Diffusion effects in

enzymes immobilized
in a porous matrix.
Introduction
Uricase enzyme
Also known as urate oxidase (UO)
An enzyme not found in humans
Exists as a tetramer of identical
subunits
A unique oxidase; does not require
a metal atom or an organic co-factor
for cataysis
Introduction
Calcium alginate
Water-insoluble, gelatinous, and
cream-colored substance
Used for the entrapment of enzymes
and in forming artificial seeds in
plant tissue culture
Alginate refer to salts of alginic
acid
Introduction
Introduction
Problem
Uric acid is degraded by uricase enzyme
immobilized in porous Ca-alginate beads.
Experiments conducted with different bead sizes
result in the following rate data:

a) Determine the effectiveness factor for particle

sizes Dp = 0.5 cm and Dp = 0.7 cm.
Problem
b) The following data were obtained for Dp = 0.5
cm at different bulk uric acid concentra- tions.
Assuming negligible liquid film resistance,
calculate Vm and Ks for the enzyme. Assume no
substrate or product inhibition.
Solution:
Given:
Part A

Required:
Effectiveness factor at Dp=0.5cm and Dp=0.7cm

Working equation:
Solution:
Part A
250

200

Rate, v (mg/l.h)
150

100

50

0
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
Bead diameter, Dp (cm)

Smaller particles have smaller diffusional limitation

Reaction rate with no diffusional limitation = 200mg/L-h

Solution:
Part A
Solution: 1.2

Part A
1

0.8

,efficiency
0.6

0.4

0.2

0
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
Bead diameter
Solution:
Part B Assumptions:
Michaelis-Menten kinetics
Resistance due to the liquid film is
negligible
Bulk concentration = Interfacial substrate
concentration
Solution:
Part B In Michaelis-Menten Kinetics:
Vm reaction rate at which all enzyme
molecules are saturated with the substrate

Ks the concentration of the substrate at

which the rate of the enzyme is half the
maximum rate
 Solution: Michaelis-Menten plot
Part B 50

45

40

35

30
S0 v
v
25
(mg UA/l) (mg UA/l-hr)
20
10 10 15
25 20 10
50 30 5

100 40 0

200 45
0 50 100 150 200 250 300

S0
250 46
 Solution:
0.1200

Part B 0.1000

0.0800

1/v
0.0600
1/S0 1/v
(mg UA/l) (mg UA/l-hr)
0.0400
0.1000 0.1000
0.0400 0.0500 0.0200
y = 0.8217x + 0.0175
0.0200 0.0333 R = 0.9995
0.0100 0.0250 0.0000

0.0050 0.0222
0.0000 0.0200 0.0400 0.0600 0.0800 0.1000 0.1200

0.0040 0.0217 1/S0

Solution:
Part B From linear regression:

y = 0.8217x + 0.0175

where: y-intercept = 0.0175 = 1/VM

Solving,
VM = 57.14 mg UA/l-hr
Solution:
Part B From linear regression:

y = 0.8217x + 0.0175

where: slope = 0.8217 = KM /VM

Solving,
KM = (57.14 mg UA/l-hr)*(0.8217)
KM = 46.95 mg UA/l
Thanks!