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ISOLATION OF ACTIVE INVERTASE AND EFFECT OF PH ON ENZYMATIC ACTIVITY Aliza Silverio, Regina Soriano, Jeremae Tan, Hazel Joy

Tequillo and Michelle Tolentino Group 7 2F-Pharmacy 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 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 biological catalysts which can alter or speed up a chemical reaction, without itself being chemically changed at the end of the reaction. Not all enzymes are protein in nature thus they are called Ribozymes which is a RNA molecule that has catalytic activity. Determination of the effect of PH on enzyme activity is the primary objective of the experiment. 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. Its official name is -fructofuranosidase (EC 3.2.1.26) and is classified as an hydrolase. Generally, invertase can break peptide bonds and specifically hydrolyzes sucrose to glucose and fructose. Dinitrosalicyclic acid (DNS) Assay method is utilized to monitor the enzymatic activity of invertase. 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. Figure 1. Bakers yeast This was allowed to stand for 20 minutes at room temperature. After sedimentation occur the supernatant liquid was collected and used in the succeeding experiment.

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. (3) to determine the optimum pH at which enzyme is more active.

EXPERIMENTAL 1.)Extraction yeast of Invertase from

0.25 g Bakers yeast was dissolved in distilled water to make a 250-Ml solution.

Figure 2. Invertase 2.) Preparation of Denatured Enzyme solution. 100 mL of enzyme stock solution was incubated in a boiling water bath for ten minutes.It was cooled.The supernatant liquid was collected .This was used as the denatured enzyme stock solution.

Figure 4. Test tubes with buffer solutions 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.

Figure 3. Denatured Enzyme solution 3.) Effect of pH on Invertase activity 6 numbered test tubes were prepared.2.90 Ml appropriate 0.1M buffer solution was added as described below. The test tubes were labeled accordingly. Table 1. The pH of Buffer Solution per

Figure 5. Test tubes in figure 4 with DNS reagent 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 characteristic red-brown color. The solutions were allowed to cool.

Test Tube
Tube no. 1 2 3 4 5 6 pH 2 3 5 8 9 11

Figure 6. Blank Solutions with denatured enzyme 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.

In DNS assay, rate of reaction of enzyme activity is monitored colorimetrically by measuring the amount of reaction products which are reducing sugars equimolar mixture of glucose and fructose that react with DNS reagent. With the use ofSpectrophotometer, absorbance at 540 nm was observed. And arrived at the conclusion that: Absorbance Color intensity of solution Amount of DNS reduced Amount of reaction products Amount of substrate consumed Rate of reaction Concentrated HCl was used for the complete hydrolysis of glycosidic bonds. 0.5 N KOH was used to neutralized excess acids. 1% DNS reagent is composed of Dinitrosalicylic acid will act as the oxidizing agent, Na2SO3 which stabilizes the red color, NaOH which increases the reactivity of sugars and changes the PH of the reaction vessel along; with the ANS production; halting the invertase reaction

RESULTS AND DISCUSSION

Figure 7. REDUCTION OF SUCROSE Invertase having the official name of -fructofuranosidase (EC 3.2.1.26) is classified as an hydrolase and are catalyzing hydrolysis of the terminal nonreducing -fructofuranoside residues. Generally, they are able to break peptide bonds and can split sucrose to glucose and fructose in the presence of invertase.

Figure 9. Glucose Standard Curve Figure 8. REDUCTION OF GLUCOSE AND FRUCTOSE The reduction of glucose and fructose using the Dinitrosalicyclic acid (DNS) Assay method. This method is utilized to monitor the enzymatic activity of invertase.. This served as the basis of the experiment. Concentration of the solution depend on the absorbance in 540 nm. pH dependence of enzyme activity is a consequence of acid-base behavior or changing degree of ionization of groups in the enzyme, in the substrate, or in both.

the glucose standard curve. We used the formula x=y-b/m to solve for it.

References
1. Boyer, Rodney (2006) Concepts in Biochemistry 3rd Edition. John Wiley and Sons Inc. U.S.A. 2. Nigam A. and Ayyagari A. (2007). Lab Manual in Biochemistry, Immunology and Biotechnology. McGraw-Hill Publishing Company. India Figure 10. Effects of pH on Invertase 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. pH Amount of Acid- Absorbance. Hydrolyzed Sucrose(mg/mL) 2 2.71x10-3 .044 3 2.57x10-3 .031 5 2.41x10-3 .015 8 3.58x10-3 .129 -3 9 3.96x10 .166 11 2.96x10-3 .0685 Table 2. Results on the Effect pH on Invertase activity The concentration was solved using the slope (-.22) and intercept(97.48) of 3. Crisostomo, A.C. et al. (2010). Laboratory Manual in General Biochemistry. C & E Publishing Inc. Philippines. 4. Seager, S.L. and Slaubaugh, M.R.(2011). Organic and th Biochemistry for Today 7 Edition. Brooks/Cole, Cengage Learning. U.S.A. 5. http://www.worthingtonbiochem.com/introbiochem/effects ph.html date accessed: January 23, 2012.

6. http://www.buzzle.com/articles/ph
-effect-on-enzymes.html accessed: January 24, 2012. date

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