Objectives

Introduction

Because of the existence of two types of linkages, the alpha-1,4 and the alpha-1,6, different structures are possible for starch molecules. An unbranched, single chain polymer of 500 to 2000 glucose subunits with only the alpha-1,4 glucosidic bonds is called amylose. On the other hand, the presence of alpha-1,6 glucosidic linkages results in a branched glucose polymer called amylopectin. The degree of branching in amylopectin is approximately one per twenty-five glucose units in the unbranched segments. Another closely related compound functioning as the glucose storage in animal cells is called glycogen, which has one branching per 12 glucose units. The degree of branching and the side chain length vary from source to source, but in general the more the chains are branched, the more the starch is soluble.

Starch is generally insoluble in water at room temperature. Because of this, starch in nature is stored in cells as small granules which can be seen under a microscope. Starch granules are quite resistant to penetration by both water and hydrolytic enzymes due to the formation of hydrogen bonds within the same molecule and with other neighboring molecules. However, these inter- and intra-hydrogen bonds can become weak as the temperature of the suspension is raised. When an aqueous suspension of starch is heated, the hydrogen bonds weaken, water is absorbed, and the starch granules swell. This process is commonly called gelatinization because the solution formed has a gelatinous, highly viscous consistency. The same process has long been employed to thicken broth in food preparation.

Depending on the relative location of the bond under attack as counted from the end of the chain, the products of this digestive process are dextrin, maltotriose, maltose, and glucose, etc. Dextrins are shorter, broken starch segments that form as the result of the random hydrolysis of internal glucosidic bonds. A molecule of maltotriose is formed if the third bond from the end of a starch molecule is cleaved; a molecule of maltose is formed if the point of attack is the second bond; a molecule of glucose results if the bond being cleaved is the terminal one; and so on. As can be seen from the exercises in Experiment No. 3, the initial step in random depolymerization is the splitting of large chains into various smaller sized segments. The breakdown of large particles drastically reduces the viscosity of gelatinized starch solution, resulting in a process called liquefaction because of the thinning of the solution. The final stages of depolymerization are mainly the formation of mono-, di-, and tri-saccharides. This process is called saccharification, due to the formation of saccharides.

Since a wide variety of organisms, including humans, can digest starch, alpha-amylase is obviously widely synthesized in nature, as opposed to cellulase. For example, human saliva and pancreatic secretion contain a large amount of alpha-amylase for starch digestion. The specificity of the bond attacked by alpha-amylases depends on the sources of the enzymes. Currently, two major classes of alpha-amylases are commercially produced through microbial fermentation. Based on the points of attack in the glucose polymer chain, they can be classified into two categories, liquefying and saccharifying.

Because the bacterial alpha-amylase to be used in this experiment randomly attacks only the alpha-1,4 bonds, it belongs to the liquefying category. The hydrolysis reaction catalyzed by this class of enzymes is usually carried out only to the extent that, for example, the starch is rendered soluble enough to allow easy removal from starch-sized fabrics in the textile industry. The paper industry also uses liquefying amylases on the starch used in paper coating where breakage into the smallest glucose subunits is actually undesirable. (One cannot bind cellulose fibers together with sugar!)

On the other hand, the fungal alpha-amylase belongs to the saccharifying category and attacks the second linkage from the nonreducing terminals (i.e. C4 end) of the straight segment, resulting in the splitting off of two glucose units at a time. Of course, the product is a disaccharide called maltose. The bond breakage is thus more extensive in saccharifying enzymes than in liquefying enzymes. The starch chains are literally chopped into small bits and pieces. Finally, the amyloglucosidase (also called glucoamylase) component of an amylase preparation selectively attacks the last bond on the nonreducing terminals. The type to be used in this experiment can act on both the alpha-1,4 and the alpha-1,6 glucosidic linkages at a relative rate of 1:20, resulting in the splitting off of simple glucose units into the solution. Fungal amylase and amyloglucosidase may be used together to convert starch to simple sugars. The practical applications of this type of enzyme mixture include the production of corn syrup and the conversion of cereal mashes to sugars in brewing.

Thus, it is important to specify the source of enzymes when the actions and kinetics of the enzymes are compared. Four types of alpha-amylases from different sources will be employed in this experiment: three of microbial origin and one of human origin. The effects of temperature, pH, substrate concentration, and inhibitor concentration on the kinetics of amylase catalyzed reactions will be studied. Finally, the action of the amylase preparations isolated from microbial sources will be compared to that from human saliva.

