Chapter 3: Enzymes

Prof. Zakaria Al-Qodah

Department of Chemical Engineering

Faculty of Engineering Technology

2nd Semester (2019-2020)

Presentation Outline: Lectures 1-4

 Introduction to Enzymes

 Kinetics of Enzyme-Catalyzed Reactions

 Effects of Environmental Conditions on Kinetics

 Inhibition of Enzyme Catalyzed Reactions

 Effect of pH and temperature

3.1 Introduction
 Enzymes are proteins with Mu. M. (15000 - several millions
daltons)

 Act as catalysts for the majority of cellular reactions

 Ribozymes are some RNA that can catalyzed some cellular reactions

 More than 2000 enzymes are known

 Enzymes are named by adding the suffix – ase to the end of the
substrate, such as urease

 Some enzymes need cofactors or coenzymes to become active

holoenzyme = apoenzyme + cofactor or/and coenzyme

 Isoenzymes are enzymes of different molecular forms but catalyzed

the same reaction
Classification of enzymes
Cofactors and coenzymes
3.2 How enzymes work
In multi-substrate reactions the enzyme has the following effects:

 Proximity effect Orbital steering

 Induced Fit


 3.3 Enzyme Kinetics
The first model for single-substrate-enzyme catalyzed reaction was
developed by Henri 1902 and Michaelis -Menten 1913 .

Simple enzyme-catalyzed reaction are named as M-M or saturation

kinetics, which is based on batch constant volume reactors with [So]
and [Eo] are known

An enzyme solution has fixed number of active sites

At high [So] all the active sites are busy or the enzyme is saturated

 Saturation kinetics involves two steps: The 1st step is reversible, the
2nd is irreversible (no product accumulation)

 3.1
3.3.2 Mechanistic Models for Simple Enzyme Kinetics
There are two major approaches to develop models for simple enzyme
catalyzed reactions:

 The rapid equilibrium approach (the first step in equation 3.1)

 The quasi-steady-state approach [ES] becomes constant)
 Both approaches share the following first three equations:

 Rate of product formation: (3.2)

 The rate of variation of [ES]: (3.3)

 Enzyme mass balance: (3.4)

 After this point the two approaches will differ in their assumptions
The rapid equilibrium approach
Henri and M. M. assumed a rapid equilibrium between the enzyme and
substrate to form an [ES] complex

 The equilibrium constant is: (3.5)

 Since [E] = [E0] - [ES] if enzyme is conserved, then:

(3.6)

(3.7)
where, which is the dissociation constant of the ES.
Substituting Eq. 3.7 into Eq. 3.2 yields:

(3.8)
where Vm = k2[E0].
Effect of [S] on the rate of enzyme catalyzed reaction ν

 Vm is the maximum rate, at very high [S] called M.M. constant

 At this value the addition of S will not increase the rate


 K m is M.M. constant

Km
 Low value of Indicates
that the enzyme has high affinity
toward the substrate

If we use a rate

v = ½ Vm =
½ Vm K m
This gives which equals
The quasi-steady-state approximation approach

 In many cases the previous assumption is not valid

 G. E. Briggs and J. B. S. Haldane first proposed using the quasi-steady-

state assumption based on experimental conditions where [E0] << [S0].

 As seen [ES] = constant or d[ES]/dt = 0.

 By applying the quasi-steady-state assumption to Eq. 3.3, we find:

 (3.9)

 Substituting [E] = [Eo] – [ES] from Eq. 3.4 in Eq. 3.9 yields:

 (3.10)

 Solving Eq. 3.10 for [ES]:

 (3.11)

 Substituting Eq. 3.11 into Eq. 3.2 yields

 or (3.12)

 Where
 Since Km results from the more general derivation, it will be used
3.3.3. Determining Rate Parameters for M. M Kinetics

 The determination of values for Km and Vm with high precision can be

difficult.
 Experimental data are obtained from initial-rate experiments

 The product (or substrate concentration) is plotted against time.

 The initial slope of this curve is estimated:

 Many such experiments can be used to generate many pairs of v and [S] data

 These could be plotted as in Fig. 3.3, but the accurate determination of Km

from such a plot is very difficult

 Consequently, other methods of analyzing such data have been suggested.

