ChE 3220 Measurements Lab

Enzyme Kinetics: Kinetics of Sucrose Hydrolysis

via the Initial Rate Method1
Department of Chemical Engineering and Materials Science
Wayne State University

Adapted from the work by Nam Sun Wang, Department of Chemical Engineering, University of Maryland
College Park, MD 20742-2111

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1. Objective
The purpose of this experiment is to determine the kinetic parameters for invertase
hydrolysis of sucrose and the effects of enzyme concentration using the initial rate
method.
2. Problem Statement
You will develop the procedures as well as conduct the necessary experiments to
obtain the following.
(a) A calibration curve for absorbance versus sucrose concentration.
explain the linearity (or non-linearity) of the curve.

Discuss and

(b) A plot of the initial rate of reaction (-rs) vs. substrate concentration (S). In this plot,
make sure you show the experimental data points and a model curve representing the
Michaelis-Menten equation drawn with your experimentally determined Vmax and KM
values. Your discussion should address any discrepancies in the data or poor matching
of your model curve with the data points.
(c) A plot of Vmax versus enzyme concentration.
concentration on maximum rate.

Discuss the effect of enzyme

(d) Compare your experimental values of the Michaelis-Menten kinetic constants to

values published in the literature. Discuss the effect (if any) of enzyme concentration on
maximum rate (Vmax)
3. Introduction and Theory
Sucrose, commonly known as table sugar, is a disaccharide composed of an -Dglucose molecule and a -D-fructose molecule linked by an -1,4-glycosidic bond.
When this bond is cleaved in a hydrolysis reaction, an equimolar mixture of glucose and
fructose is generated. This mixture of monosaccharides is called invert sugar, which is
derived from the fact that sucrose (not from hydrolysis products) rotates plane polarized
light to the right i.e., dextrorotatory, +66.5 , whereas the products of the hydrolysis
reaction rotate plane polarized light to the left i.e., levorotatory, -20 for the mixture
(+52.5 for D(+)-glucose and -92 for D(-) fructose).
Sucrose can be hydrolyzed in the presence of an enzyme called invertase
Sucrose + H2O

glucose + fructose

(1)

The official name for invertase is -fructofuranosidase (EC 3.2.1.26). Note that sucrose
can be hydrolyzed relatively easily. The reaction can proceed even without the
presence of invertase, when sucrose is in an acidic environment.

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Invertase is mainly used in the food industry, where fructose is preferred over sucrose
because it is sweeter and does not crystallize as easily. However, the use of invertase
is rather limited because another enzyme, glucose isomerase, can be used to convert
glucose to fructose more inexpensively. For health and taste reasons, its use in food
industry requires that invertase be highly purified.
A wide range of microorganisms produce invertase and can, thus, utilize sucrose as a
nutrient.
Commercially, invertase is biosynthesized chiefly by yeast strains of
Saccharomyces cerevisiae or Saccharomyces carlsbergensis. Even within the same
yeast culture, invertase exists in more than one form. For example, the intracellular
invertase has a molecular weight of 135,000 Daltons, whereas the extracellular variety
has a molecular weight of 270,000 Daltons.
In contrast to most other enzymes, invertase 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 55 C.
The reaction of the enzyme (E) and sucrose (S) can be broken into three individual
reactions as follows:
E

k1

ES

ES

k2

ES
E

(2)

However, determining the reaction rate constant (k) for each of these reactions is
dependent upon the concentration of the enzyme-substrate complex (ES).
Unfortunately, we cannot measure the concentration of this enzyme-substrate complex
in any meaningful way, and thus, we cannot solve for k1, k-1 and k2.
However, if the reaction rate constants are replaced by KM and Vmax and either rapid
equilibrium or pseudo-steady equilibrium is assumed, then one can derive the
Michaelis-Menten equation:

rs

VMAX S
KM S

(3)

which is dependent only on the rate of disappearance of the substrate (-rs), and the
substrate concentration (S).
Rearrangement of the Eq. (3) into the form y = m.x + b is straight forward, and is seen
in the Lineweaver-Burk, Eadie-Hofstee and Hanes-Woolf plots. Since the initial rate
method assumes that at a short time interval, say 5 minutes, at the beginning of the
reaction, the reaction rate does not substantially change, then:

