Enzyme Engineering &
Technology
Lecturer Dr. Kamal E. M. Elkahlout
Assistant Prof. of Biotechnology
1
CHAPTER 3
Immobilized enzymes and their
uses
2
Enzyme reactors
• An enzyme reactor consists of a vessel, or series of
vessels, used to perform a desired conversion by
enzymatic means (Figure 5.1).
• Factors that affect choice of reactor for a process:
• 1) Costs associated with substrate(s), downstream
processing, labor, depreciation, overheads and process
development.
• 2) Costs concerned with building and running the
enzyme reactor.
• 3) Form of the enzyme of choice (i.e. free or
immobilized).
• 4) Kinetics of the reaction.
• 5) Chemical and physical properties of an
immobilization support (particulate, membranous or
fibrous, density, compressibility, robustness, particle
size and regenerability).
•
•
•
•
6) Scale of operation.
7) Possible need for pH and temperature control.
8) Supply and removal of gases .
9) Stability of the enzyme, substrate and product.
(a) Stirred Tank Reactors
• Batch reactors generally consist of a tank
containing a stirrer (stirred tank reactor, STR).
• The tank is normally fitted with fixed baffles
that improve the stirring efficiency.
• In batch reactor all of the product is removed,
as rapidly as is practically possible, after a
fixed time.
• The enzyme and substrate molecules have
identical residence times within the reactor.
• a) Stirred tank batch reactor (STR), which contains all of the
enzyme and substrates) until the conversion is complete;
• b) batch membrane reactor (MR), where the enzyme is held
within membrane tubes which allow the substrate to diffuse
in and the product to diffuse out. This reactor may often be
used in a semicontinuous manner, using the same enzyme
solution for several batches;
• c) packed bed reactor (PBR), also called plug -flow reactor
(PFR), containing a settled bed of immobilised enzyme
particles;
• d)continuous flow stirred tank reactor (CSTR) which is a
continuously operated version of (a);
• e) continuous flow membrane reactor (CMR) which is a
continuously operated version of (b);
• f) fluidized bed reactor (FBR), where the flow of gas and/or
substrate keeps the immobilised enzyme particles in a
fluidized state.
• In some circumstances there may be a need for further
additions of enzyme and/or substrate (i.e. fed -batch
operation).
• Disadvantages:
• The operating costs are higher than for continuous
processes due to the necessity for the reactors to be
emptied and refilled both regularly and often.
• There are considerable periods when such reactors are
not productive.
• It also makes uneven demands on both labor and
services.
• STRs can be used for processes involving nonimmobilized enzymes.
• Batch reactors also suffer from pronounced batch-tobatch variations.
• Reaction conditions change with time.
• May be difficult to scale-up due to the changing power
requirements for efficient fixing.
• Advantageous features:
• Simplicity both in use and in process development.
• For this reason they are preferred for small-scale
production of highly priced products, especially where
the same equipment is to be used for a number of
different conversions.
• They offer a closely controllable environment that is
useful for slow reactions, where the composition may
be accurately monitored, and conditions (e.g.
temperature, pH, coenzyme concentrations) varied
throughout the reaction.
• They are also of use when continuous operation of a
process proves to be difficult due to the viscous or
intractable nature of the reaction mix.
• All reactors would additionally have heating/cooling
coils.
• (interior in reactors (a), and (d), and exterior, generally,
in reactors (b), (c), (e) and (f)).
• Stirred reactors may contain baffles in order to
increase (reactors (a), (b), (d) and (e) or decrease
(reactor (f)) the stirring efficiency.
• The continuous reactors ((c) -(f)) may all be used in a
recycle mode where some, or most, of the product
stream is mixed with the incoming substrate stream.
• All reactors may use immobilized enzymes.
• In addition, reactors (a), (b) and (e) (plus reactors (d)
and (f), if semipermeable membranes are used on their
outlets) may be used with the soluble enzyme.
(b) Membrane reactors
• The main requirement for a membrane reactor (MR) is
a semipermeable membrane which allows the free
passage of the product molecules but contains the
enzyme molecules.
• A cheap example of such a membrane is the dialysis
membrane used for removing low molecular weight
species from protein preparations.
• The usual choice for a membrane reactor is a hollowfiber reactor consisting of a preformed module
containing hundreds of thin tubular fibers each having
a diameter of about 200 μm and a membrane thickness
of about 50 μm.
• Membrane reactors may be used in either batch or
continuous mode and allow the easy separation of the
enzyme from the product.
• They are normally used with soluble enzymes, avoiding
the costs and problems associated with other methods
of immobilization and some of the diffusion limitations
of immobilized enzymes.
• If the substrate is able to diffuse through the
membrane, it may be introduced to either side of the
membrane with respect to the enzyme.