Double-reciprocal plot (Lineweaver–Burk plot).
 Equation 3.12 can be linearized in double-reciprocal form:

(3.13)

 The plot gives good estimates on Vm, but not necessarily on Km

 Data points at low values of [S] influence the slope and intercept more
than those at high [S]
Eadie–Hofstee plot

Vm [ S ]
From equation 12, v
Km  [ S ]

• Eq. 14 is obtained:

•
(14)
plot v versus v/[S] gives a line
of slope –Km and y-axis
intercept of Vm

Can be subject to large errors since both coordinates contain

v, but less bias on points at low [S]
 Hanes–Woolf plot

Rearrangement of equation 12 yields

(15)

slope
intercept

 This plot is used to determine

Vm more accurately.
Batch kinetics
 by integration of Eq. 3.12 yield:

 (3.16)

 Rearranging Eq. 3.16 gives:

 (3.17)

 A plot of 1/t ln[S0]/[S] versus {[S0] - [S]}/t results in a line

 Slope of -1/Km and intercept of Vm/Km.

 Enzyme concentration
 Purified enzyme preparations [E0] can be expressed in terms of mol/l or g/l.

 The concentration of enzyme in crude preparation, is in terms of “units.”

 A “unit” is the amount of enzyme that gives a predetermined amount of catalytic

activity under specific conditions.

 One unit would be formation of one μmol product per minute at a specified
pH and temperature with a substrate concentration much greater than the
value of Km.

 The specific activity is the number of units of activity per amount of protein.

 Only enzyme that remains catalytically active will be measured.

 The denatured enzyme will have no activity.

Interpretation of Km and Vm
 Km (or K-m) is an intrinsic parameter, Vm is not

 Km is solely a function of rate parameters and is expected to

change with temperature or pH.

 However, Vm is a function of the rate parameter k2 and the initial

enzyme level, [E0].

 As [E0] changes, so does Vm.

 k2 can be readily calculated if [E0] is known

 Allosteric enzymes
 Some enzymes have more than one substrate binding site.

 The binding of one substrate to the enzyme facilitates binding of other

substrate molecules.

 This behavior is known as allostery or cooperative binding, and regulatory

enzymes show this behavior.
Allosteric enzymes
 The rate expression in this case is:

 (3.18)

 where n = cooperativity coefficient and n > 1 indicates positive

cooperativity.

 The cooperativity coefficient can be determined by rearranging Eq.

3.18 as

 (3.19)

 By plotting lnv/(Vm - v) versus ln[S] as:

Inhibited enzyme kinetic
 Certain compound may bind to enzymes and reduce their activities ~
enzyme inhibitors

 Enzyme inhibitions may be irreversible ( heavy metal: lead, cadium,

mercury–form a stable complex with enzyme and reduce enzyme
activity) or reversible (EDTA and citrate)

 Three major class of reversible enzyme inhibition- competitive,

noncompetitive and uncompetitive inhibitions.

 Competitive- Inhibitor analogus to the substrate compete with

substrate for active site of the enzyme
 Competitive Inhibition
 The competitive enzyme inhibition scheme can be described
as:

 Assuming rapid equilibrium and with the definition of:

(3.21)
 Competitive Inhibition
 We can develop the following equation for the rate of
enzymatic conversion:

 (3.22)

 or (3.23)

 where

 Competitive inhibition can be overcome by high concentrations of

substrate.
Competitive Inhibition

The net effect of competitive inhibition is an increased value of

Km, app and, therefore, reduced reaction rate.
Noncompetitive Inhibition

 Noncompetitive inhibitors bind on site other than the active site,

reduce enzyme affinity to the substrate
 Can be described by Eq. 3.24:

(3.24)
 With the definition of:

 (3.25)

 We can develop the following rate equation:

 (3.26)

 or
 (3.27)

 where
 Noncompetitive Inhibition
 The net effect of noncompetitive inhibition is a reduction in Vm.

 High substrate concentrations would not overcome this noncompetitive

inhibition.

 Other reagents need to be added to block binding of the inhibitor to the

enzyme.

 In some forms of noncompetitive inhibition Vm is reduced and K-m is

increased. This occurs if the complex ESI can form product.
Uncompetitive Inhibition
Inhibitors binds to the ES complex only and have no affinity for
the enzyme itself.