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rs

dS
dt

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reduces to

rs

S
t

(4)

where we know the initial substrate concentration (S), the time interval, and we can
obtain the final inverted substrate concentration through the calibration curve ( S).
From this methodology the values for KM and Vmax may be obtained, thus allowing the
quantification of the enzyme effectiveness. From the definition of KM, we have that the
lower the KM, the better the enzyme at binding to the substrate. From the definition of
Vmax, it is noted that the higher the Vmax, the faster the enzyme-substrate complex goes
to product. Therefore, a low KM and a high Vmax are desired in a good enzyme for a
specific substrate.
4. Materials
4.1 Equipment
Test tubes
Thermometers
Balance
Beakers
Pipettes
Pipettes are available in wide range of sizes. Graduations should be checked to
determine the volume capacity of a give pipette. It is also necessary to determine if
the graduations use the tip of the pipette or not in order to get accurate
measurements.
Hot plates
Spectrophotometer

4.2 Reagents (Prepared by the TA)

3,5-dinitrosalicylic acid (DNS) reagent
Materials:

1) 3,5-dinitrosalicylic acid
2) NaOH
3) Distilled water (DI-water)
4) Rochelle salts (Sodium potassium tartrate tetrahydrated)
5) Phenol (melt at 50 C)
6) Sodium metabisulfite
7) Phenolphthalein solution
8) 0.1N HCl solution

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Preparation:
1) Dissolve 10.6g of 3,5-dinitrosalicylic acid and 19.8g of NaOH into 1416mL of DIwater in a stirred beaker. Add 306g of Rochelle salts, 7.6mL (8.132g) of phenol
and 8.3g of sodium metabisulfite;
2) Titrate a 3mL sample of the previous solution with phenolphthalein and 0.1N HCl
solution. It should take 5-6 mL of 0.1N HCl solution. Add NaOH into the preceding
DNS solution if required: 2g of NaOH for each additional 1mL of 0.1N HCl
solution required beyond 6mL.
Phosphate Buffered Saline (PBS) solution
Materials:

1) Phosphate Buffered Saline (pH 7.4) from Sigma

2) Distilled water (DI-water)

Preparation: 1) Dissolve 5 PBS packages in 1 L of Di-water, which will yield 0.05 M

solution.
The PBS solution can be prepared from BP399-1 Phosphate Buffered Saline 10X
Solution (0.1M), diluting this solution to 0.05M.
1 g/L Invertase Stock Solution
Materials:

1) Invertase from Bakers yeast, 30 units/mg solid

2) PBS solution

Preparation:
1) Mix 0.1 g dried invertase with 100mL of 0.05 M PBS solution (pH 7). Mix well.
This should provide enough stock solution for all groups. Keep refrigerated.
Note: Invertase must be stored in the refrigerator.
4.3 Reagents (used by the students)
DNS reagent for the analysis of reducing sugars
PBS solution
Concentrated HCl (37.3% , 11.9M)
5M KOH solution
1 g/L standard (+) Sucrose solution
Sucrose solutions: 50, 150 and 200 g/L
0.03 g/L invertase solution

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5. Experimental Procedure
5.1. Sucrose Assay by the Dinitrosalicylic (DNS) Colorimetric Method
Unlike other carbohydrates, sucrose is the only non-reducing common disaccharide.
Consequently, most tests for sugar detection utilizing such reagents as Benedict's
solution, Fehling's solution and DNS (3,5-dinitrosalicylic acid) solution result in negative
readings for sucrose. However, these methods can still be applied if sucrose is first
hydrolyzed in an acid solution to yield glucose and fructose. This method is a
straightforward modification of the original DNS method for glucose analysis. When
sucrose is hydrolyzed in an acid solution or by the action of the enzyme invertase, it
yields equimolar amounts of D(+)-glucose and D(-)-fructose. The reducing sugars
glucose and fructose both react with the DNS reagent, whereas sucrose does not.
5.2. Procedure Concerning the use of DNS
The reducing agent, DNS, when reacted, will go through a color change, from a light
orange/yellow to a nearly opaque blackish brown. The amount of color change will be
determined using a spectrophotometer, which will read the absorbance of light passed
through the sample at a wavelength of 550 nm. These absorbance readings will range
in intensity. However, these absorbance readings are meaningless unless they can be
calibrated to the amount of sucrose reacted. Therefore, in the first part of this lab, the
goal is to generate a calibration curve, which relates absorbance to g/L of sucrose.
Accuracy is of key importance because any error in the calibration curve will be
propagated throughout the experiment. For this reason, at least two separate 1 g/L
standard solutions will be required for reproducibility. The concentrated HCl is
assumed to hydrolyze all the sugar present. While the KOH would be used to stop the
reaction (the reaction should be completed by 5 minutes, so the base is just used to
return the pH to normal), the DNS is still required as a color change agent. In later
steps, the DNS will be used for both color change and to stop the reaction.
Calibration Curve:
1) Prepare at least 2 standard sucrose solutions (1.0 g/L). Dilute each standard to
make 0.0, 0.2, 0.4, 0.6, 0.8, and 0.9 g/L* solutions. Use DI-water for the dilutions;
2) Place 1 ml of each sample into test tubes;
3) Inside of the hood, add 1 drop (20 l), of concentrated HCl solution to each sucrose
solution;
4) Place test tube in an agitated water bath at 55 C for 5 minutes;
5) Remove test tubes from the agitated water bath and add 3 drops (0.05 ml), of 5 M
KOH solution to neutralize the acid;
6) Add 3 ml of DNS reagent to each test tube and mix. Place in the agitated water
bath for more than 5 minutes, and then, cool to room temperature;
7) Read absorbance at 550 nm;
8) Generate a calibration curve to correlate the absorbance to the amount of sucrose
hydrolyzed.

