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EFFECT OF pH ON THE ACTIVITY OF DENATURED AND NON-DENATURED INVERTASE FROM BAKERS YEAST (Saccharomyces cerevisiea) Patacsil, J.M.

, Perez, A., Pua, M.A., Ramirez, D.R., *Sagun, E. 2D-Pharmacy, Faculty of Pharmacy, University of Santo Tomas Abstract Enzymes are biomolecules which serve as catalysts on the different reactions that occur inside an organisms body. In this experiment, the enzyme invertase was extracted from the Bakers yeast (Saccharomyces cerevisiea). The sample was divided into two, wherein the second solution was subjected to heat in order to denature the enzyme. Through the use of DInitrosalicylic (DNS) colorimetric method, the reaction rates of the enzyme-catalyzed reactions were determined. A UV-Vis spectrophotometer was used to measure the absorbance value at 540nm. A graph of sucrose standard curve was formed by plotting the concentration (mg/mL) against the absorbance value at 540nm. Different solution with varied pH (2, 3, 5, 7, 8, 11) were added to each test tube containing denatured or non-denatured invertase. The data gathered were multiplied from the equation of the sucrose standard curve to get the concentration of the acid-hydrolyzed sucrose. The optimum pH was indicated by the peak observed on the graph of pH vs. amount of acid-hydrolysed sucrose (mg/mL). Optimum pH is defined as the pH wherein the enzyme is most active. Ideally, invertase is most favourable over a broad pH range of 3.5-5.5 with optimum pH near 5. (1)Results show that invertase exhibits high activity at pH 000. The group has therefore concluded that enzyme is affected by pH. Greatly high or low pH may cause a loss on the activity of the enzyme. The effect of pH on the enzyme activity is the same whether invertase is denatured or non-denatured.
Introduction An enzyme is a protein molecule which serves as a biological catalyst. Its basic function is to increase the rate of the different cellular reactions occurring within an organisms body. It is a target-specific protein wherein, it binds to specific substrate in order to produce products. Enzymes are regulated from a state of low activity to high activity and vice-versa. A defect on a single enzyme may cause a cascade of (2) reactions that may harm the body. Invertase is an enzyme derived from yeast. Its official name is betafructofuranosidase (EC3.2.1.26), this implies that the reaction catalysed by this enzyme is the hydrolysis of the terminal nonreducing betafructofuranoside residues in betafructofuranosides. It causes sucrose to split into glucose and fructose. Sucrose can be hydrolysed easily and proceeds in an acidic environment without the aid of invertase. Many microoraganims produce invertase and utilize sucrose as a nutrient. It is biosynthesized by yeast strains of Saccharomyces cerevisiae and Saccharomyces carlsbergensis. Invertase can exist in more than one form, even if it is within the same yeast culture. It can exhibit high activity over a broad range of pH (3.5-5.5), with the optimum near pH 5. Its enzyme activity o (3) reaches a maximum at about 55 C. Saccharomyces cerevisiae, or commonly known as Bakers yeast, is a unicellular fungus. Saccharomyces when translated means, sugar fungus. It is commonly found in the wild growing one the skins of grapes and other fruits. It is classified under the kingdom fungi because of the presence of chitin on its cell wall. It is also

considered as a fungi because it is a unicellular organism which cannot form a fruiting body. They are able to breakdown food through aerobic and anaerobic fermentation and could reproduce both asexually and sexually. It is the one responsible for the production of ethanol in alcoholic drinks. It also causes bread dough to expand due to the presence of the by-product, (4) carbon dioxide. This experiment aims to extract invertase from Bakers yeast and to determine the effects of changes in pH on the reaction rates of enzyme-catalyzed reaction. Materials and Methods The invertase was isolated from the Bakers yeast (Saccharomyces cerevisiae). The supernatant was collected which served as the enzyme stock solution. A part of this solution was subjected to heat for the denaturation of the enzyme. The following laboratory apparatuses were used in the experiment: test tubes, pipettes, beakers, volumetric flasks, paraffin film and hot plate. The following reagents were added to the solution: sucrose solution (10 g/L) and dinitrosalicylic reagent. The Dinitrosalicylic acid, chemically known as 3,5-dinitrosalicylic acid, is a yellow compound with a molecular formula of C7H4N2O7. It is insoluble in water, but soluble in (5) alcohol and benzene. The 3,5-Dinitrosalicylic acid (DNS) reagent is a widely used reagent for the estimation of reducing sugars, such as glucose. A change in color of DNS signifies that sugar is present in the solution. This color change could be used to quantify the actual amount of sugar by comparing it to a set of permanent standards. It could also be used as a color reagent in reactions of alpha-amylase. DNS reagent (6) changes its color when maltose was released. UV-Vis spectrophotometer was used to measure the intensity of light before and after passing through a sample. A hydrolysedsucrose standard curve was plotted by using (7) Dinitrosalicylic Colorimetric Method.