Reaction scheme is:

 (3.28)


Uncompetitive Inhibition

 With the definition of

 (3.29)

 We can develop the following equation for the rate of reaction:

 (3.30)

 (3.31)
Uncompetitive Inhibition
 The net effect of uncompetitive inhibition is a reduction Vm and K-m

 Reduction in Vm has a more pronounced effect than the reduction in K-m

 The net result is a reduction in reaction rate.

Substrate inhibition
 High substrate concentrations may cause inhibition in some
enzymatic reaction

 (3.33)
Substrate Inhibition

 With the definition of

 (3.33)

 the assumption of rapid equilibrium yields:

 (3.34)

 A double-reciprocal plot
Substrate Inhibition
 At low substrate concentrations, [S]2/KS1 << 1, and inhibition effect
is not observed:
 (3.35)

 or (3.36)

 At high substrate concentrations, K-m /[S] << 1, and inhibition is dominant.

 (3.37)

 or (3.38)

 The substrate concentration resulting in the maximum reaction rate can

be determined by setting dV/d[S] = 0.
 (3.39)
Summery of inhibited enzyme kinetics
 3.3. 5 pH effect on enzyme kinetics


 Different types of enzyme (acidic, neutral, basic enzymes)

Each enzyme has an optimum pH

 The pH optimum for an enzyme is usually determined experimentally

 Effect of pH
 Certain enzymes have ionic groups on their active sites, and these
ionic groups must be in a suitable form (acid or base) to function..

 Variations in the pH cause changes in the ionic form of the active site
and changes in the activity of the enzyme and hence ν

 Changes in pH may also alter the three-dimensional shape of the

enzyme.

 Enzymes are only active over a certain pH range

 The pH of the medium may affect the maximum reaction rate, Km, and
the stability of the enzyme

 The substrate may contain ionic groups, and the pH of the medium
affects the affinity of the substrate to the enzyme.
 Effect of pH
 The following scheme may be used to describe pH dependence of the
enzymatic reaction rate for ionizing enzymes.

 (3.40) (3.41)

 We can derive the following rate expression:

 (3.41) or (3.42)


 Effect of pH

Where

 Upon differentiating equation 3.41 and put it equal to zero will

obtain pH optimum of the enzyme is between pK1 and pK2.
 = ½ (pK1 + pK2)

For ionizing substrate, the following scheme and rate expression can
be developed

 (3.44) (3.45)
 3.3. 5 Temperature effect on enzyme kinetics


 The rate of enzyme-catalyzed reactions increases with temperature

up to a certain limit.

 Then enzyme activity decreases with temperature because of enzyme

denaturation.
 The pH optimum for an enzyme is usually determined experimentally
 3.3. 5 Temperature effect on enzyme kinetics
 The ascending part of Fig. 3.15 is known as temperature activation

The rate varies according to the Arrhenius equation in this region.

 (3.46a, b)

A plot of ln v versus 1/T results in a line of slope -Ea/R.

The descending part of the Figure is known as temperature inactivation

or thermal denaturation.

The kinetics of thermal denaturation can be expressed as:

 (3.47) (3.48)

where [E0] is the initial enzyme conc., kd is the denaturation constant

 3.3. 5 Temperature effect on enzyme kinetics
kd also varies with temperature according to the Arrhenius equation

 (3.49)

 (3.50)

 The activation energies of enzyme-catalyzed reactions are within the 4

to 20 kcal/g mol range (mostly about 11 kcal/g mol).
 Deactivation energies Ed vary between 40 and 130 kcal/g mol (mostly
about 70 kcal/g mol).
That is, enzyme denaturation by temperature is much faster than
enzyme activation.
A rise in temperature from 30o to 40o C results in a 1.8-fold increase in
enzyme activity, but a 41-fold increase in enzyme denaturation.
Variations in temperature may affect both Vm and Km .
Insoluble enzymes
 Enzymes are often used to attack large, insoluble substrates such
as wood chips.

 Enzyme access to the reaction site on these biopolymers is often

limited by enzyme diffusion.

 The number of potential reactive sites exceeds the number of

enzyme molecules.

 This situation is opposite that of the typical situation with soluble

substrates,

 (3.51a) where (3.51b)

 and (3.51c)

 The previous equation assumes slow binding of enzyme (i.e., [E]<< [E0]),
Presentation Outline: Lectures 5-8
 Immobilized Enzyme Systems

 Methods of Enzyme Immobilization

 Diffusional Limitations:

 Effectiveness of Immobilized Enzymes

 Overview of Industrial and Medicinal Enzymes

 Biocatalyst Immobilization - Definition
The containment of enzyme solution within a confined space with
retention of its catalytic activity for the purposes of:

 Increasing enzyme concentration

 Retaining and re-using enzyme in processing equipment.