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* 50 g/L sucrose solution means that for every liter of water, 50g of sucrose would be
added, as opposed to placing 50g of sucrose in a volumetric flask and adding enough
water to reach the 1 liter mark.
5.3. Enzyme Kinetics of Invertase via Initial Rate Determination
In this experiment, the kinetics of invertase is investigated with the method of initial
reaction rates. In this method, the reaction rate can be easily correlated to the
conditions existing at the beginning of the reaction, since one has great control over the
initial condition. The enzyme-substrate mixture is allowed to react for a specified
amount of time. The rate of reaction can be monitored by measuring the amount of
reaction products, i.e., an equimolar mixture of glucose and fructose. The amount of
reducing sugars produced is determined colorimetrically with DNS.
You are required to do experimental procedures Part A and Part B, and perform a
Michaelis-Menten analysis of the data.
Part A Effect of Substrate Concentration:
In Part A, the enzyme invertase will be used to react with sucrose instead of HCl as was
done in the development of the calibration curve. As seen in Table 1, different sucrose
concentrations will be used, while the enzyme concentration remains the same. It is
important to note that the column marked as Initial Sucrose Concentration is the
concentration in the tube at reaction time = 0, just after the addition of the invertase,
while the absorbance measurement will give the final inverted sucrose concentration at
reaction time = 10 min.
It is also important to note that between tube G and tube H, the 50 g/L sucrose
solution changes to a 200 g/L sucrose solution, to achieve an Initial Sucrose
Concentration higher than 25 g/L. Each tube, A-N, will begin reacting with the addition
of 3mL of invertase solution and will stop reacting with the addition of the 3mL of DNS.
Each tube must react for exactly 10 minutes with the enzyme and 5 minutes with the
DNS. It is up to the students to develop the best scheme to allow this to occur. It is
also important to shake each tube well after addition of the invertase and DNS, to
ensure that both reactions are well mixed. During the first few minutes of color
development, students should check for banding of DNS. Banding will appear as
color development at the top of the tube, with no color development on the lower
portion of the tube. If this is caught early enough, a quick shake can ensure
appropriate mixing. If this is not caught until after the color development has taken
place, mixing will only dilute the color and result in an inaccurate reading.
1) Prepare a working enzyme solution (0.03 g/L) by diluting the 1 g/L stock solution
with 0.05 M PBS solution at pH 7**;
2) Prepare a set of sucrose solutions (in DI-water) at various concentrations. The
mixtures indicated in Table 1 are suggested;
3) Note starting time and add 0.03 g/l invertase solution to each test tube. See Table 1
for the required amounts for adding;

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4) The reaction mixture is allowed to incubate for exactly 10 minutes at 40 C. Because