List of Reagents and Instruments

A. Equipment

• Erlenmeyer flasks
• Beakers
• Graduated cylinder
• Pipets, 1ml, 10ml
• Test tubes
• Temperature bath
• Thermometer
• Balance
• Syringe
• Filter holder and filter paper
• Spectrophotometer
• Brookfield viscometer

B. Reagents

• Enzymes
  • Bacterial amylase solution, 3000 SKB units/ml
  • Fungal amylase powder, 40,000 SKB units/g. (Concentration of the fungal amylase solution to be used in class: 75g/l)
  • Amyloglucosidase solution, 75 AG units/ml
  • Human salivary amylase
• Corn starch
• HCl Stopping Solution, 0.1N HCl
• Iodine Reagent Stock Solution (in aqueous solution) See Note 1.
  • Iodine: 5 g/l
  • KI: 50 g/l
• Potassium Phosphate Buffers
  • KH₂PO₄ (monobasic phosphate) (FW=136.1)
  • K₂HPO₄·3H₂O (dibasic phosphate) (FW=228.23)
• CaCl₂·2H₂O, 0.1M solution
• Reagents for the analysis of reducing sugars

Procedures

1. Prepare a 20 g/l starch solution.
   1. Mix 20 g of soluble potato starch in approx. 50 ml of cold water.
   2. While stirring, add the slurry to approx. 900 ml of gently boiling water in a large beaker.
   3. Mix well and cool the gelatinized starch solution to room temperature.
   4. Add more water to bring the total volume to 1 liter.
   5. Put a few drops of the starch solution on a glass plate. Add 1 drop of the iodine reagent and see that a deep blue color is developed. The blue color indicates the presence of starch in the solution.
2. Effect of the pH
   1. Prepare 0.1M pH buffer solutions ranging from pH=4.5 to pH=9 in increments of one pH unit. (Note that phosphate buffer is only good for ph=4.5--9 due to the dissociation constant.) Before coming to the lab, review how to make a pH buffer solution in a freshman chemistry textbook and calculate the relative amounts of KH₂PO₄ (monobasic phosphate) and K₂HPO₄·3H₂O (dibasic phosphate) needed to make these phosphate buffer solutions.
   2. Add an equal volume of one of the above buffer solutions to 5.0ml of the 20g/l starch solution prepared in Step 1. The resulting solution should contain 10g/l of starch in a buffered environment.
   3. Start the enzymatic digestion process by adding 0.5 ml of the bacterial amylase solution; shake and mix.
   4. Let the hydrolysis reaction proceed for exactly 10 minutes at 25ºC.
   5. Add 0.5 ml of the reacted starch solution to 5ml of the HCl stopping solution (0.1N)
   6. Add 0.5 ml of the above mixture to 5ml iodine solution to develop color. Shake and mix. The solution should turn deep blue if there is any residual, unconverted starch present in the solution. The solution is brown-red colored for partially degraded starch, while it is clear for totally degraded starch.
   7. Measure the absorbance with a spectrophotometer at 620nm. See Note 2.
   8. Carry out the same procedure for the other starch solutions buffered at different pH's. (Use your time wisely; all the solutions can be handled simultaneously if you are familiar with the procedure. Slightly stagger the sequential sample withdrawal so that there is enough time for sample preparation and handling.)

            0.5ml            |    |0.5ml  |    |0.5ml  |    |       O.D.
           sample soln--->---|    |--->---|    |--->---|    |--->---at 620 nm
                             |    |       |    |       |    |
                             |____|       |____|       |____|
                               5ml          5ml          5ml
                           starch soln  0.1N HCl soln  iodine soln
      