Tube Bla 1 2 3 4 No. nk Sucro 0 0.2 0.5 0.7 1.0 se 5 0 5 0 standa rd solutio n (mL) Distille 1.5 1.2 1.0 0.7 0.5 d 0 5 0 5 0 water (mL) Table1. Amount of sucrose standard be poured on each test tube Methodology Sucrose Assay Using Colorimetric Method

5 1.2 5

6 1.5 0

0.2 5

solution to

Dinitrosalicylic

A set of test tubes containing the amount of sucrose indicated in Table 1 was prepared. Three drops of concentrated HCl was added to each tube. The test tubes were o incubated to 90 C water bath for 5 minutes. An amount 0.15 mL of 0.5 M KOH was added to neutralize the solution. And 2.80mL of 0.1M buffer solution at pH 5 was also mixed. Three millilitres of DNS reagent were added. The test o tubes were immersed in 95 C water bath for 10 minutes until a red-brown characteristic color was obtained. The absorbance at 540nm was measured after cooling the solutions. The hydrolysed sucrose standard curve was constructed by plotting A540 against the concentration (mg/mL). Extraction of Invertase from Bakers Yeast and preparation of denatured invertase A 0.25 g of Bakers yeast was weighed and dissolved in distilled water to make 250-mL solution. The solution was allowed to stand for 20 minutes at room temperature. The supernatant was collected when sedimentation occurred. One hundred millilitres of the enzyme stock solution was incubated in a boiling bath for 10 minutes. The solution was allowed to cool. The supernatant was collected when frothing occurred. It served as the denatured invertase stock solution. Both the denatured and nondenatured were refrigerated for 48 hours to decrease the activity of invertase.

Determination of invertase activity

the

effect

of

pH

on

Twelve test tubes were labelled and filled with the appropriate amount of 0.1 M buffer solution as described in Table 2. For the first 6 test tubes, 0.10 mL of the enzyme stock solution was added. For the remaining 6 test tubes, 0.10 mL of the denatured enzyme was added. All test o tubes were incubated in 60 water bath for 5 minutes. 1.5 mL of Sucrose solution was added o and was again incubated in 60 C water bath for 5 minutes. 3 mL of DNS reagent was added to each test tube. The test tubes were immersed in o 95 C water bath for 10 minutes until a red-brown color was formed. The solutions were allowed to cool. The absorbance at 540 nm was measured using a UV-Vis Spectrophotometer. The amount of sucrose hydrolysed using hydrolyze-sucrose standard curve constructed in the dinitrosalicylic colorimetric method was determined. Tube No 1 (a) 0.1 2 (a) 0.3 3 (a) 0.5 4 (a) 1.7 5 (a) 1.9 6 (a) 1.11

Figure1. Reaction mediated by invertase The enzyme invertase catalyzes the hydrolysis of the disaccharide sucrose, to form monosaccharides, glucose and fructose. This reaction is shown in Figure 1. Glucose and fructose are reducing sugars. In their open form, they have aldehydes and ketones which could act as reducing agents and will be converted into carboxylic acids when oxidized. Sucrose doesnt have an open form. Its ketones and aldehydes are tied into rings making the sucrose unable to act as a reducing agent. In the reaction shown in figure 1, only some of the portion of sucrose will react to make glucose and fructose. The amount of sucrose reacted can be determined by adding the half of the glucose and fructose present since the ratio (10) is 1:2.

Amount of Buffer solution (mL) *(a) indicated that the test tube contained denatured enzyme

Table2. Amount of 0.1M Buffer Solution on each test tube of Denatured and NonDenatured Invertase
Results and Discussions Enzymes are not consumed in the reactions they catalyse. Thus, it could be used repeatedly and only a small amount of enzyme (8) is needed to catalyse a reaction. There are several factors which affect the rate of the enzymatic reaction, this are: temperature, pH, substrate concentration and the presence of inhibitors or activators. Substrate concentration and temperature speeds up the activity of the enzyme, while inhibitors may slow down or stop the reaction.

