TURLA, NYGEN KEITH LOUISE D.

(18-05544)
BS Chemical Engineering
ChE 415 BIOCHEMICAL ENGINEERING

1. Kinetic Parameters Evaluation

Eadie (1942) measured the initial reaction rate of hydrolysis of acetylcholine (substrate) by
dog serum (source of enzyme) and obtained the following data:

Substrate Concentration Initial Reaction Rate

mol/L mol/L min
0.0032 0.111
0.0049 0.148
0.0062 0.143
0.0080 0.166
0.0095 0.200

Evaluate the Michaelis-Menten kinetic parameters by employing (a) the Langmuir plot, (b) the
Lineweaver-Burk plot, (c) the Eadie-Hotsfee plot, and (d) non-linear regression procedure.

a. Langmuir plot

CS 1 K CS
= CS + M Plot C S vs
r r max r max r

Cs Cs/r
0.0032 0.028829
0.0049 0.033108
0.0062 0.043357
0.008 0.048193
0.0095 0.0475

Langmuir Plot
0.06

0.05

0.04
y = 3.3133x + 0.0191
Cs/r

0.03 R² = 0.8837

0.02

0.01

0
0 0.002 0.004 0.006 0.008 0.01
Cs

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 1 KM
= 3.3133 = 0.0191
r max r max

1 K M = (0.0191)r max = (0.0191)(0.3018)

r max =
3.3133
K M  0.0058 mol/L
r max  0.3018 mol/L min

b. Lineweaver-Burk Plot

1 KM 1 1 1 1
= + Plot vs
r r max CS r max CS r

1/Cs 1/r
312.5 9.009009
204.0816 6.756757
161.2903 6.993007
125 6.024096
105.2632 5

Lineweaver-Burk Plot
10
9
8
7
6 y = 0.0172x + 3.6342
1/r

5 R² = 0.9146
4
3
2
1
0
0 50 100 150 200 250 300 350
1/Cs

1 KM
= 3.6342 = 0.0172
r max r max

1 K M = (0.0172)r max = (0.0172)(0.2752)

r max =
3.6342
K M  0.0047 mol/L
r max  0.2752 mol/L min

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c. Eadie-Hotsfee Plot

r r
r = −K M + r max Plot vs r
CS CS

r/Cs r
34.6875 0.111
30.20408 0.148
23.06452 0.143
20.75 0.166
21.05263 0.2

Eadie-Hotsfee Plot
0.25

0.2

0.15
r

0.1 y = -0.0043x + 0.2645

R² = 0.6584
0.05

0
0 5 10 15 20 25 30 35 40
r/Cs

r max = 0.2645 mol/L min

− K M = −0.0043

K M = 0.0043 mol/L

d. Nonlinear regression

r maxCS
r=
K M + CS

Cs rexp
0.0032 0.111
0.0049 0.148
0.0062 0.143
0.0080 0.166
0.0095 0.200

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Assumed K M = 0.005

Assumed rmax = 0.300

Cs rexp rcalc (rexp-rcalc)2

0.0032 0.111 0.107969 9.18642E-06
0.0049 0.148 0.138922 8.2403E-05
0.0062 0.143 0.156647 0.000186248
0.008 0.166 0.175654 9.31963E-05
0.0095 0.2 0.188064 0.000142472
rmean = 0.1536  (rexp-rcalc)2 = 5.13505E-4

Nonlinear Regression
0.25

0.2

0.15
r

rexp
0.1
rcalc
0.05

0
0 0.002 0.004 0.006 0.008 0.01
Cs

Using Excel Solver

Set Objective:  (rexp-rcalc)2 = 5.13505E-4

To: min
By changing variables: K M and rmax

Thus:

rmax = 0.3018

K M = 0.0057

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Summary

Kinetic Parameters
Michaelis constant Maximum Reaction Rate
Plot Type
KM r max
(mol/L) (mol/L min)
Langmuir 0.0058 0.3018
Lineweaver-Burk 0.0047 0.2752
Eadie-Hotsfee 0.0043 0.2645
Nonlinear Regression 0.0057 0.3018

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 2. Deriving rate equation

Suppose that the following sequence describes the reactions of two different substrates
catalyzed by one enzyme:

a. Derive the rate equation by making the Michaelis-Menten assumption.

b. If the assumption of S1 is much higher than that of S 2 , how can the rate equation be
simplified? Meaning k 3  k 4
Hints:
• Set up rate equation based on the rate limiting step.
• Additional 2 relations from equilibrium reactions.
• Setup enzyme conservation.
• One by one, aim to express rate equation in terms of C S1 , CS 2 , Vm , and rate
constants noting that Vm = k 5 (E 0 ) .

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 3.

In order to measure the enzyme activity and the initial rate of reaction, 5 mL of cellobiose (100
mumol/mL) and 44 mL of buffer solution were placed in a stirred vessel. The reaction was
initiated by adding 1 mL of enzyme (beta-glucosidase) solution which contained 0.1 mg of
protein per mL. At 1, 5, 10, 15, and 30 minutes, 0.1 mL of sample was removed from the
reaction mixture and its glucose content was removed. The results were as follows:

Time Glucose Concentration

Min μmol/mL
1 0.05
5 0.23
10 0.38
15 0.52
30 1.03

a. What is the activity of the beta-glucosidase in units/mL of enzyme solution and in

units/mg protein? A unit is defined as the enzyme activity which can produce 1 mumol
of product per minute.
b. What is the initial rate of reaction? μmol/mL min

Note:
• mumol = μmol
• 1 unit activity = 1 μmol product/min

Hints:
• Initial rate is the slope.
• To compute activity, how much enzyme was used? Relate to the initial reaction rate.

1.2

1 y = 0.033x + 0.0391
Glucose concentration

0.8
μmol/mL

0.6

0.4

0.2

0
0 5 10 15 20 25 30 35
time (in min)

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 4. Batch Reactor and CSTR

A carbohydrate (S) decomposes in the presence of an enzyme (E). The Michaelis-Menten

kinetic parameters were found to be as follows:
K M = 200 mol / m 3
r max = 100 mol / m 3 min
a. Calculate the change of substrate concentration with time in a batch reactor. The initial
substrate concentration is 300 mol / m 3 .
b. Chemostat (continuously stirred-tank reactor) runs with various flow rates were carried
out. If the inlet substrate concentration is 300 mol / m 3 and the flow rate is 100
cm 3 / min , what is the steady-state substrate concentration of the outlet? The reactor
volume is 300 cm 3 . Assume that the enzyme concentration in the reactor is constant
so that the same kinetic parameters can be used. (Hint: Generation is zero in the
overall mass balance for CSTR.)

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