Understanding enzyme action on immobilised substrates
Peter J Halling1, Rein V Ulijn2 and Sabine L Flitsch3
With increasing interest in automated synthesis and screening
protocols, solid supported chemistry and biochemistry are
attractive technologies. Studies with surface-immobilised
substrates have been carried out to analyse enzyme
accessibility, kinetics and thermodynamics. Several interesting
new methods have been developed to monitor enzyme action
on substrates attached to a solid phase such as polymer beads
glass or gold surfaces. These include fluorescence
measurements, MALDI-TOF mass spectrometry, and the use
of quartz crystal microbalances to measure weight changes of
immobilised molecules directly on the surface. Approaches
that allow spatial resolution in single beads have also been
reported. The ability of enzymes to reach the inside of beads is
becoming better characterised and new supports have been
developed that allow improved accessibility. The equilibrium
position of reactions on the solid surface can be substantially
shifted compared with reactions in solution, and this can be
usefully exploited using hydrolases in reverse. Research is also
starting to tackle the way in which kinetics are modified when
the substrates are surface immobilised.
Addresses
1
Department of Pure & Applied Chemistry, University of Strathclyde,
Glasgow G1 1XW, UK
2
School of Materials, University of Manchester, Manchester M1 7HS,
UK
3
School of Chemistry, University of Manchester, Manchester M1 7HS,
UK
Corresponding author: Halling, Peter J (p.j.halling@strath.ac.uk)
Current Opinion in Biotechnology 2005, 16:385–392
This review comes from a themed issue on
Protein technologies and commercial enzymes
Edited by Bernhard Hauer and Brian K Kay
Available online 6th July 2005
0958-1669/$ – see front matter
# 2005 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.copbio.2005.06.006
Introduction
Traditionally, enzyme action is studied with both catalyst
and substrate in solution. The use of immobilised
enzymes is also quite familiar, particularly in light of
their applications as practical biocatalysts. In this case,
the dissolved substrate molecules must diffuse to find the
enzyme attached to a solid phase. This review is con-
cerned with a third type of system, in which the substrate
moieties are attached to a solid phase. Now the dissolved
enzyme molecules must diffuse to the sites where the
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substrate is bound. A comparison is illustrated in
Figure 1.
Several enzymes have evolved naturally to attack solid-
phase substrate groups, such as cellulases and lysozymes.
These will have special features aiding them to function
this way, such as separate surface-binding domains.
Enzymes specially adapted to work on solid-phase sub-
strates will not be dealt with here, but recent reviews can
be found [1–3].
More recently, enzymes that are normally used with
dissolved substrates have been used to catalyse reactions
on substrates attached to a solid phase, such as a polymer
bead or a modified glass or gold surface. There are
several motivations for studying such systems. With
increasing interest in automated synthesis and screening
protocols, solid supported chemistry and biochemistry
are attractive technologies. Thus, enzymes are useful for
selected steps in solid-phase chemical synthesis. Their
selectivity and ability to act under mild conditions may
be exploited in the removal of protecting groups [4],
cleavage of linkers at the end of a synthesis [5], or to give
selectivity in a particular coupling step [6,7,8–12].
There are several reports of on-bead screening of
enzyme substrates made by combinatorial solid-phase
synthesis [13]. More generally, enzyme assays can con-
veniently be carried out with solid-phase substrates
[14,15,16–22], especially on microarrays for the rapid
identification of substrates or inhibitors in parallel. There
might also be relevance to many processes in vivo, in
which substrates are attached to surfaces in living cells
and tissues [23,24]. The attack on immobilised substrates
by enzymes normally acting in solution may be signifi-
cant, especially in various disease states such as arthritis
[25].
Most published studies have concentrated on exploring
the scope of applications in synthesis or screening. Rates
and/or yields of biocatalytic reactions on solid-phase
supports are generally disappointing compared with reac-
tions in solution, as some authors have noted. (Yield can
sometimes, but not always, be improved by using a large
excess of enzyme.) Such low rates and yields have
prompted more detailed fundamental studies of the
kinetics and thermodynamics of biocatalytic reactions
on immobilised substrates. These studies form the core
topic of the present review, which discusses several
issues: how to monitor enzyme catalysis on solid phase;
how to define and measure kinetic and binding constants
when the substrate is attached to a solid phase; how
accessible is the substrate to the enzyme; and how will
Current Opinion in Biotechnology 2005, 16:385–392
386 Protein technologies and commercial enzymes