Figure2. Reaction of DNS in the presence of reducing sugar producing ANS To easily monitor the hydrolysis of sucrose to fructose and glucose, 3,5Dinitrosalicylic reagent, an oxidizing agent, was used. When DNS reacts with the reducible sugar, 3-amino-5-dinitrosalicylic acid (ANS) was produced. Since sucrose doesnt react with

DNS, it could be used for the assay of invertase reactivity and the amount of ANS produced will be determined through spectroscopy. DNS is a yellow reagent, sucrose stock solution is colorless, and ANS is a brownish substance which was observed in the experiment. ANS absorbs most at 540nm, thus, absorbance value was measured on the said wavelength. Since the hydrolysis of sucrose was followed by the reaction with DNS, the amount of ANS produced was twice the amount of sucrose reacted. ANS concentration can be known using the Beer-Lamberts law A=lc. Where A is the absorbance, c is the concentration, l is the path length (usually equal to 1.0), and is the molar extinction coefficient which is constant for each (10) substance. The standard curve produced on the sucrose assay has a slope equal to the value of . Thus the formula A/ can be used to (10) determine the amount of sucrose hydrolysed. The amount of Hydrolyzed Sucrose was known using the formula C1V1=C2V2, where: C1- concentration of the sucrose solution (0.1mg/mL) V1- volume of sucrose standard solution (varies per test tube) C2- Unknown V2- total volume of the solution (7.5 mL) This was used to form the graph shown on Figure 3. This figure shows the hydrolysed sucrose standard curve. The line represents the best representation for the points plotted which were known by using linear regression of the graph using a scientific calculator. The points did not give a supposed to be straight line because of some errors done by the researchers in doing spectrophotometry. The graph could also be made by using Microsoft Excels chart tools. Table 3 shows the tabulated form of the computed concentration of the hydrolysed sucrose and the absorbance value of each test tube. Figure 4 shows the theoretical structure of the sucrose standard curve. It can be observed that all the points should be near the best straight line. Compared to the hydrolysed sucrose standard curve obtained, the lines are different from each other. The theoretical line is located in quadrant I while the sucrose standard obtained from the experiment was located in quadrant 4. This difference may be caused by some errors committed by the researchers.

0 0 -0.05
Absorbance (540 nm)

0.01

0.02 y = -7.3848x R = -0.672

0.03

-0.1 -0.15 -0.2

Concentration of Hydrolyzed Sucrose (mg/mL)

Figure3. Hydrolyzed Sucrose Standard Curve


Test Tube Hydrolyzed Absorbance Sucrose (A540) (mg/mL) Blank 0 0 1 0.003 -0.165 2 0.007 -0.153 3 0.010 -0.166 4 0.013 -0.073 5 0.017 -0.024 6 0.020 -0.146 Table3. Amount of hydrolysed sucrose and its absorbance value Computations: Amount of hydrolysed sucrose (mg/mL) of blank test tube: (0.1)(0)=(x)(7.5) x= 0 mg/mL Amount of hydrolyzed sucrose (mg/mL) of test tube 1: (0.1)(0.25)=(x)(7.5) x= 0.003 mg/mL Amount of hydrolyzed sucrose (mg/mL) of test tube 2: (0.1)(0.5)=(x)(7.5) x= 0.007 mg/mL Amount of hydrolyzed sucrose (mg/mL) of test tube 3: (0.1)(0.75)=(x)(7.5) x= 0.01 mg/mL Amount of hydrolyzed sucrose (mg/mL) of test tube 4: (0.1)(1)=(x)(7.5)

x= 0.013 mg/mL Amount of hydrolyzed sucrose (mg/mL) of test tube 5: (0.1)(1.25)=(x)(7.5) x= 0.017 mg/mL Amount of hydrolyzed sucrose (mg/mL) of test tube 6: (0.1)(1.50)=(x)(7.5) x= 0.020 mg/mL

Computations for Denatured Invertase: Equation: y=-7.3848x Amount of acid-hydrolyzed sucrose (mg/ml) of test tube 1 (): x= 0.069/-7.3848 x= -0.009 Amount of acid-hydrolyzed sucrose (mg/ml) of test tube 2 (): x= 0.207/-7.3848 x= -0.028 Amount of acid-hydrolyzed sucrose (mg/ml) of test tube 3 (): x= 0.314/-7.3848 x= -0.043 Amount of acid-hydrolyzed sucrose (mg/ml) of test tube 4 (): x= 0.319/-7.3848 x= -0.043 Amount of acid-hydrolyzed sucrose (mg/ml) of test tube 5 (): x= 0.087/-7.3848 x= -0.012 Amount of acid-hydrolyzed sucrose (mg/ml) of test tube 6 (): x= 0.364/-7.3848 x= -30.897 The result of the effect of pH on nondenatured enzyme shows that there is only a small effect on the amount of sucrose hydrolysed. However, it could be observed that from pH to pH the value of sucrose hydrolysed increases. From pH to pH equal amount of sucrose was hydrolysed and from pH to pH less sucrose were hydrolysed. It can also be observed that there was a drastic decrease on the amount of sucrose hydrolysed on the last pH.

Figure4. Theoretical Sucrose Standard Curve


Effect of pH on the activity of denatured and non-denatured invertase activity: The calculated linear regression on Figure 3 was used to know the amount of the acid-hydrolysed sucrose by substituting the variables with their corresponding values. The variable y represents the absorbance value and x represents the concentration of the acid hydrolysed sucrose. Table 4 and 5 shows a tabulated form of the computed values from the given equation on Standard Sucrose Curve (Figure 3).