• Otherwise it must be within the same compartment as
the enzyme, a configuration that imposes a severe
restriction on the flow rate through the reactor, if used
in continuous mode.
• Due to the ease with which membrane reactor systems
may be established, they are often used for production
on a small scale (g to kg), especially where a multienzyme pathway or coenzyme regeneration is needed.
• They allow the easy replacement of the enzyme in
processes involving particularly labile enzymes and can
also be used for biphasic reactions.
• The major disadvantage of these reactors concerns the
cost of the membranes and their need to be replaced
at regular intervals.
• The kinetics of membrane reactors are similar to those
of the batch STR, in batch mode, or the CSTR, in
continuous mode.
• Deviations from these models occur primarily in
configurations where the substrate stream is on the
side of the membrane opposite to the enzyme and the
reaction is severely limited by its diffusion through the
membrane and the products' diffusion in the reverse
direction.
• Under these circumstances the reaction may be
even more severely affected by product inhibition
or the limitations of reversibility than is indicated by
these models.
Continuous flow reactors
• The advantages of immobilized enzymes as processing
catalysts are most markedly appreciated in continuous
flow reactors.
• In these, the average residence time of the substrate
molecules within the reactor is far shorter than that of
the immobilized-enzyme catalyst.
• This results in a far greater productivity from a fixed
amount of enzyme than is achieved in batch processes.
• It also allows the reactor to handle substrates of low
solubility by permitting the use of large volumes
containing low concentrations of substrate.
• The constant reaction conditions may be expected to
result in a purer and more reproducible product.
• There are two extremes of process kinetics in relation
to continuous flow reactors;
• A) The ideal continuous flow stirred tank reactor
(CSTR), in which the reacting stream is completely and
rapidly mixed with the whole of the reactor contents
and the enzyme contacts low substrate and high
product concentrations;.
• B) The ideal continuously operated packed bed reactor
(PBR), where no mixing takes place and the enzyme
contacts high substrate and low product
concentrations.
• The properties of the continuously operated fluidized
bed reactor (FBR) lie, generally, somewhere between
these extremes.
• An ordered series of CSTRs or FBRs may approximate,
in use where the outlet of one reactor forms the inlet
to the next reactor, to an equivalent PBR.
(c) Packed bed reactors
• The most important characteristic of a PBR is that
material flows through the reactor as a plug; they are
also called plug flow reactors (PFR).
• Ideally, all of the substrate stream flows at the same
velocity, parallel to the reactor axis with no back mixing.
• All material present at any given reactor cross -section
has had an identical residence time.
• The longitudinal position within the PBR is, therefore,
proportional to the time spent within the reactor.
• All product emerging with the same residence time
and all substrate molecule having an equal opportunity
for reaction.
• The conversion efficiency of a PBR, with respect to its
length, behaves in a manner similar to that of a well stirred batch reactor with respect to its reaction time.
• Each volume element behaves as a batch reactor as it
passes through the PBR.
• Any required degree of reaction may be achieved by
use of an idea PBR of suitable length.
• In order to produce ideal plug -flow within PBRs, a
turbulent flow regime is preferred to laminar flow, as
this causes improved mixing and heat transfer normal
to the flow and reduced axial back-mixing.
• Consequent upon the plug -flow characteristic of the
PBR is that the substrate concentration is maximized,
and the product concentration minimized, relative to
the final conversion at every point within the reactor;
the effectiveness factor being high on entry to the
reactor and low close to the exit.
• This means that PBRs are the preferred reactors, all
other factors being equal, for processes involving
product inhibition, substrate activation and reaction
reversibility.
• They are easily fouled by colloidal or precipitating
material.
• The design of PBRs does not allow for control of pH,
by addition of acids or bases, or for easy
temperature control where there is excessive heat
output, a problem that may be particularly
noticeable in wide reactors (> 15 cm diameter).
• Deviations from ideal plug-flow are due to backmixing within the reactors, the resulting product
streams having a distribution of residence times.
• In an extreme case, back-mixing may result in the
kinetic behavior of the reactor approximating to that of
the CSTR (see below), and the consequent difficulty in
achieving a high degree of conversion.
• These deviations are caused by channeling, where
some substrate passes through the reactor more
rapidly, and hold-up, which involves stagnant areas
with negligible flow rate.
• Channels may form in the reactor bed due to excessive
pressure drop, irregular packing or uneven application
of the substrate stream, causing flow rate differences
across the bed.
• The use of a uniformly sized catalyst in a reactor with
an upwardly flowing substrate stream reduces the
chance and severity of non-ideal behaviour.
(d) & (e) Continuous flow stirred
tank reactors
• This reactor consists of a well -stirred tank
containing the enzyme, which is normally
immobilized.
