Biochemical Engineering

7RCHE31

Dr. Sudhir Ranganath

Contact Hours/Week: 3 (Lecture) Credits: 3.0

CIE Marks: 50
Total Lecture Hours: 39 SEE Marks: 50
UNIT III
ENZYME CATALYZED REACTIONS
Mechanism & Michealis-Menten kinetics model
& estimation of kinetic parameters

Batch & continuous enzyme reactor kinetics

Multi-substrate enzyme reaction kinetics & estimation of

kinetic parameters

Kinetics of enzyme inhibition & estimation of kinetic

parameters

Effects of temperature, pH & shear

Immobilized enzymes & industrial applications

Diffusion in Enzymes Immobilized on a Porous Matrix
When enzymes are immobilized on internal pore surfaces of a porous matrix,
substrate diffuses through the tortuous pathway among the pores and reacts
with the enzyme immobilized on the pore surfaces.

Diffusion and reaction are simultaneous in this case.

Assumptions
Enzyme is uniformly distributed in a spherical
support particle

Reaction kinetics is expressed by Michaelis-

Menten kinetics

No partitioning of the substrate between the

exterior and the interior of the support.
Then, we write the following equation, stating that diffusion is equal to reaction
rate at steady state:

#$ [&] $ #[&] +, [&]

!" + = …… (1)
#($ ( #( -, .[&]
Diffusion in Enzymes Immobilized on a Porous Matrix
()
With boundary conditions ! = [!$ ] at & = ' and = 0 at & = 0, where,
(*

,- is the effective diffusivity of the substrate within the porous matrix.

Eq (1) can be written in dimensionless form by defining

the following dimensionless variables:

[)] * 23
!̅ = [)/ ]
, &̅ = 0
, 1= [)/ ]

(4 )̅ 6 ()̅ 04 73 )̅
(*̅ 4
+ 0 (*̅ = ̅
)/ 89 ):;
……. (2) or

< 6 !̅ 2 < !̅ 6
!̅
+ =>
<&̅ 6 ' <&̅ 1 + !̅⁄1

73 /23
where, > = ' = Thiele modulus
89
Diffusion in Enzymes Immobilized on a Porous Matrix
With boundary conditions of "̅ = 1 at %̅ = 1 and ' "̅⁄' %̅ = 0 at %̅ = 0, eq (2) can
be numerically solved to determine the substrate profile inside the matrix.
The rate of substrate consumption is equal to the rate
of substrate transfer through the external surface of the
support particle at steady state into the sphere:

. 1[3]
%) = *) = 4,- /0 15 at % = -

Under diffusion limitations, the rate per unit volume is

usually expressed in terms of the effectiveness factor
as follows:

7 [3 ]
%) = 6 : 8;[39 ] ……… (3)
8 9

The effectiveness factor is defined as the ratio of the reaction rate with
diffusion limitation (or diffusion rate) to the reaction rate with no diffusion
limitations.
Diffusion in Enzymes Immobilized on a Porous Matrix
The value of the effectiveness factor is a measure of the extent of diffusion
limitation.

For ! ≈ 1, conversion is limited by the reaction rate and diffusion limitations are
negligible.

The factor is a function of $ and %

as depicted.

For a zero-order reaction rate ie.,

(% → 0), ! ≈ 1 for a large range of
Thiele modulus values such as
1 < $ < 100.

The first order rate ie., (% → ∞), ! ≈ ($, %) and ! is approximated to the
following equation for high values of $.

- 0 0
!= [ − ] ……….. (4)
. 123. .
Diffusion in Enzymes Immobilized on a Porous Matrix
When internal diffusion limits the enzymatic reaction rate, the rate constant
values are not true intrinsic rate constants, but apparent values. To obtain true
intrinsic rate constants in immobilized enzymes, diffusion resistances should
be eliminated by using smaller particles, a high degree of turbulence
around the particle, and high substrate concentrations.

When designing an immobilized enzyme

system using a particular support, the main
variables are Vm and R, since the substrate
concentration, Km and De are fixed.

High E content leads to high E activity but

low !.

Low E content leads to lower E activity and

high !.
For maximum conversion rates, the particle size must be smaller than (#$ ≤
10 µm) and enzyme loading should be optimized.
Problem 15
Assume that for an enzyme immobilized on the surface of a nonporous
support material, the external mass transfer resistance for substrate is not
negligible as compared to the reaction rate. The enzyme is subject to
substrate inhibition.
(a) Are multiple states possible? Why or why not?
(b) Could the effectiveness factor be greater than one?
Solution to Problem 15a
At steady state, reaction rate = mass transfer rate.

#$ [&' ]
For substrate inhibition, we have: ! = & ,
= ./ &0 − &' =2
)*$ + &' + )'
&-

Solving graphically, we get the following graph:

389:; = <= = ∞
For [37 ] when the enzyme is
inhibited and <= has an
intermediate value, multiple A
steady states are possible.
5
B
Multiple steady states occur
6
as the enzyme changes from
being reaction controlled to
being diffusional controlled. <= is low
C

[34 ] [37 ]
Solution to Problem 15b
The effectiveness factor is defined as the ratio of the reaction rate with
diffusion limitation (or diffusion rate) to the reaction rate with no diffusion
limitations.

Yes. Diffusional limitations can decrease the substrate concentration such that
it is no longer inhibitory.

Thus the apparent reaction rate will be greater than the intrinsic reaction rate
for ["# ] less than ["% ].
Problem 16
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:

Bead Dia, Dp (cm) 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
Rate, v (mg/L.h) 200 198 180 140 100 70 50 30

(a) Determine the effectiveness factor for Dp = 0.5 cm & Dp = 0.7 cm.
(b) The following data were obtained for Dp =0.5 cm at different bulk uric
acid concentrations. Assuming negligible liquid film resistance, calculate
Vm and Km for the enzyme. Assume no substrate or product inhibition.

