EXTRACTION OF INVERTASE FROM YEAST AND EFFECT OF pH ON INVERTASE ACTIVITY Katrina Marie Duron,Azalea Damaris Encarnacion,Patricia Mikkaela Feliciano,Ciela

Kadeshka A. Fuentes, Bea Trixia B. Gales, Ethel Princess A. Gepulango, Group 3 2F Pharmacy Pharmaceutical Biochemistry Laboratory
ABSTRACT An Enzyme is a protein, which is capable of starting a chemical reaction, which involves the formation or breakage of chemical bonds. Enzyme activity varies due to different physical and chemical factors. Examples of the things that alter the rate of reaction of the enzymatic activity are pH and addition of chemicals or reagents. In this experiment, effect of PH on invertase activity and changing in pH were done to determine the effects of physical and chemical factors to the invertase activity of glucose. Dinitrosalicyclic acid (DNS) Colorimetric 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 (optimumpH) was observed by plotting the amount of glucose formed versus pH. INTRODUCTION Invertase is a yeast derived enzyme which is usually found in plants. It has the official name of beta-fructofuranosidase, which implies that the reaction catalyzed by this enzyme is the hydrolysis of the terminal non reducing betafructofuranoside residues in betafructofuranosides. Invertase is also used in the confectionery industry where fructose is preferred over sucrose because it is sweeter and does not crystallize easily. It exhibits relatively high activity over a broad range of pH (3.5--5.5), with the optimum near pH=4.5. The enzyme activity reaches a maximum at about 55C. Enzymes are all proteins, simple or conjugated. Most of the globular proteins are involved in metabolic functions. They are high molecular weight compounds made up principally of chains of amino acids linked together by peptide bonds. They all share the same property of protein. They are antigenic. They are denatured by such agents as elevated temperature and extreme pH values. Their physical state and catalytic function depend distinctly upon a number of physical factors such as pH, temperature, and ionic strength. All enzymes are functionally specific to varying degrees. Enzyme activity is the rate of the catalyzed reaction. When it is plotted against either pH or temperature, the curve usually has a peak or the optimum 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. This is graphically illustrated in Figure 1.

Figure 1. Effect of pH on reaction rate 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.

The optimum pH value will vary greatly from one enzyme to another, as Table 1 shows:
Table 1. pH for Optimum Activity
Enzyme pH Optimum

Lipase (pancreas) Lipase (stomach) Lipase (castor oil) Pepsin Trypsin Urease Invertase Maltase

8.0 4.0 - 5.0 4.7 1.5 - 1.6 7.8 - 8.7 7.0 4.5 6.1 - 6.8

Figure 2.Chemical Structure of DNS Dinitrosalicylic acid (D.N.S.A. or 3:5dinitrosalicylic acid) is a (yellow) reagent used to determine sugar content especially glucose. The DNS technique is employed in order to estimate sugar present in the blood, in the cerebrospinal fluid and in other human bodily fluids. This method tests for the presence of free carbonyl group (C=O), the so-called reducing sugars. METHODOLOGY

Amylase (pancreas) 6.7 - 7.0 Amylase (malt) Catalase 4.6 - 5.2 7.0

A. Extraction of Invertase from Yeast

Each of these physical and chemical parameters must be considered and optimized in order for an enzymatic reaction to be accurate and reproducible. A spectrophotometer was used to measure the amount of photons (the intensity of light) absorbed after it passes through a sample solution. With the spectrophotometer, the amount of a known chemical substance (concentrations) can also be determined by to producing a variety of wavelengths because different compounds absorb best at different wavelengths, measuring the intensity of light detected. Figure 3. Invertase from Yeast Bakers yeast weighing 0.5g was dissolved in distilled water to make 500.0 ml solution. The solution was allowed to stand for 20 minutes at room temperature. Supernatant was collected as few sedimentation occurred. The supernatant served as enzyme stock solution that will be used for the succeeding experiments.

B. Preparation of Denatured Invertase Stock Solution

Figure 4. Denatured Invertase For the preparation of denatured invertase stock solution 200mL of enzyme stock solution was incubated in a boiling water bath for about 10 minutes. The solution was allowed to cool down. The solution was served as the denatured enzyme stock solution. C. Glucose Assay using Dinitrosalicylic Colorimetric Method 5 test tubes including the b l a n k t e s t tube were prepared for sucrose assay using dinitrosalicylic colorimetric method. Designated ml of glucose s t a n d a r d s o l u t i o n and ml of distilled water were placed in each test tubes. Table 2. Test tube preparation for Dinitrosalicylic Colorimetric Method. Tube No. mL glucose standar d solution mL distilled water Blank 0 1 0.25 2 0.50 4 1.00 6 1.50

incubated in a 90C water bath for 5 minutes.0.15 ml of 0.5 KOH was placed to the test tubes for the neutralization of the solutions. 2.80 ml of 0.1M buffer pH 5 was placed on the test tubes. 3ml of DNS reagent was added to the test tubes and were immersed in a 95 C water bath for 10 minutes or until the redbrown color developed. The solutions were allowed to cool down. Absorbance at 540 plotting nm was measured using spectrophotometer. Hydrolyzed- glucose standard curve was constructed by 540A against concentration. D. Effect of pH on Invertase Activity Six numbered test tubes were prepared.2.9 ml 0.1 M buffer solution with different pH was added in each test tube: Table 3. Test tube Preparation for Effect of pH on Invertase Activity. Tube 1 2 3 4 5 No. pH of buffer solution 2 3 5 7 9

6 11

Enzyme stock solution with amount of 0.1 ml was added to each test tube.

