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Amora, Jorel Paul Ansbert Apostol, Paolo Miko R. Ayapana, Patricia Bianca C. Balaga, Vincent P. Bernal Group 2 2B Medical Technology Biochemistry Laboratory ABSTRACT
Enzymes are proteins that act as catalysts by lowering the activation energy in specific chemical reactions. A specific type of enzyme, invertase, hydrolyses sucrose into glucose and fructose. Dinitrosalicylic acid (DNS) assay method is utilized to monitor the enzymatic activity of invertase. Invertase was subjected to different pH of buffer solution and was observed under 540 nm absorbance. After analysis, an optimum level was observed by plotting absorbance versus pH.
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. It is a biological catalyst with three characteristics. First, its basic function 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 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. Since enzymes are proteins, they are very sensitive to changes in pH. Each enzyme has its own optimum range for pH where it will be most active. This is the result of the effect of pH on a combination factors: (1) the binding of the enzyme to a substrate, (2) the catalytic activity of the enzyme, (3) the ionization of the substrate and (4) the variation of protein structure. Extreme pH values result in loss of activity for most enzymes. Moreover, there is a most favorable pH for enzymethe point where enzyme is most active which is called the optimal pH. Invertase or beta-fructofuranosidase, is an enzyme derived from yeast. It catalyzes the hydrolysis of sucrose, which is a table sugar, down into the simple sugars glucose and fructose. The resulting product, also known as inverted sugar syrup. Invertase cleaves the O-C
(fructose) bond. Though the enzyme is inclined to higher levels of activity at low temperatures, it is best when used at about 60°C, because sucrose will split faster at higher temperatures. Baker’s yeast is the common name for the strains of yeast commonly used as a leavening agent in baking bread and bakery products, where it converts the fermentable sugars present in the dough into carbon dioxide and ethanol. DNS or Dinitrosalicylic acid, an oxidizing agent, was used as an assay to monitor enzyme activity. In the DNS assay, the rate of reaction is monitored colorimetrically by measuring the amount of reaction products that reacts with DNS reagent. DNS reagent contains dinitrosalicylic acid, which stabilizes the red color, NaOH that increases the reactivity of sugars and changes the pH of the reaction vessel halting the invertase reaction. The absorbance was read at 540 nm. The objectives of the experiment are  Extract invertase from baker’s yeast and  Determine the effects of changes in pH and temperature on reaction rates of an enzyme-catalyzed reaction.
EXPERIMENTAL A. Compound Tested
For extraction of Invertase from yeast: 0.25 g Baker’s yeast For Effect of pH on Invertase activity: enzyme stock solution
The principle involved in DNS assay. After cooling. Glucose and fructose) to form 3-amino-5-nitosaliclylic acid (ANS).00 2. Extraction of Invertase from Yeast Invertase was extracted from Baker’s Yeast. Data for Standard Curve Dinitrosalicylic acid (DNS) Assay was used to monitor enzyme activity. is that DNS (3. Then.1 M buffer solution and 0. 2. doing so will develop the characteristic red-brown color. Sucrose Assay Using Dinitro- salicylic Colometric Method Amount of acidhydrolyzed sucrose (mg/mL) 0. DNS does not react with sucrose because it is a non reducing sugar. Effect Activity of pH on Invertase In six test tubes.3 2 0. The mixture was allowed to stand for 20 minutes before collecting the supernatant which served as the enzyme stock solution. they are able to break peptide bonds and can split sucrose to glucose and fructose B. 3 drops (~0.11 Table 1: the pH of Buffer Solution per Test Tube .05 ml) of concentrated HCl was added to each tube. Afterwards.298 0.00 Corrected absorbance 0. Accordingly. A 2. IUPAC name 2-hydroxy-3. It was mixed thoroughly. The test tubes were immersed in 95°C water bath for 10 minutes to develop the characteristic red-brown color. 