Tangonan, Visitacion D.

February 27, 2019

Biochemical Engineering

2.1. In order to measure the enzyme activity and the initial rate of reaction, 5 mL of cellobiose
(100mumol/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 measured. 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 ß-glucosidase in units/mL of enzyme solution and in umts/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 the reaction?

Solution:

Total Volume = cellobiose + buffer solution + ß-glucosidase

= (5 + 44 + 1) mL
= 50 mL

The equation obtained from the graph below is

y = 0.033x + 0.039

m = 0.033 mumol/mL min

1.65 𝑚𝑢𝑚𝑜𝑙/𝑚𝑖𝑛
a. Activity of the ß-glucosidase (units/mL) = 1 𝑚𝐿 𝑜𝑓 𝑒𝑛𝑧𝑦𝑚𝑒

= 1.65 unilts/mL of enzyme

Activity of the ß-glucosidase (units/mg) = r × total volume

= 50 × 0.033
1.65 𝑚𝑢𝑚𝑜𝑙/𝑚𝑖𝑛
= 𝑚𝑔
0.1 × 0.1 mL
𝑚𝑙

= 165 units/mg protein.

Tangonan, Visitacion D. February 27, 2019
Biochemical Engineering

b. Based on the graph below, the initial rate is 0.033 mumol/mL.

Concentration vs. Time Graph

1.2

y = 0.033x + 0.0391
1 R² = 0.9973
Concentration (µmol/mL)

0.8

0.6

0.4

0.2

0
0 5 10 15 20 25 30 35
Time (min)

2.5. Eadie (1942) measured the initial reaction rate of hydrolysis of acetylcholme (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-Hofstee plot
d) the non-linear regression procedure.
Tangonan, Visitacion D. February 27, 2019
Biochemical Engineering

Solution:
A. Langmuir Plot

Langmuir Plot
Cs Cs/R 0.06
y = 3.3133x + 0.0191
0.0032 0.028829 0.05 R² = 0.8837
0.0049 0.033108 0.04

Cs/R
0.0062 0.043357 0.03

0.008 0.048193 0.02

0.0095 0.0475 0.01

0
0 0.002 0.004 0.006 0.008 0.01
Cs

y = 3.313305715x + 0.01912464593
1
m= = 3.313305715
rmax
Thus,
1
rmax =
3.313305715
= 0.301813381 mol L • min
KM
b= = 0.01912464593
rmax
Thus,
K M = b × rmax
= (0.01912464593)(0.301813381)
= 5.772074049 × 10 -3 mol L
Tangonan, Visitacion D. February 27, 2019
Biochemical Engineering

B. Lineweaver-Burk Plot

1/Cs 1/V Lineweaver Burk Equation Graph

312.5 9.009009 10
y = 0.0172x + 3.6342
204.0816 6.756757 8
R² = 0.9146
161.2903 6.993007
6
125 6.024096

1/V
105.2632 5 4

0
0 50 100 150 200 250 300 350
I/Cs

1 1 K 1
  m
V Vmax Vmax C s

y = 0.01719105838x + 3.63421308
1
b= = 3.63421308
rmax
Thus,
1
rmax =
3.63421308
= 0.2751627321 mol L • min
KM
m= = 0.01719105838
rmax
Thus,
K M = m × rmax
= (0.01719105838)(0.2751627321)
= 4.730338591 × 10 -3 mol
L
Tangonan, Visitacion D. February 27, 2019
Biochemical Engineering

C. Eadie-Hofstee Plot

Eadie-Hosftee Plot
R/Cs R 0.25
34.6875 0.111
0.2
30.20408 0.148
23.06452 0.143 R
0.15
20.75 0.166 0.1 y = -0.0043x + 0.2645
21.05263 0.2 R² = 0.6584
0.05

0
0 10 20 30 40
R/Cs

r
r = -K M + rmax
Cs
y= m x +b

y = -4.273141382 × 10 -3 x + 0.2644954792
b = rmax = 0.2644954792 mol L • min

m = -K M = 4.273141382 × 10 -3 mol L