Experiment 2: Starch Hydrolysis by Amylase

Theoretical Background Polymers of carbohydrates are called polysaccharides, and make up some of the most important naturally occurring compounds [1]. They have thousands of monosaccharide units linked to each other by oxygen bridges. They include starch, glycogen, and cellulose, all three of which yield only glucose when completely hydrolyzed [2].

A

B

β – 1, 4 – glycosidic bond

Figure 1. Starch (amylose) (A) and cellulose (B)

Starch occurs naturally in plants, which use it to storage glucose units for energy. It is often found in seeds and tubers (e.g., potatoes). It consists of two kinds of polymers of glucose. The simpler kind is called amylose, and it makes up about 20% of starch. It is basically a chain of glucose units linked by α – 1,4 – glycosidic bonds. During digestion, the oxygen bridges are hydrolyzed and the glucose units are broken up. 80% of starch is a water insoluble fraction called amylopectin [2], which is a branched chain polysaccharide with again α – 1,4 – glycosidic bonds. At approximately every 25 glucose units, a branching of glucose units, exists. Upon treatment with acid or under the influence of enzymes, the components of starch are hydrolyzed progressively to dextrins (mixture of low melting polysaccharides, made up of 3 – 8 glucose units), maltose and finally D-glucose [3].
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An enzyme usually catalyzes a single chemical reaction or a set of closely related reactions.Starch obtained by animals from plants is stored in the animal body in the form of glycogen. Starch is one of the major nutrients in the human died. Amylase. By comparing this amount with the graph constructed with known concentrations (calibration curve). Starch + I2  blue-black color Amylase enzyme Proteins which catalyze the chemical reactions are called enzymes. Salivary amylase. to determine the concentration of a solution While the determination of the concentration of a solution. whereas enzymes must work in mild conditions in cells. an enzyme secreted by the salivary glands and the pancreas. Enzymes are highly specific both in the reaction catalyzed and in their choice of reactants. The products of the reaction then leave the active site. Non-biological catalysts work at wide ranges of temperature and pH. the concentration of the unknown solution can be determined [9]. a slit through which a 2 . freeing it up for more similar reactions to take place. A spectrophotometer measures the transmission or absorption of liquids or solids as a function of wavelength [8]. begins the polysaccharide digestion in mouth. the spectrophotometer shines light at a set of color (at different wavelengths) through the sample and measures the percent of light that is absorbed by the sample. which are called substrates. The digestion process is completed in the small intestine by pancreatic amylase [5]. A spectrophotometer consists of a white light source (light of all visible wavelengths). which is also known as ptyalin. a prism or diffraction grating that separates the light into different wavelengths. rapidly hydrolyzes amylopectin and amylose [2]. Spectrophotometry A solution appears colored because it absorbs certain wavelengths of light in the visible spectrum and reflects the others [7]. To catalyze a reaction. a substrate must have a matching shape into the active site of the enzyme [2]. between 32 0C and 40 0C and at a pH between 6. a source of energy. to determine the absorption spectrum of a pure substance in solution 2.5 [6]. Digestive processes in both plants and animals convert starch to glucose.5 and 7. Its presence in foods and other substances can be detected by the blue-black color produced when iodine solution is added to a sample of the material to be tested [5]. Biologists use the spectrophotometer for two different purposes: 1.

= log 10 (100 / % T) APPARATUS Equipment 1. so it is usually converted into absorbance which is proportional to solute concentration. Beaker. Pasteur pipettes 4. but the absorbance is measured always [7]. and a recording device that displays the amount of transmitted light energy digitally or on a dial. however. is not proportional to solute concentration. Plastic cuvettes Buffers and Chemicals 3 . a photosensitive tube which measures the energy of light transmitted through the solution (I). Test tube rack 6. % T = (I / I0) x 100 ABS. Test tubes 2. a sample solution holder.narrow beam of the desired wavelength passes (the incident light. The arrows indicate the pathway of light [7] Transmittance is the ratio of the transmitted light energy (I) to the incident light energy (I 0). 250 mL 5. Figure 2: Schematic diagram of the components of a spectrophotometer. Digital spectrophotometers have readouts for both percent transmittance and absorbance. Pipettes 3. percent transmittance is 100X ratio (I/I0).I 0). Transmittance.