Figure 1 There are obvious attractions to methods that enable the

‘optical sectioning’ of beads. This is offered by various
(a) microscopic techniques in which an image is constructed
Substrate and enzyme soluble of a thin section inside an intact sample, formed by
S collecting just photons that originated in that section,
without physical cutting. Typically, the level of the
E
section can be moved up and down to create a full
three-dimensional image. Such methods allow investiga-
(b)
Immobilised enzymes tors to study where enzyme activity takes place within the
S interior of single beads. Reaction rates and extent can
differ in different parts of the bead, because of varied
E E E enzyme accessibility, for example. One such method —
confocal fluorescence microscopy — gave artefacts and
(c)
cannot be used to monitor reactions throughout a bead.
Solid-phase (immobilised) substrates Kress et al. [26] used confocal Raman microscopy to
monitor the release of a CN-labelled peptide by protease
E action, on various bead types. This method has the
potential to follow a reaction at different locations within
Current Opinion in Biotechnology
the bead. Bosma et al. [27] recently demonstrated that
two-photon fluorescence microscopy could be used for
Different systems for enzyme action. (a) Both enzyme (blue) and
spatial and temporal quantification of protease-catalysed
substrate (red) are free in solution. (b) The enzyme is immobilised and peptide hydrolysis on polyethylene glycol acrylamide
the substrate is in solution. (c) The substrate is immobilised and the (PEGA) beads. The reaction was stopped at various times
enzyme in solution. and the liberated amino groups labelled with a fluoro-
phore (Figure 2).

the reaction equilibrium be affected. We also present a
brief survey of some recently developed applications.
New methods to monitor enzyme action on
immobilised substrates
The extent of reaction has normally been monitored by
analysis of the material released into solution — either
directly by the enzymatic reaction or by subsequent
cleavage of a linker. Such approaches typically release
only low concentrations of material, so that analysis may
be laborious and/or inaccurate. Some new methods ana-
lyse directly what is present on the surface, and may even
give spatial resolution.
Salisbury et al. [14] and Zhu et al. [15] independently
describe the use of a coumarin substrate attached to the
solid phase in such a way that an immobilised fluorophore
is generated at the site of any enzymatic reaction
(Figure 3). This approach was applied with an array of
substrates on a planar surface, which could be used to
screen the activity and specificity of proteases [14,15]
and other hydrolases [15] (esterase, phosphatase and
epoxide hydrolase). We have been able to show that such
an approach can also give real-time monitoring of reaction
rates at different sites within a bead, using two-photon
microscopy with optical sectioning (A Lalouni et al.,
unpublished).

Figure 2

Thermolysin
Fmoc-Phe-Phe- H2N-Phe-

Dansyl
labelling
O
S NH-Phe-
O
N

Current Opinion in Biotechnology

Spatially resolved kinetics of thermolysin-catalysed hydrolysis on PEGA1900. The proteolysis reaction was stopped at various times and the
liberated amino groups labelled with dansyl fluorophore. Two-photon microscopy images of the dansylated product are shown on the right
(images were taken at 5, 10, 20,45, 60, 90, 120 and 240 min). (Figure based on the work of Bosma et al. [27].)

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 Enzyme action on immobilised substrates Halling, Ulijn and Flitsch 387

Figure 3

Enzyme Fluorogenic
recognition moiety Fluorescent
X O O HX O O

Enzyme
O O
X = O or NH

Current Opinion in Biotechnology

The generation of an immobilised fluorophore by enzyme action. A surface-immobilised coumarin is rendered non-fluorescent by covalent
modification of an amino or hydroxyl group. An enzyme able to recognise the modification can catalyse its removal and thus induce
fluorescence. Fluorescent regions generated on a planar surface indicate where enzyme and recognition moieties are complementary.
(Figure based on the work of Salisbury et al. [14] and Zhu et al. [15].)