the initial rate is being measured, the length of reaction must be controlled as
accurately as possible. Note: the reaction is stopped by the addition of DNS reagent
(next step);
5) Add 1 mL of reacted sample to 3mL of DNS reagent in a second set of test tubes.
Immerse the test tubes in an agitated water bath for 5 minutes at 55 C to develop
the characteristic red-brown color;
6) Measure the absorbance at 550 nm after cooling. Although the standard activity is
measured at 55 C, if not specifically requested, room temperature will be used for
convenience. Pre-incubate the solutions at the desired temperature for 5 minutes
and then quickly proceed to determine absorbance;
7) The amount of sucrose hydrolyzed is calculated from a calibration curve which
relates the measured absorbance to an equimolar mixture of glucose and fructose.
** In order to keep the absorbance readings under 3000 (a.u.), the working solution of
invertase will be at 0.03 g/L. Also, be sure that any time the invertase should be
diluted with phosphate buffered saline (PBS) solution, NOT WATER. PBS has the
same pH and ionic concentration that the enzyme would encounter in a cell. It ensures
that the enzyme is in a friendly environment and does not denature, or change activity.
Note: One international unit of activity is defined as the amount of enzyme needed to
hydrolyze 1 mole of sucrose to invert sugar per minute at pH = 4.5 and 55 C. A stock
solution of 1 g/L can be prepared first; dilute the stock solution 1:25 with a buffer to
obtain a working solution. If the enzyme preparation does not have the desired activity,
the concentration of the working solution may be adjusted accordingly with different
dilution factors to obtain a final solution of similar activity.
Table 1: Effect of Substrate Concentration
50 g/L (A-G)
0.03 g/L
PBS
Test
200 g/L (H-N)
Invertase
solution
Tube #
sucrose
solution
(mL)
solution (mL)
(mL)
A
0.0
3.0
3.0
B
0.5
2.5
3.0
C
1.0
2.0
3.0
D
1.5
1.5
3.0
E
2.0
1.0
3.0
F
2.5
0.5
3.0
G
3.0
0.0
3.0
H
0.3
2.7
3.0
I
0.5
2.5
3.0
J
1.0
2.0
3.0
K
1.5
1.5
3.0
L
2.0
1.0
3.0
M
2.5
0.5
3.0
N
3.0
0.0
3.0

Initial Sucrose
Concentration
(g/L)
0.00
4.17
8.30
12.50
16.67
20.83
25.00
10.00
16.70
33.30
50.00
66.70
83.30
100.00

Absorbance
(a.u.)

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Part B Enzyme Activity versus Enzyme Concentration:

The procedure in Part B is similar to that in Part A, except that the enzyme
concentration is varied while the sucrose concentration is held constant. However,
several controls are used in Part B. Tube A is a control for the DNS reaction with PBS
and sucrose. Tube I is a control for DNS reacting with PBS and invertase. Tube J is a
control to determine whether the DNS effectively stops the reaction. This is why,
immediately after the addition of the 3 mL of sucrose which starts the reaction, the 3 mL
of DNS are added to stop the reaction. If the reaction is successfully stopped, no color
development should be seen. If the DNS fails to stop the reaction, tube J should be
identical to tube H.
1) Prepare 3 mL enzyme solutions of various concentrations ranging from 0 to 0.03g/L.
See Table 2. Note that Test Tube #A is used to check the background absorbance
in the absence of enzyme. Test Tube #I is used to detect the residual reducing
sugar in the enzyme preparation. In addition, Test Tube #J is used to verify whether
the addition of DNS reagent indeed stops the hydrolysis reaction;
2) After the starting time is noted, 3 mL of 150 g/L sucrose solution is added in quick
succession to each of the test tubes marked #A - #J at the same fixed interval (e.g.,
10 seconds should be more than adequate). Add 3 mL of DNS reagent to the last
test tube marked #J immediately after the enzymatic reaction is initiated;
3) The reaction mixture is allowed to happen for exactly 10 minutes at 40 C. Because
the initial rate is being measured, the length of reaction must be controlled as
accurately as possible;
4) Add 1 mL of reacted sample to 3mL of DNS reagent in a second set of test tubes.
The addition of an alkaline DNS reagent should effectively stop the sucrose
hydrolysis reaction. This fact should be indicated by the result from Test Tube #J.
Shake each test tube well to mix the reagent;
5) Immerse the test tubes in water at 55 C for 5 minutes to develop the characteristic
red-brown color;
6) Measure the absorbance at 550 nm after cooling. The amount of sucrose
hydrolyzed is calculated from a calibration curve which relates the measured
absorbance to an equimolar mixture of glucose and fructose. Although the standard
activity is measured at 55 C, if not specifically requested, room temperature will be
used for convenience. Pre-incubate the solution at the desired temperature for 5
minutes.

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Table 2: Effect of Amount of Enzyme

Test
Tube #
A
B
C
D
E
F
G
H
I
J

0.03 g/L
Invertase
solution (mL)
0.0
0.1
0.5
1.0
1.5
2.0
2.5
3.0
3.0
3.0

PBS
solution
(mL)
3.0
2.9
2.5
2.0
1.5
1.0
0.5
0.0
3.0
0.0

150 g/L
Sucrose
solution (mL)
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
0.0
3.0

Initial Invertase
Concentration
(g/L)
0.0000
0.0005
0.0025
0.0050
0.0075
0.0100
0.0125
0.0150
0.0150
0.0150

Absorbance
(a.u.)