 The term "immobilization" is applicable also for cells, either

microbial, plant or animal cells and even for cell organelles.

 The first discovery of this process was by Nelson and Griffin 1961,
when they find that invertase is immobilized (adsorbed) on charcoal
and able to hydrolyze sucrose.

 There are many advantages that accompany immobilized enzymes

and many methods for immobilization.
 Immobilized Enzyme Systems

Advantages

 Reduce costs of operation compared to free enzyme systems

where additional separation and purification steps are needed.

 Some immobilization methods can increase enzyme activity.

 A model system to study enzyme action in membrane-bound

enzymes that occur in the cell.

 These facts suggest that energy saving, resources saving, low

pollution process and low waste products could be achieved by
using immobilization
 Immobilized Enzyme Systems

Disadvantages

 Many immobilized enzymes exhibit lower activity compared to

free enzymes.

 More expensive to prepare than free enzymes.

 Mass transfer limitations due to immobilization methods.

 Some immobilization methods could cause inhibition

 Biocatalyst detachment attrition could be noticed

Methods of Enzyme Immobilization

Major immobilization methods.

 Matrix Entrapment of Enzymes

Entrapment involves entrapping enzymes within the

spaces of a cross-linked water-insoluble polymer
 The enzyme solution is mixed with a polymeric fluid that solidifies
into various forms, depending on application (usually small beads).

 The polymeric material is semi-permeable.

 Large molecular weight enzymes can not

diffuse out

 but smaller substrate and product molecules

can.
 Matrices for Entrapment

 Ca-alginate

 Agar

 Polyacrylamide

 Collagen

 k-carrageenin
Steps for enzyme and cell immobilization
 Selection of the suitable enzyme or cell

 2- Selection of the suitable carrier.

 3- Selection of the suitable immobilization technique.

 Selection of the suitable enzyme or cell to perform a

required reaction is carried out through several screening
researches;

 while selection of the suitable carrier and immobilization

technique depends on the type of the biocatalyst.
Steps of enzyme immobilization using Ca-alginate
 Dissolve 9 g of Na-alginate in 300 ml DW.

 Stir until all sodium alginate is completely dissolved. The final

solution contains 3% alginate by weight.

 Thoroughly suspend about 1 g of the enzyme in the alginate

solution. Let air bubbles escape.

 Drip the enzyme-alginate mixture from a burette into 1000 ml of

crosslinking solution.

 The crosslinking solution is prepared by adding an additional

0.05M of CaCl2 solution with agitation with magnetic stirrer.

 Gel formation can be achieved at room temperature

Steps of enzyme immobilization using Ca-alginate
 
Steps of enzyme immobilization using Ca-alginate
 
Carrier-binding methods
This method is based on binding of the biocatalyst with a non-
soluble carrier by a covalent bond, ionic bond, physical
adsorption or bio-specific binding.

 Several materials can be used such as:

 Polysaccharides: cellulose, dextran and agarose

derivatives.

 Proteins: gelatin, albumin.

 Synthetic polymers: Polystyrene derivatives, ion exchange

resins, polyurethane.

 Inorganic materials: glass, sand, ceramic and magnetite.

Support Bonding
Figure1: Steps of CNBr enzyme immobilization method

Figure 2: The spacer effect in enzyme immobilization.

Advantages and disadvantages of the covalent binding
method
Advantages:
The enzyme doesn't leak from the carrier.
The enzyme can be easily interacts with the substrate because it is
in the surface of the carrier.
The enzyme stability is often increased due to its strong binding
with the carrier.

Disadvantages:

 The yield activity of the enzyme is low due to using of toxic

materials during immobilization.
 The optimal immobilization conditions are difficult to be find.
 Renewable of the carrier and recovery of the enzyme is impossible.
Physical adsorption

 This method depends on the physical interaction between

the carrier and the biocatalyst (enzyme or cells).

 These forces such as hydrogen bonding, Van der Waals

force and hydrophobic interaction.

 But the enzyme binding is weaker than the covalent or

ionic bonding methods.

 So, it can be affected by the environmental conditions such

as temperature and solute concentration.

