Immobilized

Enzyme Kinetics
LECTURE 3.2
 Kinetic Properties
There is usually a decrease in specific activity of an enzyme upon
insolubilization: denaturation caused by the coupling process
Microenvironment after immobilization may be drastically different from that
existing in free solution: the physical and chemical character of the support
matrix, or interactions of the matrix with substrates or products involved in
the enzymatic reaction
The Michaelis constant may decrease by more than one order of magnitude when
substrate of opposite charge to the carrier matrix

The diffusion of substrate can limit the rate of the enzyme reaction: the
thickness of the diffusion film determines the concentration of substrate in the
vicinity of the enzyme and hence the rate of reaction
The effect of the molecular weight of the substrate can also be large. This may
be an advantage in some cases, since the immobilized enzymes may be
protected from attack by large inhibitor molecules
Effects of solute diffusion on the kinetics of immobilized
enzymes

external diffusion the transport of

substrates towards the surface, and
products away
internal diffusion the transport of the
substrates and products, within the pores
of immobilised enzyme particles
Kinetics of immobilized enzymes
Partitioning effect

The solution lying within a few molecular

diameters (10 nm) from the surface of an
immobilized enzyme will be influenced by both
the charge and hydrophobicity of the surface
The Km of an enzyme for a substrate is apparently
reduced if [S] in the vicinity of the enzyme's active
site is higher than that measured in the bulk of
the solution
 Kinetics of immobilized enzymes
A high concentration of ionizing groups may cause a partitioning of gases away
from the microenvironment with consequent effects on their apparent kinetic
parameters
It is also a useful method for protecting oxygen-labile enzymes by 'salting out' the
oxygen from the vicinity of the enzyme
Partition of hydrogen ions The pH of the microenvironment may differ
considerably from the pH of the bulk solution

Enzyme immobilized on charged

supports:
free enzyme
enzyme bound to a (+)ly charged
support; a bulk pH of 5 is needed to
produce a pH of 7 within the
microenvironment
enzyme bound to a (-)ly charged
support; a pH of 7 within the
microenvironment is produced by a
bulk pH of 9
Kinetics of immobilized enzymes
If the surface is predominantly hydrophobic
Hydrophobic molecules will partition into the microenvironment of the
enzyme and hydrophilic molecules will be partitioned out into the bathing
solution
Partition will affect the apparent kinetic constants of the enzyme

E.g. the reduction in the Km of immobilized alcohol

dehydrogenase for butanol
If the support is polyacrylamide, the Km is 0.1 mM but if a more
hydrophobic copolymer is used as the support, the Km is reduced to 0.025
mM
Kinetics of immobilized enzymes
A similar effect may be seen in the case of
competitive inhibitors

Invertase

Ki, mM free Bound to PS

(hydrophobic)
Aniline (hydrophobic) 0.94 0.39

Tris-(hydroxymethyl)- 0.45 1.20

aminomethane
(hydrophilic)
 Kinetics of immobilized enzymes
Enzymatic depolymerisation (including hydrolysis) of
macromolecules may be affected by diffusional control
Large molecules diffuse fairly slowly. After reaction, the cleaved
fragments normally retain their ability to act as substrates for the
enzyme
They are likely to be cleaved several times while they are in the
vicinity of the immobilised enzyme
This causes a significant difference in the molecular weight profiles
of the fragments produced by the use of free and immobilised
enzymes
Immobilized enzyme produces a small quantity of well-hydrolysed
low molecular weight product with the majority of the substrate
molecules unchanged
 Kinetics of immobilized enzymes
Co-immobilization of the necessary enzymes for the pathway results in a
rapid conversion through the pathway due to the localized high
concentrations of the intermediates
The reduction in the apparent lag phase is most noticeable when there are
more enzymes in the pathway

It is least pronounced where the

flux through the pathway is
controlled by the first step

Mixture of free enzymes

Co-immobilized enzymes
Diffusional Limitation in Immobilized Enzyme
System

Immobilized enzyme system normally includes

- insoluble immobilized enzyme
- soluble substrate, or product

They are heterogeneous systems

Substrate
HIGH
external diffusion

Immobilized
Sb
Enzyme

Low S concentration

DIFFUSION
DRIVING FORCE
 HIGH

Immobilized
Sb
Enzyme

DIFFUSION
DRIVING FORCE
 HIGH

Immobilized
Sb
Enzyme
REACTION

PRODUCT
external diffusion HIGH

Immobilized
Sb
Enzyme

REACTION

DIFFUSION
PRODUCT DRIVING FORCE
 Diffusional Limitation in
Immobilized Enzyme Systems
In immobilized enzyme systems,
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

