave the O-C(fructose) bond, whereas the sucrases cleave the O-C(glucose) bond. [5]

Materials and Methods The materials used in the experiment were Bakers yeast, sucrose standard solution, 100 mg/L, concentrated HCl, 0.5 M KOH, Dinitrosalicylic acid (DNS) reagent, 0.1 buffer solutions, sucrose solution 10g/L, test tubes, pipettes,beakers, volumetric flasks, paraffin film, hot plate and UV-Vis spectrophotometer. A. Extraction of Invertase Yeast 0.25 g Bakers yeast was dissolved in distilled water to make a 250 ml solution. Then 2

it was allowed to stand for 20 minutes at room temperature. Sedimentation occurred and the supernatant which served as the enzyme stock solution was collected. B. Preparation of Denatured Invertase Stock Solution 100 ml of enzyme stock solution was incubated in boiling water for 10 minutes. Then it was allowed to cool. This served as the denatured enzyme stock solution that will be used for the succeeding experiments. C. Glucose Assay Using Dinitrosalicylic Colorimetric Method

Table 2: Preparation of Standard Solution Group 1 2 3 4 5 6 7 8 Test Tube Blank 1 2 3 4 5 6 7 1% glucose solution (mL) 0.00 0.30 0.50 0.80 1.20 1.50 1.80 2.10 Distilled H2O (mL) 3.00 2.70 2.50 2.20 1.80 1.50 1.20 0.90 Figure 2: Sample transferred into cuvettes.

In the preparation of standard solution, the group was assigned with test tube number 6 which consisted of 1.50 ml of 1.00% glucose solution and 1.50 ml of distilled water. 3.00 ml of Dinitrosalicylic acid (DNS) reagent was added to 3.00 ml of the standard solution. It was then mixed using a vortex mixer. It was heated in a water bath at 95 degree Celsius for 10 minutes. Then it was cooled to room temperature. 2 sets were made. After that, the absorbance at 540 nm was taken using a spectrophotometer.

Figure 3: The spectrophotometer used to find the absorbance.

D. Effect of pH on Invertase Activity 2 test tubes both containing 1.00 ml of buffer solution at pH 7.5 was added 0.50 ml enzyme and 1.50 ml sucrose solution. A third test tube with 1.00 ml of buffer solution at pH 7.5 was added 0.50 ml denatured enzyme and 3

1.50 ml sucrose solution. The test tubes were placed in a water bath at 60 degree Celsius for 15 minutes. After that, 3.00 ml of DNS reagent was added, and then the tubes were placed back in the water bath for 10 minutes at 95 degree Celsius. It was then cooled to room temperature. The samples were transferred to a cuvette for spectrophotometry, and then the absorbance at 540 nm was measured.

The samples were transferred to a cuvette for spectrophotometry, and then the absorbance at 540 nm was measured.

Figure 5: Effect of temperature. 2 test tubes containing the stock enzyme and 1 containing the denatured enzyme. The test tubes were placed in a water bath at 90 degree Celsius for 15 minutes.

Results and Discussions A. Glucose Assay Using Dinitrosalicylic Colorimetric Method Reducing sugars like glucose and fructose can react with DNS. In the experiment, glucose reacted with DNS through an oxidation reaction and became aldonic acid or gluconic acid.[6] DNS which was yellow in color reduced to ANS (3-amino-5-nitrosalicylate) and became a darkorange colored solution. The reaction can be used for the quantification of reducing sugars since the absorbance at 540 nm is linearly dependent on the concentration or mass. Given that the concentration of the 1% glucose standard solution is 0.01 mg/ml, the amount of acid hydrolysed glucose was computed using the formula C1V1=C2V2. The values of acid hydrolysed glucose concentration is tabulated below.

Figure 4: Effect of pH. 2 test tubes containing the stock enzyme and 1 containing the denatured enzyme to be transferred to cuvettes for absorbance measuring.