Nishino et al. [28,29] report the use of a quartz crystal
microbalance to monitor in real time enzymatic catalysis
on substrates immobilised on the quartz. This method
relies on the very sensitive measurement of mass
increases and decreases on the nanogram level. Micro-
balance measurements allowed monitoring of both the
initial increase in mass as enzyme bound to the surface,
and the subsequent decrease as glucoamylase or glycogen
phosphorylase released sugar residues from their sub-
strates (Figure 4).
Yeo and Mrksich [30] have developed self-assembled
monolayers of cutinase substrates that become electro-
chemically active upon cutinase hydrolysis. Thus, biolo-
gical activities are translated into electric signals that can
allow for kinetic measurements. On the same monolayer
systems reactions could also be monitored by matrix-
assisted laser desorption time-of-flight mass spectrometry
(MALDI-TOF MS). Mrksich and colleagues [31] have
also used MALDI-TOF MS to analyse the phosphoryla-
tion of peptides by kinases and in a two-step enzymatic
modification of an oligosaccharide, which was immobi-
lised as self-assembled monolayers on gold. Furthermore,
MALDI-TOF MS was used to determine the time-
dependence of enzymatic galactosylation, which demon-
strates that this technique can provide kinetic information
on biological activities [32].
Self-assembled monolayers on gold were also used by
Houseman and Mrksich [33] for the generation of carbo-
hydrate arrays and to measure the subsequent glycosyla-
tion using a galactosyltransferase. Both the substrate and

Figure 4

Add AMP
100
Add enzyme

0
∆m (ng cm–2)

–100

–200

–300
0 10 20 30 40
Time (min)
Current Opinion in Biotechnology

Time-course of mass changes followed using a quartz crystal microbalance. The first addition was glycogen phosphorylase, which binds to the
immobilised amylopectin substrate causing the mass to increase. The second addition of AMP activates the enzyme, starting progressive
catalytic dissolution of the substrate and mass decrease. (Figure redrawn from data of Nishino et al. [29].)