3 mL of DNS reagent was added.7 4 1. the amount of sucrose hydrolyzed was determined using hydrolyzed-sucrose solution standard curve constructed in the dinitrosalicylic colometric method.50 2.50 3.5M KOH. The solution was cooled. Sucrose Assay Using Dinitrosalicylic Colometric Method To construct the hydrolyzed-sucrose curve at against concentration (mg/ml). Since invertase is classified as a hydrolase.439 0.1M buffer solution was added and was mixed using the Vortex mixer.832 Table 2.Procedures 1. The blank solutions were prepared by repeating steps 1-4. The absorbance was measured at 540nm. 2.10 mL enzyme stock solution was added to each test tube.00 1. Three mL of DNS reagent was added.783 0.5-dinitrosalicylic acid. After which they were incubated at 90°C water bath for 5 minutes to hydrolyze sucrose. the absorbance was then measured at 540 nm and the hydrolyzed-sucrose curve was constructed by plotting against concentration.50 1. pH of Buffer solution 1 0.170 0.25 grams of Baker’s yeast in distilled water to make a 250-mL solution.5 3 0. Extraction of Invertase from Yeast The extraction of invertase was prepared by dissolving 0. the rate of reaction is monitored by measuring the amount of reaction products that react with DNS reagent.90 mL appropriate 0.5-dinitrobenzoic acid) reacts with reducing sugars (eg. six test tubes were prepared with specific amounts of sucrose standard solution and distilled water covered with marble or parafilm to prevent evaporation of solvent.B. Tube No. 3. Then denatured enzyme was added instead of enzyme stock solution. All tubes in 60°C water bath were incubated for 5 minutes. Allow the mixture to be cooled. The test tubes were then immersed in a 95°C water bath for 10 minutes.80 mL 0.15 mL of 0. RESULTS AND DISCUSSIONS A.654 0. The solution was neutralized by adding 0. Lastly.
Glucose is an aldose because of its aldehyde functional group.htm1/27/13  http://www. meaning the residues of the enzyme is properly protonated or deprotonated. This is why pH affects enzyme activity. the reaction of the hydrolyzed sucrose and the activation energy is directly proportional..com/articles/ph-effecton-enzymes.edu/dept/chemeng/BiotechEnviron/IMMOB/enzymeac. Effect of pH on Invertase Activity C. Araki. Or in other words. T.2 0. When pH of a particular medium changes. Consequently.html 1/27/13 From Journals Wang. The peak of the curve represent the optimum pH (~pH 5) or the best pH condition wherein the is highest. Matsuoka.5 0..6 absorbance 0. or in both. Also. But for a significant change in pH levels. they cannot 7 9 11 identify each other.4 0. Data for effect of pH 0. a change in the structure of the enzyme affects the rate of reaction.1 0 1 3 5 pH Figure 1 Effects of pH The rate of a chemical reaction and the enzyme activity is greatly influenced by the structure of the enzyme. there will be no reaction as such. Ogawa.3 0..com/introbiochem/effectsph. pH dependence of enzyme activity is a consequence of either acid-base behavior or changing degree of ionization of groups in the enzyme.com/what-isinvertase. A graphical method of analyzing pH dependence of enzyme activity 524 (1999) . The optimum pH also shows the best conformation in which enzyme activity is the highest. in the substrate. may the pH level also affect the charge properties and shape of the substrate. T.rpi.378 0. According to the graph as well as the table above. the enzyme and the substrate may undergo denaturation. Not only on enzymes. REFERENCES From the internet  http://www.html1/27/13  http://www. In such cases.J.178 Table 3. Within a narrow pH range. The curve is bell-shaped. pH 1 3 5 7 9 11 Corrected absorbance 0.The absorbance shows the color intensity of the solution which is the amount of DNS reduced that affects the amount of reaction products and the amount of substrate consumed thereby affecting or altering the rate of reaction.buzzle.edu/~chm/vchembo ok/570enzymes.501 0.htm1/27/13  http://www.html 1/27/13  http://www.298 0. changes in the structural shapes of the enzymes and substrates may be reversible.elmhurst.576 0. The phenomenon of optimum pH explains that the slight alteration of pH affects the structure thereby altering its function. it leads to alteration in the shape of the enzyme.wisegeek. M.287 0.worthingtonbiochem.7 0.
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