E. Effect of Temperature on Invertase Activity 2 test tubes both containing 1.00 ml of buffer solution at pH 5 was added 0.50 ml enzyme and 1.50 ml sucrose solution. A third test tube with 1.00 ml of buffer solution at pH 5.0 was added 0.50 ml denatured enzyme and 1.50 ml sucrose solution. The test tubes were placed in a water bath at 90 degree Celsius for 15 minutes. After that, 3.00 ml of DNS reagent was added, and then the tubes were placed back in the water bath for 10 minutes at 95 degree Celsius. It was then cooled to room temperature.

Table 3: Concentration of Hydrolized glucose with its corresponding absorbance.

D. Effect of pH on Invertase Activity 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.

Test tube no.

Blank 1 2 3 4 5 6

Amount of Acid Absorbance Hydrolized 540 Glucose 0 2.018 0.001 2.77 0.00167 2.775 0.00267 2.83 0.004 2.9 0.005 2.925 0.006 2.775

The line that was drawn in the graph below is the best fit line. It was shown below through the slope-intercept form of y = 91.864x + 2.4464, which was calculated by the excel program. It can be observed that as the absorbance increases, the amount of hydrolyzed glucose also increases. This means that they are directly proportional to each other. By looking at the markers and comparing it to the line, the trend was not that followed because of some errors or causes during the experiment.

Figure 7: Effect of pH on Enzyme http://www.worthingtonbiochem.com/introbiochem/effectsph.html Utilizing the formula x=(y-b)/m, wherein y=absorbance, b= intercept and m= slope, we can find the amount of acid-hydrolized sucrose. Below is the computation. Test tube 1- pH 2 x = (y-2.4464)/91.864 x=-0.02336 Test tube 2 pH 3 x = (y-2.4464)/91.864 x= - 0.02631

Figure 6: Graph of Glucose Assay using DNS colorimetric method.

Test tube 3 pH 5 x = (y-2.4464)/91.864 x= -0.02544 5

Test tube 4 pH 7 x = (y-2.4464)/91.864 x=- -0.02663 Test tube 5 pH 7.5 x = (y-2.4464)/91.864 x= -0.02599 Test tube 6 pH 7.5 x = (y-2.4464)/91.864 x= -0.02663 Test tube 7 pH 8 x = (y-2.4464)/91.864 x= -0.0264 Test tube 8 pH 9 x = (y-2.4464)/91.864 x= -0.02038 Table 4: Concentration of Hydrolized glucose at a certain pH with its corresponding absorbance.

Extremely high or low pH values generally result in complete loss of activity for most enzymes. pH is also a factor in the stability of enzymes. As with activity, for each enzyme there is also a region of pH optimal stability. Optimum pH is the pH level at which the enzyme has maximum potency and efficiency to catalyze the reaction. The optimum pH value will vary greatly from one enzyme to another. The common optimal ph for invertase is 4.5. [8] However for this experiment, the optimal pH is 5.0. This can be observed from the graph. It will produce a bell-shaped line where the peak signifies the optimum pH. Enzymes, are active only within a narrow pH range usually between 5 and 9. Several factors are influenced directly by the pH in which the reaction takes place. These are as follows: the binding of substrate to the enzyme, the ionization states of the amino acid residues involved in the catalytic activity of the enzyme, the ionization of the substrate and the variation in the protein structure at extreme pH. [9]

pH

2 3 5 7 7.5 7.5 8 9

Amount of AcidHydrolyzed Sucrose (mg/mL) (x) -0.02336 -0.02631 -0.02544 -0.02663 -0.02599 -0.02663 -0.0264 -0.02038

Absorbance540 (y) 0.3 0.029 0.109 0 0.0585 0 0.021 0.574

Figure 8: Graph of the Effect of pH on Invertase

D. Effect of Temperature on Invertase Activity Temperature can affect an enzyme in two ways. One is a direct influence on the reaction rate constant, and the other is in thermal denaturization of the enzyme at elevated temperatures. [9] Utilizing the formula x=(y-b)/m, wherein y=absorbance, b= intercept and m= slope, we can find the amount of acid-hydrolized sucrose. Below is the computation. Test tube 1- 20 oC x = (y-2.4464)/91.864 x= -0.02526