3. Effect of Temperature
   1. Obtain hot water from either a faucet or a hot temperature bath. Adjust the temperatures of the temporary water baths in 500 ml beakers so that they range from 30 ºC to 90 ºC in increments of 10 ºC.
   2. Prepare the starch substrate by diluting the 20g/l starch solution prepared in Step 1 with an equal volume of pH=7.0 phosphate buffer solution. This results in a working starch concentration of 10 g/l. Add 5 ml of the starch solution to each of the test tubes.
   3. Allow the temperature of each of the starch solutions to come to equilibrium with that of the water bath.
   4. Add 0.5 ml of the bacterial amylase solution to each of the thermostated test tubes to start the reaction.
   5. Stop the reaction after exactly 10 minutes and analyze the starch content by following the procedures outlined in Step 2.
4. Effect of Heat Treatment
   1. Place 0.5 ml of the bacterial amylase solution each of eleven test tubes.
   2. Heat-treat the enzyme solution by placing all the test tubes, except one, in a hot (90ºC) water bath. The untreated enzyme is used as the control. Take out the first test tube from the heat after one minute and quickly bring it to room temperature by immersing it in a cool water bath. Remove the second test tube after 2 minutes, the third after 3 minutes, and so on.
   3. Add 5 ml of the 10 g/l buffered (pH=7.0) starch solution to each of the test tubes containing the enzymes.
   4. Carry out the hydrolysis reaction at room temperature and analyze the sample after exactly 10 minutes by following the procedures outlined in Step 2.
   5. Mix an equal volume of the CaCl₂ solution to the enzymes and repeat the same procedures to investigate the heat stabilization of the enzymes in the presence of Ca²⁺ ions.
   6. This set of studies can be done quickly if the procedures are synchronized. If time permits, try 0.5 ml samples of the amyloglucosidase and 0.5 ml samples of the fungal amylase solution. Compare the sensitivity to heat for these related enzymes. Hint: The liquefaction step in the production of high-fructose corn syrup is carried out at about 105ºC.
5. Activity of Human Salivary Amylase Obtain enough saliva to repeat the pH effect study as in Step 2.
6. Enzyme Specificity Use 0.5 ml of the cellulase left over from the previous experiment. Follow a similar procedure to determine the decrease in the starch concentration as outlined in Step 2. Measure the rate with buffered starch solution at pH=4.0 and 7.0.
7. Effect of Substrate Concentration
   1. Add 0.5 ml of the bacterial amylase solution to 50 ml of a 10g/l starch solution buffered at pH=7.0. Note that less enzyme per ml of substrate is used in this part of the experiment than the previous parts. The objective here is to slow down the reaction so that multiple sampling is possible with reasonable accuracy before all the starch is consumed.
   2. Take samples periodically to monitor both the decrease in the starch concentration and the increase in the reducing sugars until most of the starch is hydrolyzed. The starch concentration is measured with the same steps outlined above and the sugar concentration with the dinitrosalicylic colorimetric method used in the previous experiment.
   3. Continuously monitor the viscosity of the substrate-enzyme mixture with a viscometer. Generate a calibration curve for the viscosity as a function of the starch concentration. Note that this part of the study is fruitful only when the starch solution is extremely thick.
8. Effect of Enzyme Sources
   1. Repeat Procedure 7 with 0.5 ml of the fungal amylase solution.
   2. Repeat Procedure 7 with 0.5 ml of the amyloglucosidase.
   3. Repeat Procedure 7 to study the joint action of a mixture of 0.167 ml of bacterial amylase, 0.167 ml of fungal amylase solution, and 0.167 ml of amyloglucosidase.
   4. This entire Procedure 8 can be concurrently carried out along with Procedure 7.
9. Sequential Enzymatic Treatment (Corn Syrup Production) In making industrial sugars, e.g. corn syrup, large gelatinized starch molecules are first chopped into smaller dextrins with the help of bacterial amylase. The liquefaction step is followed by saccharification with either fungal amylase or amyloglucosidase, depending on the end use of the sugar. These sequential enzymatic treatment steps will be simulated in this part of the experiment
   1. Add 0.5 ml of the bacterial amylase solution to 50 ml of the 20g/l non-buffered starch solution prepared in Step 1. Periodically place a few drops of the reaction mixture on a glass plate and add one drop of the iodine reagent. The color should finally turn red, indicating the total conversion of starch to dextrin. This liquefaction step should last for approximately 10 minutes.
   2. When the process of liquefaction is complete, adjust the pH of the starch solution to 4.7 with 1N HCl.
   3. Filter the starch solution if it is turbid. Separate the solution into two equal parts.
   4. To the first starch solution, add 0.5 ml of amyloglucosidase; to the second solution add 0.5 ml of fungal amylase solution.
   5. Measure the sugar concentrations periodically. Note that you need to use the appropriate calibration curves because one is maltose and the other is glucose. Also do not forget to reference your observation to the initial absorbance at the start of the saccharification process so that the increase in the sugar concentration can be correctly measured. This saccharification step should last for about 30-60 minutes.
   6. Taste the two sugar solutions and compare the sweetness. See Note 3.
10. Inhibition Follow Procedure 7, except that the buffered starch solution at pH-7.0 also contains hydrogen peroxide at a level of 0.5 g/l. If time permits, try hydrogen peroxide at a level of 1.0 g/l.
11. Enzyme Activity versus Enzyme Concentration Mix 0.5, 1.0, 1.5, 2.0, and 2.5 ml of enzyme solutions with 5 ml of 10g/l starch solution. Measure the starch concentration after 10 minutes as in Step 2.
12. For Curious Students Study the simple cleavage of the alpha-1,4 glucosidic bonds by using maltose as the substrate and amyloglucosidase as the enzyme. Plan ahead to see how one can perform this. For example, you need to be able to distinguish between maltose and glucose. Note that in order to use a colorimetric method, maltose and glucose must have different extinction coefficients. Can material balance be used to back out the individual concentrations?