www.sciencedirect.com Current Opinion in Biotechnology 2005, 16:385–392

388 Protein technologies and commercial enzymes
the product of the enzymatic reaction could be monitored
looking at lectin binding using fluorescence spectroscopy
and surface plasmon resonance spectroscopy.
Ability of the enzyme to get close to the
substrate sites (‘accessibility’)
Before discussing this topic it is important to define
clearly what we mean by ‘accessibility’. In this section
we deal with the ability of the enzyme to approach within
1 nm or so of the substrate moieties. Such accessibility is
essentially a problem encountered with porous solid
supports that are commonly used in solid-phase synthesis.
Porous supports generally have a high loading of func-
tional groups, which are distributed over surfaces inside
the pores so that enzymes need to penetrate the pores for
successful catalysis. These accessibility problems should
not arise where substrates are attached to a planar surface.
The issue of how substrate presentation on surfaces can
affect biocatalysis is considered part of reaction kinetics,
although it has been termed accessibility on a molecular
scale.
It was realized early on that many of the support materials
commonly used in solid-phase chemistry do not permit
enzyme access to their interior pores. Such polymers were
developed to provide high loadings of pendant groups
(i.e. effectively high surface area), which has led to very
small (effective) pore sizes. In addition, most of these
supports were designed for organic synthesis applications
and hence are hydrophobic and do not swell in aqueous
media. A major breakthrough in this area was the devel-
opment of the PEGA range of support materials first used
by Meldal [13]. PEGA-based polymer beads have been
promoted as suitable for enzyme reactions and are popu-
larly used because they are compatible both with condi-
tions used in solid-phase synthesis as well as aqueous
systems for enzyme reactions. PEG is also known to resist
non-specific protein adsorption to the surface and there-
fore limits enzyme denaturation at the solid–liquid inter-
face.
Another porous polymer that can swell both in organic
and aqueous solvents is TentaGelTM, a co-polymer of
polystyrene and PEG. In aqueous environments, Tenta-
GelTM has generally been found to be less accessible to
enzymes; however, a high-yielding enzymatic reaction on
TentaGelTM has been reported by Altreuter et al. [6]
using an organic medium (10% tetrahydrofuran in iso-
octane). The swelling of these beads is solvent-depen-
dent and must allow better access in organic media,
perhaps because of the behaviour of the polystyrene core.
The chymotrypsin catalyst used in this study was an ion-
paired complex that must be larger than the free enzyme
(22 kDa).
Of the non-swelling support materials, controlled pore
glasses (CPGs) have proved most useful and have been
Current Opinion in Biotechnology 2005, 16:385–392
used successfully with several enzymes. Kress et al. [26]
investigated the accessibility of various solid supports
(TentaGelTM, PEGA1900, and beaded CPG) to a range
of enzymes. Using confocal Raman microscopy (see
above), they showed that none of the investigated
enzymes could enter the polymer matrix of TentaGelTM.
PEGA1900 was compatible only with the two smallest
enzymes (35 kDa), while CPG with 100 nm pores
was successful even with a 90 kDa enzyme, proving its
superiority over other materials in terms of accessibility.
Bosma et al. [27] recently used two-photon fluorescence
microscopy to monitor rates of protease-catalysed peptide
hydrolysis at different locations in PEGA beads. Thermo-
lysin (33 kDa) took as long as 1 h to reach the centre of a
PEGA1900 bead, and complete conversion was observed
after 3 h. The enzyme reaction rate was an order of
magnitude less than that of the equivalent reaction in
solution. Similar reaction times were observed when
studying both enzymatic synthesis and hydrolysis reac-
tions by two different proteases of similar size, suggesting
that enzyme diffusion through the pores and not enzyme
kinetics is rate limiting [22].
Some new support materials have been presented as
offering better enzyme accessibility. By including per-
manent charges in the PEGA support the bead swelling in
aqueous media was increased and enzymes of opposite
net charge could be attracted into the support resulting in
higher catalytic yields [34,35]. A new resin (EXPO3000)
could be used in chemical synthesis steps in a relatively
compact form until selective cleavage of a silyl-based
cross-linker expands the polar resin to render it pene-
trable to enzymatic reaction [36]. Other silicon oxide
materials, organically modified xerogels, were demon-
strated to be partially accessible to small proteases such
as thermolysin and almost completely inaccessible to
penicillin G amidase (88 kDa) [37]. Finally, Basso et al.
[38] report a non-swelling organic polymer support that
has pores able to provide a reasonable loading of reactive
groups, but which also allow enzyme access.
Effect of the solid support on the equilibrium
position of the enzymatic reaction
At first sight it may seem a little surprising that the
equilibrium position of the catalysed reaction can be
affected by immobilisation of the substrate. However, a
major shift was observed when peptide hydrolysis on