 Example of surface carriers is activated carbon, silica,

alumina, clay, etc.
Effect of Immobilization Methods on the
Retention of Enzymatic Activity of Aminoacylase
Comparison between surface and entrapment
methods
 Surface adsorption only has one external mass transfer
resistance

 Passive: No adverse effects

 Week binding: Detachment is possible

 Entrapment Two mass transfer resistances

 Active and could have negative effects

 Restricted in the pores: no leaking

 Immobilized Enzyme
Reactors

Recycle packed column reactor:

- allow the reactor to operate at high fluid velocities.
- a substrate that cannot be completely processed on a single pass
Fluidized Bed Reactor:
- a high viscosity substrate solution
- a gaseous substrate or product in a continuous reaction system
- care must be taken to avoid the destruction and
decomposition of immobilized enzymes
- An immobilized enzyme tends to decompose
upon physical stirring.
- The batch system is generally suitable for the production
of rather small amounts of chemicals.
 Diffusional Limitation in
Immobilized Enzyme System
Immobilized enzyme system normally includes
- insoluble immobilized enzyme
- soluble substrate, or product

They are heterogeneous systems

Substrate
HIGH

Immobilized ILM SF
F AN
ER Sb
Enzyme TR

Low S concentration CE
EN
E R
FF
DI O N
N T I
T I O A C
N TR
A
ATT
RDIFFUSION
CE IC
R
N
CO LEC
T DRIVING FORCE
E
 HIGH

Immobilized ILM SF
F AN
ER Sb
Enzyme TR

REACTION CE
EN
E R
FF
DI O N
N T I
T I O A C
N TR
A
ATT
RDIFFUSION
CE IC
R
PRODUCT
N
CO LEC
T DRIVING FORCE
E
 HIGH

Immobilized ILM SF
F AN
ER Sb
Enzyme TR
LE
TIC
AR
-P R
RA FE
NT NS
I A
TR
DIFFUSION
DRIVING FORCE
 HIGH

Immobilized I L
F AN
M SF ER Sb
Enzyme TR
REACTION E
L
TIC
A R
A-P ER
TR SF
IN AN
TR PRODUCT
Diffusional Limitation in Immobilized
Enzyme System
In immobilized enzyme system, the overall production rate
is determined by

 Liquid film mass transfer (external diffusion)

substrate, product
 Intraparticle mass transfer (internal diffusion)
substrate, product in porous supports
 Enzyme catalysis reaction
 Diffusional Limitation in Immobilized Enzyme
System

Diffusion effects in surface-bound enzymes on nonporous

support materials. Ss
Sb

k2
E+S ES  P  E

Enzyme
Ss: substrate concentration at surface;
Liquid Film Thickness, L
Sb: substrate concentration in bulk solution.
 Diffusion effects in surface-bound enzymes
on nonporous support materials.
Assume:
 Enzyme are evenly distributed on Ss
Sb
the surface of a nonporous support
material.
 All enzyme molecules are equally
active.
 Substrate diffuses through a thin Enzyme
liquid film surrounding the support
surface to reach the reactive Liquid Film Thickness, L
surface. No Intraparticle diffusion

-The process of immobilization has not altered the enzyme

structure and the intrinsic parameters (Vm, Km) are unaltered.
Diffusion effects in surface-bound enzymes
on nonporous support materials.

To determine the significant effect of external diffusion

resistance on the rate of enzyme catalytic reaction rate:
Damköhler numbers (Da)

maximum rate of reaction Vm '

Da  
maximum rate of diffusion k L [ Sb ]
Vm ' is the maximum reaction rate per unit of
external surface area (e.g. g/cm2-s)
kL is the liquid mass transfer coefficient (cm/s)
[ Sb ] Is the substrate concentration in bulk solution (g/cm3)
 Diffusion effects in surface-bound enzymes
on nonporous support materials.

maximum rate of reaction Vm '

Da  
maximum rate of external diffusion k L [ Sb ]

When Da >> 1, the external diffusion rate is limiting;

Da << 1, the reaction rate is limiting;
Da ≈ 1, the external diffusion and reaction
resistances are comparable.
Diffusion effects in surface-bound enzymes
on nonporous support materials.
The external diffusion rate Js (g/cm2-s):

J s  k L ([ Sb ]  [ S s ])

kL is the liquid mass transfer coefficient (cm/s).