Assume the enzyme catalyzed

reaction rate follows Michaelis-Menten
type kinetics.
Enzyme

Liquid Film Thickness, L

Ss: substrate concentration at the surface;

Sb: substrate concentration in bulk solution.
 Diffusion Effects in Surface-bound Enzymes
on Nonporous Support Materials

Assume: Ss
Sb
-Enzyme are evenly distributed on the
surface of a nonporous support
material.
-All enzyme molecules are equally
active.
Enzyme
-Substrate diffuses through a thin
liquid film surrounding the support Liquid Film Thickness, L
No intraparticle diffusion
surface to reach the reactive surface.
-The process of immobilization has not altered the enzyme
structure and the intrinsic parameters (Vm, Km) are unaltered.
Mass transfer coefficient
(cm/s)

Substrate
conc.
(g/cm3)
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:
Damkhler 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).
If the product formation rate is :
Vm '[ S s ]
v
'

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

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
When the system is strongly reaction limited,
[Sb] [Ss]
the overall reaction rate is equal to the rate:

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

where
Vm'
K m,app K m 1
k L ([ Sb ] K m )
Km,app is increased. It is a function of mixing speed and Sb.
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)
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.
Enzyme
are bound
on surface

At steady state, the reaction

rate=mass transfer rate
Diffusion Effects in Surface-bound Enzymes
on Nonporous Support Materials
k2
E+S ES P E
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.
Example 3.4
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

To increase the overall reaction rate

with external diffusion limitation

maximum rate of reaction Vm '

Da
maximum rate of diffusion k L [Sb ]
-Increase .

-Increase .
 Diffusion Effects in Enzymes
Immobilized in a Porous Matrix

Assume:
- Enzyme is uniformly distributed in a
spherical support particle.
- The reaction kinetics follows Michaelis-
Menten kinetics.
- There is no external diffusion limitation,
(no partitioning of the substrate between
exterior and interior of support).
 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.

Ex. Spherical support particles

Sr
A steady state: Diffusion rate =reaction rate
Equation 3.56 can be written in dimensionless form by defining
the following dimensionless variables:
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.
 reaction rate with intraparticle diffusion limitation

reaction rate withoutdiffusion limitation.

Under diffusion limitations, the
rate per unit volume is usually
expressed in terms of the
effectiveness factor as follows:
For a zero-order reaction rate (b 0), h1
for a large range of Thiele modulus values
such as 1 <f<100. For a first-order reaction
rate (b ), h=(f,b) and h is approximated to
the following equation for high values of f.

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 limitation. The value of
the effectiveness factor is a measure of the extent of
diffusion limitation. For h<1, the conversion is diffusion
limited, whereas for h1 values, conversion is limited by the
reaction rate and diffusion limitations are negligible. The
factor is a function of f and b as depicted in Figure 3.20.
 Low enzyme content will result in
lower enzyme activity per unit
volume but a high effectiveness
factor.

For maximum conversion rates,

particle size should be small (Dp
10 mm) and enzyme loading should
be optimized.

As depicted in the example in Fig.

3.21, Dp 10 mm and enzyme
loadings of less than 10 mg/cm3 are
required for high values of the
effectiveness factor (h 0.8).
 Under internal diffusion limitations, the rate per unit volume is
expressed in terms of the effectiveness factor as follows:
Vm" [ S s ]
rs
K m [Ss ]
is .
Vm" is the maximum velocity per volume of the support.
Km is the M-M constant.
[ S s ] is the substrate concentration on the surface of the support.
reaction rate with intraparticle diffusion limitation

reaction rate withoutdiffusion limitation.
1 the rate is .
1 the rate is .
At specific conditions (T, P) for a fixed system,

To increase the intra-particle mass transfer rate:

- the size of immobilized enzyme particle

- the porosity or specific surface area of the particle

Kinetics of immobilized enzyme
Enzyme Reactors
Membrane reactors have been used quite recently as have
applications of whole cell processes.

Retention of the cells within the reactor may be achieved by

membrane separation or by the same immobilization methods
that are used for isolated enzymes (34).

In principle, the cell itself can be regarded as a form of native

immobilization of enzymes.

\Biosensors are a very special form of carrier-fixed biocatalysts.

The major goal behind immobilization is the recovery of the

biocatalysts,

separation from products and reactants, and

subsequent reuse
-in batch or continuous processes.

Subsequent reuse in batch or continuous processes is especially

important for a reduction of the catalyst costs.