PEGA1900 was studied in detail (Figure 5) [7,39]. When
phenylalanine (attached to PEGA1900) was incubated
with the protease thermolysin and an Fmoc-protected
amino acid such as Fmoc-glycine, the dipeptide was
formed in 99% yield as opposed to the 2.3% yield found
for the equivalent reaction in solution. This was not a
simple mass action effect, as the same concentrations of
free Fmoc-Gly were used in both cases. Two possible
explanations for the equilibrium shift were discussed: a
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Figure 5
(a)
H3N+–Phe–O–
(b)
H3N+–Phe–OMe
Thermolysin-catalysed peptide synthesis on PEGA polymers compared with aqueous solution. A saturated aqueous solution at pH 7.4 of
N-(9-fluorenyl methoxy carbonyl) glycine (Fmoc-Gly) was used in both cases. The percentage equilibrium conversion to peptide is shown
for the phenylalanine substrate (a) attached to PEGA1900 or (b) in solution [7]. There is a significant shift in equilibrium towards peptide synthesis
when the reaction is carried out on solid support.
hydrophobic effect of transferring the Fmoc-glycine out
of the aqueous phase and a suppression of the ionisation
of the amine as a result of mutual repulsion in the
immobilised state. More detailed studies [39] indicated
that the hydrophobic effect appeared to be dominant in
these experiments, contributing as much as a 10 000-fold
increase in the equilibrium ratio of amide to amine. The
effect was greatest for amino acids with the most hydro-
phobic sidechains and/or protecting groups.
This effect may explain poor yields observed in hydro-
lyses on solid-phase substrates, where an unfavourable
equilibrium could be a real possibility even in aqueous
media, contrary to normal expectation.
The kinetics of enzymatic reaction
It has commonly been noted that even when the enzyme
can gain access to the substrate molecules, the observed
rates seem to be poor compared with the corresponding
reaction in solution. Many applications use large enzyme
concentrations, long reaction times or both, and several
recent papers provide new observations of this type
[11,12,40]. Some recently reported work that makes
use of new analytical methods specially tailored to look
at reactions on solid-phase supports has investigated the
reasons behind the reduced rates.
Turning to the analysis of kinetic data, Gutierrez et al.
[41,42] make an important theoretical point about
many solid-phase enzyme assays. They note that these
operate under conditions of enzyme excess, while a
significant fraction of the immobilized substrate is present
as the complex with the enzyme. As a result, the kinetic
regime is quite different from most conventional solution
reactions. Gutierrez and colleagues have developed
model equations that describe the expected behaviour,
and which point to important consequences for how
quantitative enzyme assays should be performed in this
solid-state format. These conditions undoubtedly apply
in many assay applications, including that with HIV
protease described by the authors. They might also apply
in some preparative applications, where very high
enzyme concentrations are used in an attempt to obtain
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Enzyme action on immobilised substrates Halling, Ulijn and Flitsch 389
+ Fmoc-Gly
Fmoc–Gly–Phe–O– 99%
Fmoc–Gly–Phe–OMe 2.3%
Current Opinion in Biotechnology
good yields. Of course the objective in a preparative use
should be a genuine catalytic reaction, where many moles
of substrate are converted for each mole of enzyme. In
this case the kinetics will be closer to conventional
behaviour, but immobilization of the substrate will still
cause some differences, which we are currently analysing
in more detail.
Nishino et al. [28,29] report the analysis of quartz
crystal microbalance traces (see above and Figure 4) to
determine kinetic parameters for the enzyme binding to
the substrate and for catalysis. In the first study with
glucoamylase, kinetic constants were estimated by fitting
an overall model. In the second study, the very low
activity of glycogen phosphorylase in the absence of its
activator AMP was exploited to measure enzyme binding
independently. Hence, the parameter estimates are not
dependent on the correctness of the full kinetic model.
The kinetic constants obtained could be directly com-
pared with solution assays. The kcat values estimated for
both glucoamylase and glycogen phosphorylase were in
good agreement with values previously determined for
the same enzymes with dissolved substrates. The pre-
viously published Km for glucoamylase was some four
orders of magnitude greater than the enzyme–substrate
dissociation constant calculated from binding and disso-
ciation rate constants. The authors point out that these
should not be expected to be equal, because the system is
very far from the regime (k 1  kcat) where this is found.
However, calculating the expected Km for the simple
Michaelis–Menten steady-state model from their rate
constants gives a value two orders of magnitude greater
than the literature value for the reaction in solution. A
similar calculation for the phosphorylase also gives a Km
value two orders of magnitude larger than the solution
value. There are clearly complexities in the kinetics of
these systems still to be understood. It should be noted
that both these enzymes are adapted to work on very large