Notes

1. Dilute the stock solution 1:100 to obtain a working solution. Other dilutions may be used, depending on the enzyme activity. Iodine does not dissolve much in water. Iodine (I₂) alone or iodide (I^-) alone does not color starch. It is the triiodide complex ( I₃^-, formed by I₂+I^-) that gives off the blue color when it is incoporated into the coil structure of starch.
2. Remember to take care of the background absorbance caused by the colored iodine solution. The true absorbance should be roughly proportional to the starch concentration. The enzyme solution may have to be diluted first if all the starch present in the sample is digested and all the color disappears in 10 minutes. The most reliable results are obtained when the decrease in the absorbance is approximately 20-70% of the absorbance of the original, undigested starch solution. To measure the amount of starch digested, you need to know the absorbance corresponding to the initial undigested starch solution by following the same procedure with a sample in which plain water in lieu of the enzyme solution is added to the starch solution.
3. Be sure you do not contaminate your sugar solution during the various stages of the reaction. Do not taste the sugar solutions and risk your health if you are not confident about your lab techniques. However, do not expect others to trust your results if you cannot even convince yourself. All glassware used in this biochemical engineering laboratory should always be much cleaner than the eating utensils on your dinning table. The reagent or analytical grade chemicals we use are also much purer than the food grade ones. Thus, as long as they are not poisonous or toxic and as long as you do not contaminate them when weighing, the intake of a small amount of them should not cause you any harm. Furthermore, as sterility will be stressed in the later part of the course when microorganisms are introduced, the glassware used then should be thoroughly aseptic, certainly cleaner than your finger. If you have hesitation in eating and drinking from any of the glassware or spatula that you use, I suggest that you get into the habit of really cleaning them before using them in the experiment. Although I am not encouraging you to go around and lick everything in sight, you should develop a good, aseptic laboratory habits so that you know you will not hesitate to do so if needed. The small amount of HCl added to adjust the pH to 4.7 should not affect you at all; many carbonated drinks are much more acidic than this.
4. Do as many experiments as you wish or as time and supplies/materials permit. You can effectively cover all the procedures by teaming up with a few other classmates and exchanging data at the end of the lab period. (Be sure to give the proper credit, or blame for that matter, to your lab partners. Also be sure you know what your lab partners have done.) However, you must prepare your own lab report.