• The substrate stream is continuously pumped into
the reactor at the same time as the product stream
is removed.
• If the reactor is behaving in an ideal manner, there
is total back-mixing and the product stream is
identical with the liquid phase within the reactor
and invariant with respect to time.
• Some molecules of substrate may be removed
rapidly from the reactor, whereas others may
remain for substantial periods.
• The reaction S>>>>>>>>>P is assumed, and substrate
molecules that have long residence times are converted
into product.
• The average residence time of the product being
greater than that for the substrate.
• The composition of the product stream is identical with
that of the liquid phase within the reactor.
• These reactors may be operated for considerably longer
periods than that determined by the inactivation of
their contained immobilized enzyme, particularly if
they are capable of high conversion at low substrate
concentrations.
• This is independent of any enzyme stabilization and is
simply due to such reactors initially containing large
amounts of redundant enzyme.
• In general, there is little or no back -pressure to
increased flow rate through the CSTR.
• Such reactors may be started up as batch reactors until
the required degree of conversion is reached, when the
process may be made continuous.
• CSTRs are not generally used in processes involving
high conversions but a train of CSTRs may approach the
PBR performance.
• This train may be a number (greater than three) of
reactors connected in series or a single vessel divided
into compartments.
• In order to minimize back-mixing CSTRs may be used
with soluble rather than immobilized enzyme if an
ultrafiltration membrane is used to separate the
reactor output stream from the reactor contents.
• This causes a number of process difficulties,
including concentration polarization or inactivation
of the enzyme on the membrane but may be
preferable in order to achieve a combined reaction
and separation process or where a suitable
immobilized enzyme is not readily available.
(f) Fluidized bed reactors
• These reactors generally behave in a manner
intermediate between CSTRs and PBRs.
• They consist of a bed of immobilized enzyme which is
fluidized by the rapid upwards flow of the substrate
stream alone or in combination with a gas or secondary
liquid stream, either of which may be inert or contain
material relevant to the reaction.
• A gas stream is usually preferred as it does not dilute
the product stream.
• There is a minimum fluidization velocity needed to
achieve bed expansion, which depends upon the size,
shape, porosity and density of the particles and the
density and viscosity of the liquid.
• This minimum fluidization velocity is generally fairly low
(about 0.2 -I.0 cm s-1) as most immobilized-enzyme
particles have densities close to that of the bulk liquid.
• In this case the relative bed expansion is
proportional to the superficial gas velocity and
inversely proportional to the square root of the
reactor diameter. Fluidising the bed requires a large
power input but, once fluidized, there is little
further energetic input needed to increase the flow
rate of the substrate stream through the reactor.
• At high flow rates and low reactor diameters almost
ideal plug -flow characteristics may be achieved.
• However, the kinetic performance of the FBR
normally lies between that of the PBR and the CSTR,
as the small fluid linear velocities allowed by most
biocatalytic particles causes a degree of back-mixing
that is often substantial, although never total.
• The actual design of the FBR will determine
whether it behaves in a manner that is closer to
that of a PBR or CSTR.
• It can, for example, be made to behave in a manner
very similar to that of a PBR, if it is baffled in such a
way that substantial backmixing is avoided.
• FBRs are chosen when these intermediate
characteristics are required, e.g. where a high
conversion is needed but the substrate stream is
colloidal or the reaction produces a substantial pH
change or heat output. They are particularly useful
if the reaction involves the utilization or release of
gaseous material.
• The FBR is normally used with fairly small immobilized
enzyme particles (20-40 mm diameter) in order to
achieve a high catalytic surface area.
• These particles must be sufficiently dense, relative to
the substrate stream, that they are not swept out of
the reactor.
• Less-dense particles must be somewhat larger.
• For efficient operation the particles should be of nearly
uniform size otherwise a non-uniform biocatalytic
concentration gradient will be formed up the reactor.
• FBRs are usually tapered outwards at the exit to allow
for a wide range of flow rates.
• Very high flow rates are avoided as they cause
channeling and catalyst loss.
• The major disadvantage of development of FBR
process is the difficulty in scaling-up these reactors.
• PBRs allow scale-up factors of greater than 50000
but, because of the markedly different fluidization
characteristics of different sized reactors, FBRs can
only be scaled-up by a factor of 10 -100 each time.
• In addition, changes in the flow rate of the
substrate stream causes complex changes in the
flow pattern within these reactors that may have
consequent unexpected effects upon the
conversion rate.
Immobilized Enzymes
•Immobilized enzymes are enzymes which are
attached in or onto the surface of an insoluble
support
•Immobilized enzymes have several advantages over the soluble
enzyme:
– Convenience: Miniscule amounts of protein dissolve in the
reaction, so workup can be much easier. Upon completion,
reaction mixtures typically contain only solvent and reaction
products.