molecule substrates, albeit more or less dissolved ones.
A detailed study of kinetics was reported by Altreuter
et al. [6] for an enzymatic solid-phase synthesis of
dipeptides in organic solvents. For comparison, the
Current Opinion in Biotechnology 2005, 16:385–392
390 Protein technologies and commercial enzymes
equivalent reaction in solution was included in the study
and was found to be typically 1–2 orders of magnitude
faster, similar to the reduction in reaction rates observed
by Bosma et al. [27]. Salisbury et al. [14] compared the
relative rates for different peptide substrates in solution
and on the solid phase. They found reasonable agree-
ment, although they did not attempt to compare absolute
rates.
Applications
A wide variety of new applications of enzyme action on
solid-phase substrates has been described during the
review period [8–12,18,22,23,40,43–45], of which only
an outline survey will be given here.
With the advent of microarray technology, substrates
immobilised on flat glass or gold surfaces are becoming
more important. In such cases good accessibility of the
enzyme to the immobilised substrate is expected, and
indeed, complete enzymatic conversions have been
described in several studies [30,32]. The main issue here
is compatibility of the surface toward non-specific protein
adsorption and, consequently, steric blockage of substrate
molecules and/or loss of enzyme activity. This is usually
addressed by modification of the surface with protein-
resistant molecules such as PEG (or by pre-coating the
surface with an inert protein such as bovine serum albu-
min [14].
Self-assembled monolayers (SAMs) of functionalised
alkanethiols on gold provide structurally well-defined
substrates which have several advantages in studying
bio-interfacial science: they can be bio-inert; ligand
density can be controlled by mixing functionalised with
non-functionalised alkanethiols in precise ratios; functio-
nalisation of alkanethiols is straightforward; and several
analytical methods can be used to monitor the reaction,
such as radiolabelling, mass spectrometry (MALDI) [31]
and surface plasmon resonance spectroscopy. Interest-
ingly, it was found that yields of enzymatic reactions were
dependent on ligand density and were affected by steric
crowding of the ligand: yields of galactosylation reactions
of SAMS catalysed by bovine galactosyltransferase
increased with an increase in ligand density up to 70%
and then decreased dramatically with larger densities [46].
The described shift in equilibrium position from amide
hydrolysis in solution to synthesis on solid support was
exploited in the protease-catalyzed synthesis of several
phenylalanine dipeptides on PEGA1900 [7] and on syn-
beads [38]. These reactions were remarkably successful
and complete conversions to dipeptides were observed in
several cases. It was found that by tuning the reaction
conditions, both acylation and hydrolysis reactions were
feasible. It was demonstrated that protease enantiospe-
cificity could be exploited in such systems to access both
diastereoisomeric dipeptides via either acylation or
Current Opinion in Biotechnology 2005, 16:385–392
hydrolysis reactions starting from racemic mixtures of
amino acids [8]. The feasibility of acylation reactions
on PEGA supports is not limited to proteases. It was
demonstrated that several lipases could be used for the
kinetic resolution of racemic esters on solid-phase using
an acylation/deacylation capture and release strategy.
The reactions exhibit enantiospecificity that is consistent
with the operation of a parallel kinetic resolution process
[10]. Synthetic reactions on PEGA beads are not exclu-
sive to hydrophobic substrates, as demonstrated recently
by the synthesis of disaccharides by a glycosynthase
(51 kDa) on PEGA1900 [9]. It was recently demonstrated
that the primary specificity of proteases could be studied
by looking at the protease-catalyzed coupling of fluores-
cent-labelled amino acids to PEGA1900-immobilised
amino acids instead of the hydrolysis of peptides. This
method greatly simplified the screening process [22]. In
screening for enzyme selectivity, it is expected that gel
arrays will increasingly replace two-dimensional surfaces
for several reasons, including higher loading possibilities
and the avoidance of protein denaturation at the solid–
liquid interface. One recent application demonstrated a
bead array variant of the DNA chip where the polymerase
chain reaction (PCR) cycling took place on oligonucleo-
tide primers that were immobilized on individual beads
[40].
With the increased understanding of enzyme reactions
with immobilised substrates it is likely that these systems
will find new applications beyond those in screening and
solid-phase chemistry described above. For example, Yeo
and Mrksich [30] recently demonstrated the possibility of
using an enzymatic conversion to switch a surface from
being redox inactive to redox active. A recent paper by
Chilkoti’s group [47] describes the use of an atomic force
microscopy tip to deposit DNase onto immobilised DNA
on a gold surface, thus allowing for hydrolysis of DNA in
nano-sized patterns (enzymatic nanolithography).
Conclusions
Valuable new methods have been described to follow
enzyme action on immobilised substrates. We can expect
further improvement to offer greater ease, simplicity,
