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ENCH 535 Principles of Biochemical Engineering

Tutorial 5

1. The enzyme fumarase (E) has the following kinetic constants:

1 2
+ +
k-1

k1 = 109 M-1 s-1 k-1 = 4.4104 s-1 k2 = 103 s-1

a. What is the value of the half-saturation constant for this enzyme?


b. At an enzyme concentration of 10-6 M, what will be the initial rate of product formation at
a substrate concentration of 10-3 M?

2. Amyloglucosidase from Endomycopsis bispora is immobilized in polyacrylamide gel.


Activities of immobilized and soluble enzyme are compared at 80C. Initial rate data
measured at a fixed substrate concentration are listed below.
Enzyme activity, M/min mL
Time (min)
Soluble Enzyme Immobilized Enzyme
0 0.86 0.45
3 0.79 0.44
6 0.70 0.43
9 0.65 0.43
15 0.58 0.41
20 - 0.40
25 0.46 0.39
30 0.41 0.38
40 - 0.37

What is the half-life for each form of enzyme?

3. The intrinsic kinetic parameters of an immobilized -galactosidase from Aspergillus oryzae in


polyacrylamide gel are: vmax = 250 moles of hydrolyzed lactose / min g catalyst, and
KM = 58 mM. The diffusion coefficient of lactose in the gel is 5.110-2 cm2/min. Determine
the maximum size of catalyst particle that can be used to obtain a global effectiveness factor
greater than 90%. The density of the polyacrylamide gel is 1.1 g/mL.

4. An enzyme is embedded uniformly within spherical particles at a concentration E0 = 10 M.


When these particles are mixed with various substrate concentrations S0, and the initial
reaction rate v0 is measured, it is found that the rate is proportional to substrate concentration
for the conditions tested, with v0/S0 = 0.65 s-1. In a second preparation using the same
particles, it is determined that there is double the amount of active enzyme per particle (E0 =
20 M), and this time v0/S0 = 1.00 s-1.

a. Calculate the ratio of the two effectiveness factors, (E0 = 20 M)/(E0 = 10 M).
b. Calculate the ratio of the two Thiele moduli, (E0 = 20 M)/(E0 = 10 M).
Solution to Tutorial 5

1. Known: reaction mechanism; rate constants; and enzyme and substrate concentration.

Assumptions: same assumptions as in the derivation of the Michaelis-Menten equation,


either quasi-steady state or fast equilibrium.

Analysis:

First, we noted that the reaction mechanism is the same mechanism assumed in the derivation
of the Michaelis-Menten equation. Therefore the product formation rate will be given by:

dP vmax [ S ] k2 [ E0 ][ S ]
= =
dt km + [ S ] km + [ S ]

The half-saturation constant, km, will then be a function of the individual rate constants as
shown in the derivation of the Michaelis-Menten equation:

k1
km = , if the rapid equilibrium assumption is used, or
k1

k1 + k2
km = , if the quasi-steady state assumption is used.
k1

Therefore:

k1 4.4 104 s 1
km = = 9 1 1 = 4.4 10 5 M [rapid equilibrium]
k1 10 M s

k1 4.4 104 s 1 + 103 s 1


km = = = 4.5 10 5 M [quasi-steady state]
k1 109 M 1s 1

The initial rate of product formation is then:

dP k2 [ E0 ][ S ] 103 s 1 106 M 103 M


= = 5 3
= 9.57 10 4 M / s
dt km + [ S ] 4.5 10 M + 10 M

Where the value used for km was the one calculated using the quasi-steady state assumption.
2. Known: initial reaction rate data for free and immobilized enzymes after treatment at
80C for different lengths of time.

Assumptions: Enzymatic reaction follows Michaelis-Menten mechanism. Enzyme


degradation reaction follows first order kinetics.

Analysis:

Let E0 be initial amount of enzyme before thermal treatment, and E0(t) the amount of
active enzyme after time t operating at 80C.

Recalling that the maximum reaction rate is given by (as per Michaelis-Menten
mechanism):

v max = k 2 [ E 0 ]

Assuming a first order degradation kinetics for the enzyme thermal degradation:

[ E0 (t )] = [ E0 ] e kd t
Then the maximum reaction rate at different times can be written as:

vmax (t ) = k 2 [ E0 (t )] = k 2 [ E0 ] e kd t = vmax e kd t
Recall the Michaelis-Menten equation:

v max [ S ] k 2 [ E 0 ][ S ]
v= =
k m + [S ] k m + [S ]

At a constant substrate concentration [S], the reaction rate is directly proportional to vmax,
and we can use v instead of vmax:

v(t ) = v0 e kd t ln (v(t ) ) = ln (v0 ) k d t


From a plot of ln(v) vs t, we can obtain kd:

kd = 0.106 min-1 (free soluble enzyme)

kd = 0.025 min-1 (immobilized enzyme)

The half-life time is calculated as:

E (t ) ln 2
[ E0 (t )] = [ E0 ] e kd t ln 0 = k d t t1 / 2 =
E0 kd
t1/2 = 6.5 min (free soluble enzyme)

t1/2 = 33.8 min (immobilized enzyme)

3. Known: Intrinsic kinetic parameters, effective diffusivity of substrate into the gel, and
density of the gel support.

Assumptions: spherical particles.

Analysis:

We desire to know the maximum size of the particles to achieve a given effectiveness
factor. As we dont know the substrate concentration in the bulk fluid, we can assume that
we can adjust it at will. Such that we can assume it to be small enough, such that:

kM
=
SS
Then, we can approximate the effectiveness factor as:

3 1 1
h= 0.9
tanh( )
The Thiele modulus is given by:

mol
1/ 2
1 mmol g
250 1.1 3
min g 1000 mol
= R max = R
v cm
k M De mmol 2 cm
2
1L
58 5.1 10
L min 1000 cm 3
= 9.642 R
Then,

3 1 1
0.9 R 0.137 cm
9.642 R tanh(9.642 R ) 9.642 R
4. Known: ratio of initial reaction rate to initial bulk substrate concentration for two
different enzyme loads in the particles.

Assumptions: Michaelis-Menten kinetics

Analysis:

a) The effectiveness factor is given by:

v (observed with diffusion effects)


h=
v (No diffusion effects)

The reaction rate without diffusion limitation can be calculated using the Michaelis-
Menten expression with S equal to the bulk substrate concentration (S = S0):

vmax S 0 k [ E ]S
v (no diffusion) = = 2 0 0
kM + S0 kM + S0
Then,

k 2 [ E0 ]S 0
v (no diffusion, [ E0 ] = 10M) =
kM + S0

2k 2 [ E0 ]S 0
v (no diffusion, [ E0 ] = 20M) =
kM + S0

2k 2 [ E0 ]S 0
v (obs) 2
kM + S0 v (obs) 2
= =
k 2 [ E0 ]S 0 2v (obs)1
v (obs)1
kM + S0

v (obs)1 = 0.65 s 1 S 0 v (obs) 2 = 1.00 s 1 S 0

Then,
v (obs) 2 1.00 S 0
= = = 0.769
2v (obs)1 2 0.65S 0

This means that doubling the enzyme concentration in the particles resulted in reduction
of the effectiveness factor to 76.9% the original effectiveness factor.

b) The Thiele modulus for spherical particles is defined as:

vmax k [E ]
=R =R 2 0
k M De k M De

Then,

2k 2 [ E 0 ]
R
2 k M De
= = 2 = 1.414
1 k 2 [ E0 ]
R
k M De