Enzymes immobilized on a support often show enhanced stability

when compared with the soluble form.
 When considering the use of soluble or
carrier-fixed enzymes, the following
topics we have to be addressed:
1. Additional costs for support and chemicals performing the
immobilization have to be balanced against the increase of
stability.

2. Loss of activity during the immobilization step.

3. When the catalyst is immobilized only by adsorption or

entrapment without covalent attachment, its leakage from the
carrier support has to be examined and compared with the
overall deactivation rate.
4. Mass transfer limitations for enzymes on a support may cause
problems when adjusting of the pH is necessary during the
reaction.

5. With soluble enzymes, higher volumetric activities at high

catalyst concentrations are possible, enabling conversion of poor
substrates at reasonable rates.

6. Whereas membrane reactors can be easily sterilized before use,

this is not possible for reactors with carrier-fixed enzymes.

To prevent microbial contamination, these processes are quite

often operated at higher temperatures.
7. When enzymes are to be used together with organic
solvents to increase reactant or product solubility or to
alter their kinetics, it may become necessary to
immobilize them on a support

The support will at the same time act as a water pool to

maintain the enzymatic activity.

In such systems, water-insoluble organic solvents have

less effect on the enzyme stability than water-soluble
solvents.
 Process design and operational strategies of
immoblized enzyme reactors
The final decision for a certain reactor design should be based on an
optimization process covering all relevant factors contributing
to the overall costs, including investment, catalyst
consumption, or productivity.
Comparison of Processes Using Soluble or
Carrier-Fixed Enzymes
Reactors for Immobilized Enzymes
The methods for the heterogenisation (or localization) of enzymes

by coupling them to insoluble supports or

by entrapment.

The types of reactors used for immobilized enzymes are summarized in

Figure given bellow.
Reactors for immobilized enzymes. (ac)

(a) Batch reactors with complete backmixing;

(b) Stirred-tank reactor;
(c) Fixed-bed reactor;
Fluidized-bed reactor. (df) are

Continuously operated reactors

with complete back mixing.

(gh) are the Continuously operated
reactors with plug-flow behavior.
(i) Reactor with the enzyme immobilized in or
on a membrane that may at the same time
separate two phases such as water and
organic solvent.

(j) reactor with physically separated enzyme

and organic solvent in order to prevent
denaturation of the protein
The principles developed for general heterogeneous
catalysis in synthetic chemistry are valid, resulting in well-
known reactor configurations.

Differences between enzyme catalysis and other systems

result from the nature of the biocatalyst and reaction
medium.

For example, soft particles containing the biocatalyst, such

as alginate beads, may limit the pressure drop in fixed-
bed reactors.
The decision as to specific reactor design will be based on a careful analysis of the
kinetic properties of the reaction system.

For example, if the enzyme shows a strong substrate-surplus inhibition, a

continuously operated reactor with complete backmixing working at high
conversion is advantageaous.
A reaction with strong product inhibition may utilize a batch
reactor or a plug flow reactor to achieve higher volume and
catalyst specific productivities.

An extractive bioreactor may be used if substrates and products

show different solubilities.

By using this reactor configuration, the destabilizing effect of

organic solvents may also be overcome, because the enzyme is
separated from the organic phase, which is used to extract the
insoluble product .

The aqueous phase containing the enzyme will be saturated until

the maximum solubility of with the substrate is reached.
Reactions using biocatalysts are normally performed in aqueous
solution at temperatures between 10 and 80 C and at ambient
pressure.

Due to the inhibition of some enzymes by heavy metals, the

materials of construction must not release these elements.

Reactors are operated under conditions that prevent microbial

contamination.

The reactor itself as well as the substrate may be sterilized prior to

reaction by using chemical agents (ethanol, formaldehyde,
ethylenoxide, Velcorin) or steam.
Ultraviolet rays may be used to sterilize the immobilized enzyme on its support
.

Alternatively, the immobilization may be performed under sterile conditions.

Antibacterial agents may be added to the reaction mixture to prevent

microbial growth while the reactor is running.
In some cases, the reactants may act as sterilants or
inhibitors of microbial growth, such as ketones or
alcohols.

At higher concentrations (more than 500 mmol/L),

solutions may become autosterile because of osmotic
pressure effects.

Ndustrial processes are often performed at elevated

temperatures, above 55 C, reducing the danger of
microbial contamination.
For a constant product quality and reproducibility of
downstream processing, the reactor should be operated
at constant conversion.

To overcome the deactivation per unit of time that

shows all biocatalysts as a result of denaturation
processes, either the residence time has to be increased
or fresh enzyme has to be supplied.