spatial resolution and real-time operation. The new meth-
ods have already given a better understanding of the issue
of enzyme accessibility to the bead interior, and this
should continue. The development of new supports
should deliver a range suitable for enzymatic solid-phase
reactions. The somewhat unexpected large effects on
chemical equilibrium position are well established, and
should continue to be exploited. We are still at a fairly
early stage of understanding enzyme kinetics when the
substrate is attached to a solid phase, but progress has
been made. Applications of enzyme reactions on immo-
bilised substrates continue to grow and underlie the
importance of better fundamental understanding of these
systems.
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 Enzyme action on immobilised substrates Halling, Ulijn and Flitsch 391
Update
An interesting new application has just been reported by
Su et al. [48]. Solutions of different protein kinases flowed
in microfluidic channels across a plane surface that had
bands of different immobilised peptides arrayed at right
angles to the flow. Spots of reaction identified where a
kinase recognised the peptide on display.
References and recommended reading
Papers of particular interest, published within the annual period of
review, have been highlighted as:
 of special interest
 of outstanding interest
1. Bayer EA, Belaich JP, Shoham Y, Lamed R: The cellulosomes:
multienzyme machines for degradation of plant cell wall
polysaccharides. Annu Rev Microbiol 2004, 58:521-554.
2. Boraston AB, Bolam DN, Gilbert HJ, Davies GJ: Carbohydrate-
binding modules: fine-tuning polysaccharide recognition.
Biochem J 2004, 382:769-781.
3. Watanabe K: Collagenolytic proteases from bacteria. Appl
Microbiol Biotechnol 2004, 63:520-526.
4. Kadereit D, Waldmann H: Enzymatic protecting group
techniques. Chem Rev 2001, 101:3367-3396.
5. Reents R, Jeyaraj DA, Waldmann H: Biocatalysis in polymer-
supported synthesis: enzyme-labile linker groups. Adv. Synth.
Catal. 2001, 343:501-513.
6. Altreuter DH, Dordick JS, Clark DS: Solid-phase peptide
 synthesis by ion-paired a-chymotrypsin in nonaqueous
media. Biotechnol Bioeng 2003, 81:809-817.
The first report of reaction on an immobilized substrate by an enzyme in
organic media. Chymotrypsin was solubilised by the ion-pairing method,
and catalysed aminolysis of Z-Phe-OMe by leucine residues bound to
TentaGelTM beads, with yields up to 98%. Highest initial rates were found
in 90% isooctane 10% tetrahydrofuran, and were an order of magnitude
lower than the same reaction in organic solution.
7. Ulijn RV, Baragaña B, Halling PJ, Flitsch SL: Protease-catalyzed
 peptide synthesis on solid support. J Am Chem Soc 2002,
124:10988-10989.
The equilibrium for peptide synthesis can be much more favourable when
one substrate and the product are attached to a solid support. Several
peptides could be synthesized in yields over 99% even in aqueous media,
using thermolysin catalysis. The possible reasons for this equilibrium shift
are outlined.
8. Ulijn RV, Bisek N, Flitsch SL: Enzymatic optical resolution via
acylation-hydrolysis on a solid support. Org Biomol Chem 2003,
1:621-622.
9. Tolborg JF, Petersen L, Jensen KJ, Mayer C, Jakeman DL,
Warren RAJ, Withers SG: Solid-phase oligosaccharide and
glycopeptide synthesis using glycosynthases. J Org Chem
2002, 67:4143-4149.
10. Humphrey CE, Turner NJ, Easson MAM, Flitsch SL, Ulijn RV:
Lipase-catalyzed kinetic resolution on solid-phase via a
‘capture and release’ strategy. J Am Chem Soc 2003,
125:13952-13953.
11. Kohli RM, Burke MD, Tao JH, Walsh CT: Chemoenzymatic route
to macrocyclic hybrid peptide/polyketide-like molecules. J Am
Chem Soc 2003, 125:7160-7161.
12. Wu X, Bu X, Wong KM, Yan WL, Guo ZH: Biomimetic synthesis of
gramicidin S and analogues by enzymatic cyclization of linear
precursors on solid support. Org Lett 2003, 5:1749-1752.
13. Meldal M: The one-bead two-compound assay for solid phase
screening of combinatorial libraries. Biopolymers 2002,
66:93-100.
14. Salisbury CM, Maly DJ, Ellman JA: Peptide microarrays for the
 determination of protease substrate specificity. J Am Chem
Soc 2002, 124:14868-14870.
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A peptide is joined by an amide bond to a coumarin ring that is attached
by a linker to the solid-phase surface. The conjugate is non-fluorescent,
but protease-catalysed hydrolysis of the amide liberates a strongly
fluorescent free aminocoumarin, still attached to the surface. An array
on a plane surface with different peptide sequences is used to screen
enzyme specificity with fluorescence imaging. The relative rates on
different substrates were in reasonable agreement between the surface
immobilised and solution assays.
15. Zhu Q, Uttamchandani M, Li DB, Lesaicherre ML, Yao SQ:
 Enzymatic profiling system in a small-molecule microarray.
Org Lett 2003, 5:1257-1260.
A coumarin ring has an attached linker that will allow it to be coupled to
the solid surface. An amino or hydroxyl group on the coumarin is modified
with an enzyme substrate moiety, which renders the conjugate non-
fluorescent. The conjugate is then attached to the surface in array format.
Enzyme action liberates the free amino or hydroxyl group, inducing
fluorescence. This may occur either directly (e.g. amide or phosphate