Test Tube

AcidAbsorbance hydrolyzed (A540) sucrose (mg/mL) 1 0.1 -0.009 0.069 2 0.3 -0.028 0.207 3 0.5 -0.043 0.314 4 1.7 -0.043 0.319 5 1.9 -0.012 0.087 6 1.11 -30.897 0.364 Table4. Amount of acid-hydrolyzed sucrose and Absorbance540nm based on pH (Denatured)

pH

Test Tube

pH

Acidhydrolyzed sucrose (mg/mL)

Absorbance (A540)

1 2 3 4 5 6 Table5. Amount of acid-hydrolyzed sucrose and Absorbance540nm based on pH (NonDenatured) Computations for Non-Denatured Invertase: Equation: y=-7.3848x Amount of acid-hydrolyzed sucrose test tube 1 (): x= 0.069/-7.3848 x= -0.009 Amount of acid-hydrolyzed sucrose test tube 2 (): x= 0.207/-7.3848 x= -0.028 Amount of acid-hydrolyzed sucrose test tube 3 (): x= 0.314/-7.3848 x= -0.043 Amount of acid-hydrolyzed sucrose test tube 4 (): x= 0.319/-7.3848 Amount of acid-hydrolyzed sucrose test tube 5 (): x= 0.087/-7.3848 x= -0.012 Amount of acid-hydrolyzed sucrose test tube 6 (): x= 0.364/-7.3848 x= -30.897 (mg/ml) of

Figure5. Effect of pH on the activity of Denatured and Non-denatured enzyme Figure 5 compares the effect of pH on the activity of denatured and non-denatured invertase. It could be seen that .

(mg/ml) of

(mg/ml) of

(mg/ml) of

(mg/ml) of

(mg/ml) of

Figure6. Theoretical effect of pH on the activity and Stability of invertase Theoretically an invertases activity can be determined at a pH range of 4 to 10. It was known that maximum invertase activity was exhibited at pH 5.0. Invertase was stable at pH between 4-5. This data is shown in Figure 6. (9)

The result of the effect of pH on nondenatured enzyme shows that there is only a small effect on the amount of sucrose hydrolysed. However, it could be observed that from pH to pH the value of sucrose hydrolysed increases. From pH to pH equal amount of sucrose was hydrolysed and from pH to pH less sucrose were hydrolysed. It can also be observed that there was a drastic decrease on the amount of sucrose hydrolysed on the last pH.

References

(1): Worthington Biochemical Corporation (2013). Introduction to Enzymes. [ONLINE] Available at: http://www.worthingtonbiochem.com/introbiochem/effectsph.html . [Last Accessed December 28, 2013]. (2): Charles E. Ophardt (2003). Role of Enzymes in Biochemical Reactions. [ONLINE] Available at: http://www.elmhurst.edu/~chm/vchembook/570e nzymes.html. [Last Accessed December 28, 2013]. (3): Greenwood Health Systems Inc. (2009). Invertase. [ONLINE] Available at: http://greenwoodhealth.net/np/invertase.htm. [Last Accessed December 28, 2013]. nd (4): Freeman, S.. Biological Science. 2 ed. Vol. 1. Upper Saddle River, NJ: Pearson Education, 2005. (5): Stephanie Chandler (2013). General Use of Dinitrosalicylic Acid. [ONLINE] Available at: http://www.ehow.com/about_5244996_generaluse-dinitrosalicylic-acid.html#ixzz2oe1n6aWH. [Last Accessed December 28, 2013]. (6): Whitney, P. J. and Saqib, A. A., (2011). Differential behaviour of the dinitrosalicylic acid (DNS) reagent towards mono- and di-saccharide sugars. Biomass and Bioenergy. 35 (11), pp.4748-4750 (7) Bean, K. (2012). UV Visible Spectrophotometers and Their Uses in a Laboratory. [ONLINE] Available at: http://www.labtech.co.uk/uv-visiblespectrophotometers-and-their-uses-laboratory. [Last Accessed December 28, 2013]. (8): Encyclopedia Britannica (2013). Enzyme. [ONLINE] Available at: http://global.britannica.com/EBchecked/topic/18 9245/enzyme/2123/Factors-affecting-enzymeactivity. [Last Accessed December 28, 2013]. (9): Kaur, N. and Sharma, A. D., (2005). Production, optimization and characterization of extracellular invertase by an actinomycete stain . Journal of Scientific and Industrial Research. 64, pp.515-519 (10): F. Daniels, J. W. Williams, P. Bender, R. A. Alberty, C., D. Cornwell, J. E. Harriman, Experimental Physical Chemistry , 7th Ed.McGraw-Hill, New York, NY, 2010, Exp. 37, pp236-239.