Questions

1. Plot the enzyme activity versus pH. From this curve, what is the optimal pH? Explain why enzyme activities depend on the pH. Similarly plot the enzyme activity versus temperature. Report the optimal temperature.
2. To what extent did the heat treatment affect the enzyme activities? What happens to an enzyme when it is subjected to heat?
3. What is the amylase activity in your saliva? How does it compare to those preparations isolated from microbial sources? Do they all share the same optimal pH? How does the optimal pH for the salivary amylase compare with the pH of the stomach? If the pH of the stomach is not at all favorable for amylase, has the nature made a mistake? Considering the relatively short period of time food stays in the mouse and considering the amylase activities of human saliva, there is really not much degradation of starch molecules in the mouse. Why does the nature seemingly endow amylase activities in saliva so inefficiently?
4. Did cellulase exhibit any amylo-saccharifying activities?
5. Combine the data from this experiment with those from the previous experiment and derive the rough ratio of the cellulase activity to the amylase activity. For the purpose of this comparison, you may define the activity to be the number of glucosidic bonds broken per gram of enzyme per minute. Which type of bond was easier to break, alpha-1,4 or beta-1,4? One should base his comparison on the same conditions, e.g., the same acid concentrations and temperature.
6. From the experimental data for Procedures 7 and 8, plot the starch concentration, reducing sugar concentration, and viscosity as functions of time. Do you get a closed material balance between the starch converted and the sugar generated? If not, explain this discrepancy. Is it possible to detect no starch at all and at the same time only a negligible amount of sugar during the course of the hydrolysis reaction? Make a plot of the reaction rate versus the substrate concentration. Are Michaelis-Menten kinetics applicable to this enzyme system? If so, what are the values for the model parameters? (You may need to use a Lineweaver-Burk plot or other closely related plots to derive these parameters.) If not, what model best describes what you have observed?
7. Did the presence of hydrogen peroxide affect the enzyme activities? If so, is it a competitive, a non-competitive, or uncompetitive inhibitor?
8. Is the enzyme activity directly proportional to the enzyme concentration? If not, which quantity better describes the amount of enzymes present?
9. How would 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. Repeat for a citrate buffer. (Phosphate, acetate, and citrate buffers are the most commonly encountered ones.)
10. The more accurate name for alpha-amylase is 1,4-alpha-D-glucan-glucanohydrolase (EC 3.2.1.1), it is 1,4-alpha-D-glucan maltohydrolase (EC 3.2.1.2) for beta-amylase, and exo-1,4-alpha-glucosidase or 1,4-alpha-D-glucan glucohydrolase (EC 3.2.1.3) for amyloglucosidase. What reaction does beta-amylase catalyze?
11. Comment on ways to improve the experiment.

References

1. Bailey, J.E. and Ollis, D.F., Biochemical Engineering Fundamentals, 2nd Ed., Chapter 3, McGraw-Hill, 1986.
2. Standard SKB method to determine enzyme activity: Cereal Chem., 16, 712, 1939.

Tabular Forms

                                PH OPTIMUM
                        Steps #2 #5 #6 (2 Students)

       ------------------------------------------------------------
                                 Enzyme Sources
                ---------------------------------------------------
          pH      Bacterial  Fungal      Amylo-    Saliva Cellulase
                Amylase    Amylase  Glucosidase
       ------------------------------------------------------------
         5.0                                         ---     ---
         6.0                                         ---     ---
         7.0
         8.0                                         ---     ---
         9.0                                         ---     ---
       ------------------------------------------------------------

         TEMPERATURE OPTIMUM
         Step #3 (1 Student)

       -----------------------
       Temperature   Bacterial
           (C)        Amylase
       -----------------------
           30
           45
           60
           75
           90
       -----------------------

-------------------------------------------------------------------------------
                                        Enzyme Sources
   Time of     ----------------------------------------------------------------
Heat Treatment      Bacerial               Fungal               Amylo-
    at 90C           Amylase               Amylase           Glucosidase
    (min)      ----------------------------------------------------------------
               no Ca++    /w Ca++    no Ca++    /w Ca++    no Ca++   /w Ca++
-------------------------------------------------------------------------------
      0
      4
      8
     12
     16
     20
-------------------------------------------------------------------------------

                                REACTION KINETICS
                           Steps #7 #8 #10 (2 Students)

----------------------------------------------------------------------------
                                     Enzyme Sources
         -------------------------------------------------------------------
                        Bacterial              Fungal           Amylo-
                        Amylase                Amylase        Glucosidase
Reaction -------------------------------------------------------------------
  Time       no H2O2          /w H2O2
  (min)  -------------------------------------------------------------------
          Starch  Glucose  Starch  Glucose  Starch  Glucose  Starch  Glucose
          conc.   conc.    conc.   conc.    conc.   conc.    conc.   conc.
          (g/l)   (g/l)    (g/l)   (g/l)    (g/l)   (g/l)    (g/l)   (g/l)
----------------------------------------------------------------------------
    0
    5
   10
   15
   20
   25
   30
----------------------------------------------------------------------------

               ENZYME CONCENTRATION
               Step #11 (1 Student)

       ---------------------------------------
       Enzyme          Enzyme Sources
                ------------------------------
       Volume   Bacerial  Fungal     Amylo-
        (ml)    Amylase   Amylase  Glucosidase
       ---------------------------------------
        0.5
        1.0
        1.5
        2.0
        2.5
       ---------------------------------------