Test tube 7 95 oC x = (y-2.4464)/91.864 x= -0.02631 Test tube 8 95 oC x = (y-2.4464)/91.864 x= -0.02652 Table 5: Concentration of Hydrolized glucose at a certain Temperature with its corresponding absorbance Temp. (oC) Amount of AcidHydrolyzed Sucrose (mg/mL) (x) -0.02526 -0.02336 -0.02021 -0.01993 -0.02346 -0.02402 -0.02631 -0.02652 Absorbance540 (y)

Test tube 2 30 oC x = (y-2.4464)/91.864 x= -0.02336 Test tube 3 50 oC x = (y-2.4464)/91.864 x= -0.02021 20 30 50 60 70 90 95 95

0.126 0.3 0.59 0.616 0.2915 0.24 0.029 0.0105

Test tube 4 60 C
o

x = (y-2.4464)/91.864 x=-0.01993 Test tube 5 70 oC x = (y-2.5614)/27.665 x= -0.02346 Test tube 6 90 oC x = (y-2.4464)/91.864 x= -0.02402

Like most chemical reactions, the rate of an enzyme-catalyzed reaction increases as the temperature is raised. A ten degree Centigrade rise in temperature will increase the activity of most enzymes by 50 to 100%. Variations in reaction temperature as small as 1 or 2 degrees may introduce changes of 10 to 20% in the results. In the case of enzymatic reactions, this is complicated by the fact that many enzymes are adversely affected by high temperatures. As shown in the graph, the reaction rate increases with temperature to a maximum level, then abruptly declines with further increase of temperature. [10] Hence the rate of reaction increases with temperature; within the temperature range in

which the enzyme is stable and retains its full activity. Optimum temperature is the temperature at which the enzyme has maximum potency and efficiency to catalyze the reaction. The optimum temperature of invertase is about 55oC. For this experiment, it is 60 oC. A low absorbance would mean that the enzymatic activity is slow before.

[2] Enzymes. (n.d.). Retrieved January 13, 2014, from http://www.elmhurst.edu/~chm/vchemb ook/570enzymes.html

[3] Animation: How Enzymes Work. (n.d.). Retrieved January 14, 2014, from http://highered.mcgrawhill.com/sites/0072495855/student_vie w0/chapter2/animation__how_enzymes _work.html Figure 9: Graph of the Effect of Temperature on Invertase

[4] invertase - definition of invertase by the Free References [1] Enzyme: MedlinePlus Medical Encyclopedia. Retrieved January 12, 2014, from (n.d.). Retrieved January 14, 2014, http://www.thefreedictionary.com/inver from tase http://www.nlm.nih.gov/medlineplus/e ncy/article/002353.htm [5 ] Schiweck, H., Clarke, M., & Pollach, G. (2007). Retrieved from DOI: 10.1002/14356007.a25_345.pub2 8 Online Dictionary, Thesaurus and Encyclopedia. (n.d.).

eng/Biotech[6] Ward, W. (2012). The Isolation of Invertase from Bakers Yeast. An Introduction to Protein Purification Strategies. In Protein Purification (pp. 30-50). USA. [10] Worthington Biochemical Corporation (n.d.). Temperature Effects [7] Enzyme Kinetics of Invertase. (n.d.). Retrieved January 13, 2014, from http://www.eng.umd.edu/~nsw/ench48 5/lab14.htm (Introduction to Enzymes). Retrieved January 13, 2014, from http://www.worthingtonbiochem.com/introbiochem/tempEffect s.html Environ/Projects00/temph/enzyme.html

[8] Worthington Biochemistry (n.d.). Effects of pH (Introduction to Enzymes). Retrieved January 13, 2014, from http://www.worthingtonbiochem.com/introbiochem/effectsph.ht ml

[9] Effects of Temperature and pH on Enzyme Kinetics. (n.d.). Retrieved January 13, 2014, from http://www.rpi.edu/dept/chem-