S0 (mg UA/L) 10 25 50 100 200 250

Rate, v ( mg UA/L.h) 10 20 30 40 45 46
Problem 16: Solution to (a)

Reaction rate with diffusion limitation

Effectiveness Factor, η =
Reaction rate without diffusion limitation

Maximum reaction rate 89 :; = <. > ?@,

DE
BCC
F.G
A= DE = <. >
HCC
F.G

89 :; = <. I ?@,

DE
JC
F.G
A= DE = <. K>
HCC
F.G
Problem 16: Solution to (b)
Since there is no film resistance, Sb = Ss

Need to determine rate without diffusional limitation from effectiveness factor

!"#$ "# 12 = 0.5 6/

!"#$ %&#ℎ()# *&++),&(- .&/&#"#&(- =
η "# 12 = 0.5 6/

S0 (mg UA/L) 10 25 50 100 200 250

Rate, v ( mg UA/L.h) 10 20 30 40 45 46
Rate without 20 40 60 80 90 92
diffusion limitation,
(mg UA/L.h)

Plot 1/v vs 1/S to determine Vm and Km

Problem 16: Solution to (b)

!
"#
= 0.0088 so, () = 113.63 ./ 01⁄2. ℎ

Similarly, 4) = 46.69 ./ 01⁄2

Problem 17
Two enzymes are both immobilized on the same flat, nonporous surface. For
enzyme A the substrate is S1 and for enzyme B the substrate is S2. The
product of the first reaction is S2. That is

S1 S2 P
EA EB

(a) Figure below depicts the rate of the first reaction on the surface as a
function of local concentrations of S1. If the bulk concentration of S1 is 100
mg/L and the mass transfer coefficient is 4 x 10-5 cm/s, what is the rate of
consumption of S1 for a 1 cm2 surface? What is the surface concentration
of S1?
Problem 17
(b) The rate of the second reaction is:

" #$ " ' ("* #$ +,-./01

− = =
"% "% 2* 3#$ +,-./01

where Km = 5 x 10-3 mg/cm3 and V”m = 4 x 10-6 mg/cm3s. The bulk

concentration of S2,bulk is maintained at 5 x 10-3 mg/cm3 and the mass transfer
coefficient is the same for S1 and S2. Calculate S2,surface and the rate of
formation of P (assuming all stoichiometric coefficients are one).
Problem 17: Solution to (a)
Given:
Sbulk = 100 mg/L
kL = 4 x 10-5 cm/s

To solve this graphically, we need to get the rate of diffusion at Sbulk = 100
mg/mL and S1 = 0, then ! = #$ %&'() − %+ becomes ! = #$ %&'()

Substituting Sbulk and kL, we get v = 4 x 10-6 mg/cm2s

The diffusion curve is drawn with x = 100 and y = 4. The intersection gives S1
and v which are S1 = 20 mg/L and v = 3.2 x 10-6 mg/cm2s
Problem 17: Solution to (b)
Given:
V”m = 4 x 10-6 mg/cm2s
Km = 5 x 10-3 mg/cm3
kL = 4 x 10-5 cm/s

" #$ "' ("* #$,,-./012

− = =
"% "% 3* + #$,,-./012

= 56%7 89 :;<<=> − 56%7 89 "?99;:?8@ 6A6>

" #$ "' ("* #$,,-./012

− = = = B − CD #$,,-./012 − #$,E-FG
"% "% 3* + #$,,-./012

4 x 10LM #$,,-./012 LM − 4 x 10LS #

= 3.2 x 10 $,,-./012 − 0.005
5 x 10LO + #$,,-./012

Solving, we get S2,surface = 0.0095 mg/cm3

and dP/dt = 3.02 x 10-6 mg/cm2.s

Industrial Scale Production of Enzymes
Industrial Scale Production of Enzymes
Large-scale production via fermentation
Usually over-producing strains are cultivated in fermenters
Proteases from Bacillus, Aspergillus, Rhizopus, etc
Pectinases from A. Niger
Lactases from Yeasts, Aspergillus
Lipases from Yeasts and Fungi
Glucose Isomerase from Flavobacterium
Cells & media separated (Centrifugation/Filtration)
Intracellular or extracellular Enzyme
Cell disruption & removal of DNA/RNA and cell debris
Protein precipitation
Ultrafiltration of desired Enzyme
Chromatographic separation
Crystallization & Drying
Applications of Enzymes
Name Source Application

Amylase Bacillus subtillus, A. niger Starch hydrolysis, Glucose production

Glucoamylase A. niger Saccharification of starch

Trypsin Animal pancreas Meat tendering, cell detachment for culturing

Papain Papaya Meat tendering

Glucose isomerase Flavobacterium Isomerization of glucose to fructose

Penicillinase B. subtillus Degradation of penicillin

Lignase Fungal Biopulping of wood

Lipase Rhizopus Hydrolysis of lipids, digestive aid

Invertase S.cerevisiae Hydrolysis of sucrose for further fermentation

Cellulase Trichoderma viridae (Fungus) Cellulose hydrolysis

Luciferase Photonis pyralis (Firefly) Tumor diagnosis

Pectinase A. niger Clarification of fruit juices

Lysozyme Staphylococci Anti-bacterial agent

Urease Pea seeds Artificial kidney for dialysis

Enzymes as Biosensors
Luciferase Assay
Done in live animals
Luciferase gene is obtained from Firefly
Luciferase gene expressing cancer cells are
developed
Cancer cells are injected into animals
Luciferin (Substrate) is injected into the animal
Luciferin binds to Luciferase enzyme inside the
cancer cells
Reaction results in bioluminescence
Tumor volume and progression are quantified
Thank you