The reaction rate is :
Vm '[ S s ]
v  d [ S s ] / dt  d [ P] / dt 
K m  [S s ]
Vm ' the maximum reaction rate per unit surface area.
(g/cm2-s)
Diffusion effects in surface-bound enzymes
on nonporous support materials.

According to the mass balance of substrate

at the surface :

Accumulation of substrate Ss =
substrate gain - substrate consumption

k2
E+S ES  P  E
Diffusion effects in surface-bound enzymes
on nonporous support materials.

According to the mass balance of substrate

at the surface :
Vm '[ S s ]
d [ S s ] / dt  k L ([ Sb  [ S s ]) 
K m  [S s ]
At steady state, the reaction rate is equal to
the external diffusion rate:
Vm '[ S s ]
k L ([ Sb  [ S s ]) 
K m  [S s ]
Diffusion effects in surface-bound enzymes
on nonporous support materials.

At steady state, the reaction rate is equal to

the external diffusion rate:

Vm '[ S s ]
J s  k L ([ Sb ]  [ S s ]) 
K m  [S s ]
With the equation and known Sb, KL, Vm’ or Km,
to determine numerically or graphically:
- The substrate concentration at the surface.
- The reaction rate.
 J s  k L ([ Sb ]  [ S s ])

Graphical solution for reaction rate per unit of surface area

for enzyme immobilized on a non-porous support
Diffusion effects in surface-bound enzymes
on nonporous support materials.

When the system is strongly external diffusion

(liquid film mass-transfer) limited, [Ss]≈0,
the overall reaction rate is equal to the rate:

v  k L [ Sb ] Da>>1

The system behaves as pseudo first order.

The rate is a linear function of bulk substrate concentration.

Diffusion effects in surface-bound enzymes
on nonporous support materials.

To increase the overall reaction rate

with external diffusion limitation

maximum rate of reaction Vm '

Da  
maximum rate of diffusion k L [ Sb ]
-Increase the bulk concentration of substrate.

-Increase the liquid film mass transfer coefficient k L.

The liquid film mass transfer coefficient kL:
 D2 / 3   1/ 2 
 AB   U 
k L  0.6     
 1 / 6   d p1 / 2 
   
(H. Fogler, Elements of Chemical Reaction Engineering 1999, p705)

DAB is mass diffusivity of the substrate in the liquid phase,

a function of temperature and pressure (m2/s)
ν is the kinematic viscosity (m2/s), a function of temperature.
U is the free-system liquid velocity
(velocity of the fluid flowing past the particle) (m/s).
dp is the size of immobilized enzyme particle (m).
At specific T and P, increasing U and decreasing dp increase
the liquid film mass transfer coefficient and
the external diffusion rate.
Diffusion effects in surface-bound enzymes
on nonporous support materials.

When the system is strongly reaction limited,

[Sb] ≈ [Ss]

the overall reaction rate is equal to the rate:

Vm '[ Sb ] Da << 1
v
K m, app  [ Sb ]

K m, app can be determined experimentally.

 Diffusion Effects in Enzymes
Immobilized in a Porous Matrix
- Substrate diffuses through the tortuous
pathway within the porous support to reach
the enzyme.
- Substrate reacts with enzyme on the pore
surface.
 Diffusion Effects in Enzymes
Immobilized in a Porous Matrix
Assume:
-Enzyme is uniformly distributed in a
sphere support particle.
-There is not partitioning of the substrate
between the exterior and interior of the
support.
 Diffusion Effects in Enzymes
Immobilized in a Porous Matrix
At steady state, the intraparticle diffusion rate of
substrate equals to the reaction rate in a
spherical shell:
d[S ] 2 d[S ]
De 4r  De 4r 2  v 4r 2 r
dr r  r dr r
R

v is the reaction rate

per unit volume of support (mg/cm3-s).
De is the effective diffusivity (cm2/s). r r+Δr
Diffusion Effects in Enzymes
Immobilized in a Porous Matrix
Dividing the two sides of the equation by 4r
yields
d[S ] 2 d[S ] 2
De r  De r
r  r r
dr dr  vr 2
r
d d[S ] 2
When r →0 ( De r )  vr 2
dr dr
Re-arrange this equation

d 2[S ] 2 d[S ] 2
De ( r  2r )  vr
dr 2 dr
 Diffusion Effects in Enzymes
Immobilized in a Porous Matrix
Dividing the two sides of the equation by r2, yields,

d 2 [S ] 2 d [S ] Vm" [S ]
De (  )v v
dr 2 r dr K m  [S ]

