DEPARTMENT OF CHEMICAL ENGINEERING

N ATIONAL I NSTITUTE OF T ECHNOLOGY

WINTER SEMESTER 2023-24
CH6027E BIOPROCESS ENGINEERING

Assignment 1: Fermentation, Enzyme Kinetics

1 (a) Explain the characteristics of enzymes.

(b) Discuss the industrial applications of aerobic and anaerobic fermentations.
(c) Compare and contrast submerged and solid state fermentations.

2 (a) Distinguish between aseptic operation and containment. Why are they maintained in fermentation industries?
(b) List the objectives of using agitator in a fermenter.
(c) Explain the in detail various spargers used in fermenters.
(d) List any three elements used in different grades of steels that are used in construction of vessels for fermentation.
Explain their importance.

3 (a) Briefly describe synthetic medium, its advantages, disadvantages and applications in fermentation.
(b) Explain any four methods of maintaining the desired temperature in a laboratory-scale fermenter.

4 Consider the following reaction sequence:

1 k 2 3k k
E+S −
↽−
−⇀− (ES)1 −
↽−
−⇀− (ES)2 −−→ E + P
k−1 k−2

Develop a suitable rate expression for production formation, i.e. v = k3 (ES)2 by using

(a) the equilibrium approach

(b) the quasi-steady-state approach

5 Consider the reversible product-formation reaction in an enzyme-catalyzed bio-reaction as shown below:

1 k 2 k
E+S −
↽−
−⇀− ES −
↽−
−⇀− E+P
k−1 k−2

Develop a rate expression for product-formation using quasi-steady-state approximation and show that
Vs p V
dP Km [S] − K p [P]
v= =
dt 1 + K[S]m + [P]
Kp

k−1 +k2 k−1 +k2

where Km = k1 , Kp = k−2 , Vs = k2 [E0 ] and Vp = k−1 [E0 ]

6 Chymotrypsin is a serine protease that cleaves the amide linkages in proteins and peptides. It has a binding pocket
which is selective for the aromatic residues of amino acids. The reaction occurs by the reversible formation of a
Michaelis complex, followed by acylation to give a tetrahedral acylenzyme intermediate which is then converted to
acid. The elementary reaction steps can be presented as follows:
k1 k
E + RCO−X −
↽−
−⇀ 2
− RCO−X · E −−→ RCO−E + XH
k−1

k
RCO−E −−3→ RCOOH + E

(a) Develop a rate expression for product-formation using quasi-steady-state approximation for both the intermedi-
ates.
(b) What will be the rate when k2 ≫ k3 ? Explain its physical interpretation.

CH6027E BIOPROCESS ENGINEERING Assignment - 1 Page 1 of 2

 7 The hydration of carbon dioxide is catalyzed by carbonic anhydrase as follows:
E − +
H2 O + CO2 −
↽−
−⇀
− HCO3 + H

The following data were obtained for the forward and reverse reaction rates at pH 7.1 and an enzyme concentration of
2.8 × 10−9 M:

Hydration Dehydration
1
(mM)−1 s [CO2 ] , mM 1
(mM)−1 s HCO3− , mM
 
v, v,
36 1.25 95 2
20 2.5 45 5
12 5 29 10
6 20 25 15

v is the initial reaction rate at the given substrate concentration. Calculate the forward and reverse catalytic and
Michaelis constants.

8 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 measured. The results were as follows:

Time (min) Glucose Concentration (µmol/mL

1 0.05
5 0.23
10 0.38
15 0.52
30 1.03

(a) What is the activity of the β -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?

9 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 in mol/L Initial Reaction Rate in mol/L min

0.0032 0.111
0.0049 0.148
0.0062 0.143
0.008 0.166
0.0095 0.2

Evaluate the Michaelis-Menten kinetic parameters by employing

(a) the Langmuir plot

(b) the Lineweaver-Burk plot
(c) the Eadie-Hofstee plot

10 The KM value of an enzyme is known to be 0.01 mol/L. To measure the maximum reaction rate catalyzed by the
enzyme, you measured the initial rate of the reaction and found that 10 percent of the initial substrate was consumed·
in 5 minutes. The initial substrate concentration is 3.4 × 10−4 mol/L. Assume that the reaction can be expressed by
the Michaelis-Menten kinetics.

(a) What is the maximum reaction rate?

(b) What is the concentration of the substrate after 15 minutes?

End

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