The latter is especially easy for soluble enzymes. For

carrier-fixed enzymes, a combination of both methods
is used, as discussed later.
 Immobilized Enzyme Reactors

Recycle packed column reactor:

- allow the reactor to operate at high fluid velocities.
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.
The immobilization of enzymes onto particulate carriers that may
be packed into a column (the packed-bed reactor), such as a
typical HPLC column, facilitates repetitive use of the enzyme and
also allows the automation of enzymatic assays.

Open-tubular reactors have also been constructed by covalently

immobilizing an enzyme onto the inner wall of a nylon or
polyethylene tube.

Immobilized enzyme reactors are used in conjunction with a pump,

to force a buffer, or mobile phase, through the reactor at a steady
rate, an injector located between the pump and the reactor to
allow the introduction of substrate solutions, and a detector
located close to the column exit.
The mobile phase contains all required cosubstrates and activators required
for the enzymatic reaction, but does not contain the analyte substrate.

A typical packed-bed system may use a 25-cm long reactor with a 5-mm inner-
diameter, packed with the carrier-enzyme solid phase at high pressures.

Flow rates of 0.52 mL/min and sample injection volumes of 10100 mL are
common.

Detection involves the same principles used in homogeneous enzymatic

assays, and flow-through optical absorbance and fluorescence detectors, and
amperometric and potentiometric electrochemical detectors may be
employed, with detector volumes of the order of tens of microliters being
standard.
Enzyme reactor systems may be of the continuous flow or the
stopped-flow variety.
Continuous flow systems are further categorized as open or
closed systems.

The open system, shown in Figure , continuously pumps fresh

buffer through the injector, reactor and detector, ultimately into a
waste reservoir for discarding.

This arrangement is preferred for the testing of enzyme reactors,

since unreacted substrate, cofactors and the products of the
enzymatic reactions will not be reexposed to the column.
Diagram of an open enzyme reactor system
Closed systems may be employed when buffer recycling is possible, that is
when the buffer contains high concentrations of all necessary cosubstrates,
when complete consumption of injected substrate occurs within the reactor,
and when products of the enzymatic reaction do not inhibit the immobilized
enzyme.
A closed system for immobilized oxidase enzymes is shown in Figure below.
Diagram of a closed enzyme reactor
system.
Both open and closed continuous flow systems rely on the fixed time, or
endpoint method for the determination of substrate concentrations.

At a fixed and constant flow rate, the injected volume of substrate will spend a
fixed time on the column, and this time is related to the volume of the column
(that volume not occupied by stationary phase) and the mobile-phase flow
rate.
Indicator reactions that are chemical in nature may be introduced either into
the mobile phase or at the end of the column by the method of postcolumn
reagent addition.

Postcolumn addition of reagents dilutes the column eluent, so that, when

possible, the addition of indicator reagents to the mobile phase is preferable.
The conditions under which chemical indicator
reactions are used often necessitates the use of
postcolumn addition, however.

Figure given below shows an experimental setup

for urea assays using an immobilized urease
reactor.30 The postcolumn addition of sodium
hydroxide allows the NH4 produced by the
reactor to be detected as NH3 at an ammonia gas-
sensing electrode placed in a flow cell.
Enzyme reactor system for urea based on immobilized urease
and potentiometric detection.
Stopped-flow enzyme reactor systems have been
designed for automated kinetic assays.

A diagram of a stopped-flow reactor that uses a

postcolumn chemical indicator reaction is shown in
Figure below.

In this system, the flow rate of themobile phase through

the reactor dictates the residence time of the analyte on
the column.
Stopped-flow enzyme reactor with
absorbance detection
THEORETICAL TREATMENT OF PACKED-BED
ENZYME REACTORS
Packed-bed enzyme reactors, those employing enzymes immobilized onto a particulate
phase that is subsequently packed into a column, may be characterized by their column
capacity, C, and the degree of reaction P.
The parameter C is defined by the equation.

where k is the decomposition rate constant for the enzymesubstrate complex (either k2
or kcat),

Et is the total number of moles of enzyme immobilized, and the value of is a constant
for a given reactor, and is equal to the ratio of reactor void volume to total reactor
volume (i.e., is always less then unity).