Then
d 2 [S ] 2 d[S ] Vm" [S ]
De (  )
dr 2 r dr K m  [S ]
"
Vm is the maximum reaction rate per unit volume of support
(mg/cm3-s).
De is the effective diffusivity (cm2/s).
The above equation can be written in dimensionless form
by defining the following dimensionless variables:
[S ] r Km
S ,r  , 
[S s ] R [S s ]

d2S 2 dS R 2Vm" S
 
2 r dr S s De S  
dr
d2S 2 dS S
 2
2 r dr S
dr
Vm "
  R =Thiele modules
S s De
 d2S 2 dS 2 S
 
2 r dr S  
dr

With boundary conditions of

S  1, at r  1
d S / d r  0, at r  0
This differential equation can be solved numerically.
Refer to H. Fogler, Elements of Chemical Reaction Engineering
1999, p746 for analytical solution for first order reaction.
 At steady state, the rate of substrate consumption is equal to
the rate of substrate transfer through the external surface
of the support particle into the sphere.

2 d[S ]
rs  N s  4R De
dr rR
Under diffusion limitations, the rate per unit volume is usually
expressed in terms of the effectiveness factor as follows:

"
Vm [ S s ]
rs  
K m  [S s ]
  is the effectiveness factor.
reaction rate with intraparticle diffusionlimitation

reaction rate without diffusionlimitation.
  f ( ,  )
Vm " Km
  R 
S s De [S s ]

 1 the rate is diffusion limited.

 1 the rate is reaction limited.

The relationship can be obtained empirically.

Analytical expression is available
for some special cases
 " Km
  R
Vm 
S s De [S s ]
Relationship of effectiveness factor with the size of
immobilized enzyme particle and enzyme loading
At specific conditions (T, P) for a fixed system,

To increase the intra-particle mass transfer rate:

- Decrease the size of immobilized enzyme particle

- Increase the substrate concentration

- Increase the porosity or specific surface area of the particle

- Reduce the enzyme loading to suitable value
 Electrostatic and Steric Effects in
Immobilized Enzyme Systems
The optimum pH for immobilized enzyme system will
shift from that of soluble free enzyme
The charged matrix may repel or attract substrate,
product, cofactors or H+
Electrostatic effect

The activity of enzyme toward a high-molecule-weight

substrate may be reduced.
Steric hindrance
 Summary of Diffusion Effects
- Determine the support to be non-porous or porous.
- Identify the substrate determining the reaction rate.
- Conduct mass balance of the substrate of interest.

Accumulation of substrate of interest =

rate of substrate gain - substrate consumption rate
(production formation rate, or reaction rate)
At steady state,
Rate of substrate gain = substrate consumption rate
 Summary of Diffusion Effects
In surface-bound enzymes on nonporous support
materials.
Consider external diffusion rate (liquid film mass transfer rate)

At steady state, the reaction rate per unit surface area is

equal to the rate of net substrate gain in regard to the
external diffusion.

With the equation and known Sb, KL, Vm’ or Km,

to determine graphically or numerically:
- The substrate concentration at the surface.
- The reaction rate.
 Summary of Diffusion Effects
In surface-bound enzymes on porous support
materials.

Consider intraparticle diffusion rate.

At steady state, the reaction rate per unit volume

is equal to the rate of net substrate gain in regard
to the intraparticle diffusion.
  is the effectiveness factor.
reaction rate with intraparticle diffusionlimitation

reaction rate without diffusionlimitation.

  f ( ,  )

Vm " Km
  R 
S s De [S s ]
 1 the rate is diffusion limited.
 1 the rate is reaction limited.
At specific conditions (T, P) for a fixed system,

To increase the intra-particle mass transfer rate:

- Decrease the size of immobilized enzyme particle
- Increase the substrate concentration
- Increase the porosity or specific surface area of
the particle
Electrostatic and Steric Effects in Immobilized
Enzyme Systems

- The optimum pH for immobilized enzyme

system will shift from that of soluble free enzyme
Electrostatic effect

- The activity of enzyme toward a high-molecule-

weight substrate may be reduced.
Steric hindrance