– Economical: easily removed from the reaction  reusage
– Stability: Immobilized enzymes typically have greater thermal and
operational stability than the soluble form of the enzyme
Immobilization criteria
• There are a number of requirements to achieve a
successful immobilization:
– The biological component must retain substantial
biological activity after attachment
– It must have a long-term stability
– The sensitivity of the enzyme must be preserved
after attachment
– Overloading can block or inactivate the active site
of the immobilized biomaterial, therefore, must
be avoided
Immobilization methods
a)
b)
c)
d)
adsorption
covalent binding
entrapment
encapsulation
Adsorption and Ionic binding
• Simplest immobilization method
– Mix the enzyme and support in suitable conditions
• First immobilized enzyme model: invertase on the activated charcoal
(Nelson and Griffin, 1916)
• Forces are weak so leakage is generally a problem
• Supports such as alluminium hydroxide are often utilized
• With a suitable charged matrix, ionic interactions may also be
promoted
• This technique is technically undemanding and economically
attractive
• Regeneration is easy
• Best known industrial example: amino acylase immobilized on DEAESephadex in the production of amino acids
Gel-fibre entrapment and encapsulation
Entrapment
• Enzymes may be entrapped within the matrix of a polymeric gel
– Incubate the enzyme together with the gel monomers
– Promote gel polymerization
• Polyacrylamide and polymethacrylamide gels are examples
• Gel pore size is a crucial factor
Encapsulation
• Encapsulation involves entrapping the enzymes within a semipermeable
membrane such as cellulose nitrate and nylon-based membranes
Covalent immobilization
• The most widely used method for enzyme immobilization
–
–
–

It is technically more complex
It requires a variety of often expensive chemicals
It is time-consuming
But immobilized enzyme preparations are stable and leaching is
minimal
• Enzymes are immobilized by a suitable group in the surface:
– Hydroxyl groups in supports (e.g cellulose, dextran, agarose)
– Amino, carboxyl and sulfhydryl groups in amino acids
Covalent immobilization
• The conditions for immobilization by covalent binding are much more
complicated and less mild than in the cases of physical adsorption and
ionic binding. Therefore, covalent binding may alter the conformational
structure and active center of the enzyme, resulting in major loss of
activity and/or changes of the substrate
• Covalent attachment to a support matrix must involve only functional
groups of the enzyme that are not essential for catalytic action
• Higher activities result from prevention of inactivation reactions with
amino acid residues of the active sites. A number of protective methods
have been devised:
– Covalent attachment of the enzyme in the presence of a competitive inhibitor
or substrate
– A chemically modified soluble enzyme whose covalent linkage to the matrix is
achieved by newly incorporated residues
Site-specific immobilization
Three different approaches:
(a) Gene fusion to incorporate a
peptidic affinity tag at the N- or
C-terminus of the enzyme. The
enzymes are then attached from
this affinity tag to anti-tag
antibodies on membranes
(b) Modification to incorporate a
single biotin moiety on enzymes
(see figure)
(c) Site-directed mutagenesis to
introduce unique cysteines to
enzymes. The enzymes are
attached on thiol-reactive
surfaces through the sulfhydryl
group
Properties of support material
• The form, shape, density, porosity, pore size distribution, operational
stability and particle size distribution of the supporting matrix will
influence the result
• The ideal support is cheap, inert, physically strong and stable
• Ideally, it should:
– increase the enzyme specificity (kcat/Km)
– shift the pH optimum to the desired value for the process
– discourage microbial growth and non-specific adsorption
• Some matrices may possess other properties which are useful for
particular purposes such as
– ferromagnetism (e.g. magnetic iron oxide, enabling transfer of the biocatalyst
by means of magnetic fields)
– a catalytic surface (e.g. manganese dioxide, which catalytically removes the
inactivating hydrogen peroxide produced by most oxidases)
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 ionising 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 immobilised 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 immobilised 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)aminomethane
(hydrophilic)
0.45
1.20
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
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
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
Sample
Solution
Biological
Detection Transducer
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
Enzyme
Cell
Micro
organism
Antibody
Nucleic
acids
Electroactive
substance
electrode
pH change
pH electrode
Heat
thermistor
Light
photon
counter
Mass piezoelectrical
change
device
READ-OUT
TRANSDUCER
BIOELEMENT
PERFORMANCE FACTORS
Selectivity
Linear working range
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 realtime 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 β-cyclodextrin derivative with a
nicotinamide group attached to the secondary face of a β-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 β-CD cavity along with the reacting groups...
Dendrimers as synthetic enzyme mimics
C. Liang and J.M.J. Fréchet, 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 pnitrophenolate is released into water
Membrane Proteins
 Membrane proteins account for 20–25 % 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