Aim: To study the various parameters that affect the kinetics of alpha-amylase catalyzed hydrolysis

of starch.

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.

Theory: Starchy substances constitute the major part of the human diet for most of the people in the world, as wel
as many other animals. They are synthesized natural y in a variety of plants. Some plant examples with high starch
content are corn, potato, rice, sorghum, wheat, and cassava. It is no surprise that al of these are part of what we
consume to derive carbohydrates. Similar to cel ulose, starch molecules are glucose polymers linked together by
the alpha-1,4 and alpha-1,6 glucosidic bonds, as opposed to the beta-1,4 glucosidic bonds for cellulose. In order
to make use of the carbon and energy stored in starch, the human digestive system, with the help of the enzyme
amylases, must first break down the polymer to small er assimilable sugars, which is eventual y converted to the
individual basic glucose units.

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
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 cal ed 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 cel s is cal ed 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 general y insoluble in water at room temperature. Because of this, starch in nature is stored in cel s
as smal 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 swel . This process is commonly cal ed
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 smal er sized segments. The
breakdown of large particles drastical y reduces the viscosity of gelatinized starch solution, resulting in a
process cal ed 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 cal ed 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 cel ulase. 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
commercial y 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 usual
y carried out only to the extent that, for example, the starch is rendered soluble enough to al ow 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 smal est glucose subunits is actual y undesirable.
(One cannot bind cel ulose 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 cal ed maltose. The bond breakage is thus
more extensive in saccharifying enzymes than in liquefying enzymes. The starch chains are literal y chopped
into smal bits and pieces. Final y, the amyloglucosidase (also cal ed 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 dif erent sources wil be employed in this experiment: three
of microbial origin and one of human origin. The ef ects of temperature, pH, substrate concentration, and
inhibitor concentration on the kinetics of amylase catalyzed reactions wil be studied. Final y, the action of
the amylase preparations isolated from microbial sources wil 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 Buf ers
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

Because there is a variety of kinetic studies in this experiment, work wil be divided among the entire class.
Each student wil be assigned responsibilities for dif erent sections.

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.
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
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.
1. Prepare the starch substrate by diluting the 20g/l starch solution prepared in Step 1 with an
equal volume of pH=7.0 phosphate buf er 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.
2. Al ow the temperature of each of the starch solutions to come to equilibrium with that of the
water bath.
3. Add 0.5 ml of the bacterial amylase solution to each of the thermostated test tubes to
start the reaction.
4. 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 al 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 buf ered (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 ef ect study as in Step 2.
6. Enzyme Specificity Use 0.5 ml of the cel ulase 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 buf ered 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 buf ered 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 al the starch is consumed.
2. Take samples periodical y 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.
Precaution

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 tri odide complex ( I -, formed by I +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 tobe diluted first if al the starch present in the sample is digested and al 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.

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.

9/25/2014

Starch Hydrolysis by Amylase

(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

Results :Various parameters that affect the kinetics of alpha-amylase catalyzed hydrolysis of starch
was studied .