The degree of reaction, P, varies between zero (no product formed) and unity (complete
conversion of substrate).
An equation equivalent to the MichaelisMenten equation has been
derived for immobilized enzymes in packed-bed reactor systems, and
is given in Eq.

where Q is the volume flow rate of the mobile phase. In general, this
equation predicts that for a given column capacity, the degree of
reaction, P, is inversely related to the mobile-phase flow rate, Q. That
is, the faster the analyte plug flows through the reactor, the less likely
will be its complete conversion into product.
Application of immobilized enzymes
Bioreactors
Large scale production or conversion of various compounds
 Application of immobilized enzymes
Biosensors
An analytical device which can detect and quantify specific analytes in
complex samples

Biological
Sample Detection Transducer
Solution Element
Signal
Processor

Readout
Signal
Enzyme biosensors
Electrodes detecting
gases such as O2, CO2,
NH3 and various ionic
species are
commercially available
Application of immobilized enzymes
Bioremediation
For the removal/detoxification of contaminants
E.g. Polyphenol oxidase immobilized on chitosan coated membranes
Biosensors

a compact analytical
device incorporating
a biological sensing
element with a
transducer
Biosensors
BIOELEMENT TRANSDUCER

Electroactive electrode
Enzyme
substance
Cell
pH change pH electrode

READ-OUT
Micro
Heat thermistor
organism
Light photon
Antibody
counter
Nucleic
Mass piezoelectrical
acids
change device
PERFORMANCE FACTORS

Linear working range Selectivity

Reproducibility

Response time

Lifetime

The biosensor should be cheap, small, portable and

capable of being used by semi-skilled operators
Applications of Biosensors
Health care and life sciences research applications
Glucose and urea sensors
Proteomics
Genomics
Toxicology
Oncology
Drug discovery
Process industries
Monitoring of active component or pollutants
Food and drink
Measuring Ripeness
Contaminant/Pathogen Detection
Process/Quality Control
Detection of Genetically Modified Organisms in Food
Environmental monitoring
BOD, Pesticide
Defence and security
Military; Nerve gases and explosives
Forensics; DNA identification
Beetle/Chip Sensor
Schroth P. et al., Sensors and Actuators B, 78: 1-5, 2001
Whole-beetle

Antenna

Receptor Molecules

Detection of a single, damaged

potato plant within a field of a
thousand undamaged plant
 Bioelectronic sniffer for nicotine
Mitsubayashi K. et al, Analytica Chimica Acta
A bioelectronic sniffer for nicotine in the gas phase was developed with enzyme
inhibition principle to butyrylcholinesterase activity
Micromachined sensor for lactate monitoring
C.G.J. Schabmueller et al, Biosensors & Bioelectronics 21:1770-1776, 2006

Sport Medicine
location independent, permanent real-
time measurement of the lactate
concentration during exercise

The size of the chip is

5.5 6.4 0.7 mm
 What else?
Engineers for the Japanese company Toto have designed a toilet
that analyses urine for glucose concentrations, registers weight
and other basic readings, and automatically sends a daily report
by modem to the user's doctor...

One glucose sensor that looks like a watch sits on the skin and
produces small electric shocks, which open up pores so that fluid
can be extracted to monitor tissue glucose concentrations
 Cyclodextrin as a dehydrogenase mimic
R. Kataky and E. Morgan, Biosensors & Bioelectronics, 18:1407-1417, 2003

Cyclodextrins are very attractive as biomimetic

materials; a suitably modified cyclodextrin may
bind the substrate and then catalyse a reaction,
mimicking an enzyme-catalysed reaction

The model compound: a simple -cyclodextrin derivative with a

nicotinamide group attached to the secondary face of a -CD
The nicotinamide group would act as the electron transfer
agent and the cyclodextrin would provide a suitable cavity for
the reaction to take place in
Only small, unbranched alcohols such as ethanol are small
enough to fit into the -CD cavity along with the reacting
groups...
Dendrimers as synthetic enzyme mimics
C. Liang and J.M.J. Frchet, Progress in Polymer Science, 30: 385-402, 2005

Substrate migration into the dendrimer interior: The

nanoenvironment is greatly influenced by the branching
units. These favorable conditions encourage transition state
stabilization. The resulting product is released from the
nanoreactor into the solvent

(a) Substrate drawn from water into

the hydrophobic region in close
proximity to amines
(b) Charged tetrahedral transition
state (TS) intermediate is
stabilized by the amide group
(c) TS intermediate collapses and p-
nitrophenolate is released into
water
Membrane Proteins
Membrane proteins account for 2025 % of all open
reading frames
Wide range of central functions

At least 50 % of all drug targets are membrane

proteins
PROBLEMS Insoluble in aqueous solution so hard
to work with
Model Membrane Systems
to offer a native environment for the membrane proteins
Tethered Bilayer Membranes (tBLM)
Artificial lipid bilayers attached to
solid surfaces allow the
opportunity to use several surface
sensitive techniques

Atomic force microscopy

Surface plasmon spectroscopy
Impedance spectroscopy
Quartz crystal microbalance
etc