After the test tubes were mixed, they were incubated in 60C water bath for 5 minutes. One and a half mL of sucrose solution was added and the test tubes were again incubated for 60C water bath for another 5
minutes. Three mL of DNS reagent was then added. The test tubes were immersed in 95C water bath to develop the characteristic red-brown color. The test tubes were cooled after. Blank solutions were prepared by following the first step but instead of enzyme stock solution, denatured enzyme solution was added. The absorbance was measured at 540 nm.

1.50

1.25

1.00

0.50

3 drops of concentrated HCl was added to each of the test tubes and were

RESULTS AND DISCUSSION A. Invertase from Yeast Invertase was extracted from brewers yeast and acts as a catalyst for the hydrolysis of sucrose. (Campbell&Reece,2002) 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.

The DNS also reacted to glucose , a product from invertase activity of sucrose, a product from invertase activity of sucrose, and converted to gluconic acid. The absorbance of each hydrolyzed sucrose on test tubes was identified using the UV-vis spectrophotometer. To compute for the amount of glucose formed, the equation C1V1=C2V2 was used. Using the formula the following were obtain. Test tube 1: ( )( ) Test tube 2: ( )( )

Figure 5. Breakdown of Sucrose to Glucose and Fructose through Invertase Two Stock Solution was prepared in the experiment: the enzyme stock solution and the denatured invertase stock solution (which served as the blank). B. Glucose Assay using Dinitrosalicylic Colorimetric Method In the experiment, as the dinitrosalicylic acid, a yellow dye, was incorporated in the test tubes with the presence of heat, the mixture slowly turned red- brown. This is because the conversion of the 3,5dinitrosalicylic acid to 3-amino-5nitrosalicylicacid, which contributed to the red-brown color of the mixture.

Test tube 4: ( )( ) Test tube 6: ( )( )

Table 4. Amount of Acid-Hydrolyzed Sucrose and absorbance at 540nm Amount of Glucose formed Blank 1 2 4 6 0 5.56x10 - 3 0.01 0.022 0.033 Absorbance at 540 nm

0 0.207 0.285 2.54 2.75

Figure 7. conversion of DNS

2.5 2 1.5 1 0.5 0

pH3:

pH5: Figure 8. Amount of Glucose vs. Absorbance The line drawn on the graph represented the best fit line and was computed through the linear regression function of a scientific calculator. The slope intercept form computed was found to be y=97.38x+0.22. In the graph shown for absorbance vs. amount of acid hydrolyzed sucrose, a linear trend was not identified. This is due to some possible causes. C. Effect of pH on Invertase Activity The intercept was found to be -0.22 while the slope was 97.38 The following absorbance will be used to compute for the concentration or amount of glucose formed. Table 5. pH and Absorbance of Invertase pH 2 3 5 7 9 11 pH2: Absorbance at 540nm 0.044 0.031 0.015 0.129 0.166 0.0685

pH7:

pH9:

pH11:

Table 6. pH and Amount of Glucose Formed pH 2 3 5 7 9 11 Amount of Glucose formed

0.0039 0.0034 0.0029 0.0024 2 3 5 7 9 11

http://www.eng.umd.edu/~nsw/ench485/l ab14.htm date accessed: January 13,2014 http://giapo.com/2013/09/sugar-inversionmaking-invertedsyrup/ date accessed: January 13,2014 http://greenwoodhealth.net/np/invertase.h tm date accessed: January 13,2014 http://www.worthingtonbiochem.com/introbiochem/effectsph.html date accessed: January 11,2014

Figure 9. pH vs. Amount of Glucose formed Most proteins, and therefore enzymes, are active only within a narrow pH range usually between 5 and 9. The result of the experiment was different from the ideal optimum pH of 4.5,we got the optimum pH of 9 for our invertase. This kind of error was committed because of some possible factors like the pH of the mixture was not accurate or the spectrophotometer itself committed the errors. Changes in pH may not only affect the shape of an enzyme but it may also change the shape or charge properties of the substrate so that either the substrate cannot bind to the active site or it cannot undergo catalysis. References Boyer, Rodney (2006) Concepts in Biochemistry 3rd Edition. John Wiley and Sons Inc. U.S.A. Campbell, N.A.; Reece, J.B.; Biology, 6th ed. San Francisco; Benjamin Cummings, 2002. Crisostomo, A.C. et al. (2010).Laboratory Manual in General Biochemistry. C & E Publishing Inc. Philippines http://www.buzzle.com/articles/ph-effect-onenzymes.html date accessed: January 11, 2014.