add 60 mL of 0. Add 1 drop of the iodine reagent and see that a deep blue color is developed.5 to pH = 9. 2. 3. Add 0. Add 0. Dilute to 1:100 5. Prepare 0. shake and mix. 6.5 mL of the above mixture to 5 mL iodine solution to develop color. Iodine reagent stoch solution (in aqueous solution) Iodine: 5 g/L . 7. Start the enzymatic digestion process by adding 1 mL of human salivary enzyme solution.23 g/mol) PROCEDURE Preparation of 20 g/L starch solution 1. Preparation of Enzyme Solution Dilute 1 mL of your saliva with 9 mL water.3H2O (dibasic phosphate) (MW=228. 3. while it is clear for totally degraded starch. Let the hydrolysis reaction proceed for exactly 10 minutes at 25 0C. Human salivary enzyme 2. Shake and mix.1 M pH buffer solutions ranging from pH = 4.1 g/mol) K2HPO4. Effect of Temperature 4 . Effect of pH 1. Put a few drops of the starch solution on a glass plate. The solution should turn deep blue if there is any residual. Mix well and cool the gelatinized starch solution to room temperature. The blue color indicates the presence of starch in the solution. Add more water to bring the total volume to 1 liter. Add an equal volume of one of the above buffer solutions to 5. 0. Mix 20 g of soluble potato starch in approx. Meaure the absorbance with a spectrophotometer at 620 nm. HCl stopping solution. Starch solution.1N HCl 4. 4.0 in increments of one pH unit. 5. 4. The resulting solution should contain 10 g/L of starch in a buffered environment. While stirring. Then.5% NaCl solution.1 N).0 mL of the 20 g/L starch solution prepared in Step 1. Potassium phosphate buffers KH2PO4 (monobasic phosphate) (MW=136. 900 mL of gently boiling water in a large beaker. KI: 50 g/L. 2. 5. unconverted starch present in the solution. 20 g/L 3. add the slurry to approx. 50 mL of cold water. The solution is brown-red colored for partially degraded starch.5 mL of the reacted starch solution to 5 mL of the HCl stopping solution (0.1.

DATA TABLE pH ABS T ABS REPORT OBJECTIVES 1. Add 1 mL of human salivary enzyme solution to each of the thermostated test tubes to start the reaction. 4. and citrate buffers are the most commonly used buffer. Prepare the temperatures of the temporary water baths in 250 mL beakers. acetate.1. Discuss why do you choose this optimal temperature and pH. Stop the reaction after exactly 10 minutes and analyze the starch content by following the procedures outlined in Step 3. what is the optimal pH? Explain why enzyme activities depend on the pH. Add 5 mL of the starch solution to each of the test tubes. How could you make an acetate pH buffer solution? List the required chemicals and the composition needed to make one liter of acetate buffer as a function of the pH (5 7 9 11 13). and adjust the temperatures ranging from 30 0C to 90 0C in increments of 20 0C. 2.0 phosphate buffer solution. 2. Allow the temperature of each of the starch solutions to come to equilibrium with that of the water bath. From this curve. Prepare the starch substrate by diluting the 20 g/L starch solution prepared in Step 1 with an equal volume of pH = 7. Plot the enzyme activity (absorbance) versus pH. This results in a working starch concentration of 10 g/L.) REFERENCES 5 . 3. 5. Report the optimal temperature. Similarly plot the enzyme activity versus temperature. (Phosphate.

Organic Chemisty.indiana. Organic Chemistry.htm Spectrophotometer Resources 7. 2003 http://library. Perona. M. J.1.N. Introduction to Spectrophotometry. Heitholt. Stryer.physics.. GreenWood Health Inc. 2003 http://greenwoodhealth. CHEMystery.davidson. R.html 9. Morrison.tamu-commerce. Indiana University.org/ 3.html 6..html 8.. The Columbia Electronic Encyclopedia.. 2000 http://science. 2003 http://reference.edu/perona/2000/Exp8/bkg.. A. R.. Texas A&M University. Campbell. Freeman Company.htm 5..H. 6th Ed.. Spectrophotometry. 4th Ed.. Spectrophotometer.bio.csustan. 2000 http://www. Prentice Hall. New Jersey. 2000 http://www7. Analysis of Foods for Starch and Vitamin C.. 1999 4. M. Biochemistry. Carbohydrates.T. Dzierba.allrefer. and Boyd. 2003 http://dustbunny. Amylase.edu/~dzierba/P360n/KPAD/Exps/Spectro/spectro.net/np/amylase. Davidson College. 1992 2. L. Starch. New York.edu/Courses/Bio111/Bio111LabMan/Lab%201.thinkquest. Columbia University Press.com/encyclopedia/S/starch.edu/agscience/clasnote/pls497/SpecLecture/ 6 . W.