ester hydrolysis) or indirectly (e.g. enzyme reaction produces a diol that is
oxidized by periodate and then undergoes spontaneous b-elimination).
16. Lee Y-S, Mrksich M: Protein chips: from concept to practice.
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17. Houseman BT, Huh JH, Kron SJ, Mrksich M: Peptide chips for the
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18. Hansen KK, Hansen HC, Clark RC, Bartlett PA: Identification of
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proteomics. Curr Top Med Chem 2003, 3:705-724.
20. Uttamchandani M, Chan EWS, Chen GYJ, Yao SQ: Combinatorial
peptide microarrays for the rapid determination of kinase
specificity. Bioorg Med Chem Lett 2003, 13:2997-3000.
21. Min DH, Mrksich M: Peptide arrays: towards routine
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primary protease specificity by peptide synthesis on a solid
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Peptides attached to the solid phase were labeled with a 4-cyanoben-
zamide group at the N terminus, in the portion that would be released after
protease action. The CN stretching gives a distinctive Raman signal that
can be used to assess the extent of reaction on single beads. Only MMP-
12 (matrix metalloprotease; 22 kDa) and thermolysin (35 kDa) were able to
work well on PEGA, while MMP-13 (42.5 kDa), Clostridium collagenase
(68 kDa), and NEP (neutral endopeptidase; 90 kDa) showed no activity. All
enzymes gave good cleavage on controlled pore glass particles with
nominal pore size 100 nm, while none worked with TentaGelTM.
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 Using two photon microscopy to quantify enzymatic
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After various periods of protease action, liberated amino groups on the
solid phase were derivatised with dansyl groups. The extent of reaction at
all sites inside the bead could then be determined by imaging using two-
photon fluorescence microscopy. It was found that the thermolysin
reaction at the center of a PEGA bead only reached a rate similar to that
on the surface regions after 1 h. This is presumed to reflect the time taken
for the enzyme to diffuse through the bead.
Current Opinion in Biotechnology 2005, 16:385–392
392 Protein technologies and commercial enzymes
28. Nishino H, Nihira T, Mori T, Okahata Y: Direct monitoring of
 enzymatic glucan hydrolysis on a 27-MHz quartz-crystal
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Amylopectin substrate was immobilized on the surface of a quartz crystal
microbalance, then glucoamylase was added. An initial increase in mass
could be attributed to binding of the enzyme to the surface, and a
subsequent progressive decrease to liberation of glucose into solution.
Fitting to simulated curves gave values of the rate constants for enzyme
binding, dissociation and catalytic reaction (kcat).
29. Nishino H, Murakawa A, Mori T, Okahata Y: Kinetic studies of
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The same basic approach as described previously [28] was used to
study the binding and catalytic action of glycogen phosphorylase on
immobilized amylopectin. In this case, the very low activity of this enzyme
in the absence of AMP was exploited to measure the rate constants and
extent of binding in a first step. The AMP was added, and the resulting
rate of mass decrease used to estimate kcat. Studying the effect of AMP
concentration also gave a binding constant for this activator.
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32. Su J, Mrksich M: Using mass spectrometry to characterize self-
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33. Houseman BT, Mrksich M: Carbohydrate arrays for the
evaluation of protein binding and enzymatic modification.
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PEGA-type supports were made carrying quaternary ammonium groups
to give a permanent positive charge. The charge made the beads swell
more in aqueous media and gave much better action with the 88 kDa
enzyme penicillin G amidase, presumably because the negatively
charged enzyme was attracted into the beads.
35. Basso A, Ulijn RV, Flitsch SL, Margetts G, Brazendale I, Ebert C,
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and making it suitable for an enzymatic reaction. It was shown to facilitate
reaction with the protease subtilisin (27 kDa).
37. Basso A, De Martin L, Ebert C, Linda P, Gardossi L, Ulijn RV,
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Current Opinion in Biotechnology 2005, 16:385–392
improved efficiency of solid-phase biotransformations. Chem
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The authors introduce a new type of support specifically designed for
enzymatic reactions. This rigid support has pores that allow good access
by the relatively large penicillin G amidase (88 kDa).
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The factors causing equilibrium shift with immobilised substrates were
examined in more detail to distinguish three effects: excess dissolved
substrate shows mass action; hydrophobic groups are removed from
aqueous solution; and mutual electrostatic repulsion suppresses ionisa-
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40. Dressman D, Yan H, Traverso G, Kinzler KW, Vogelstein B:
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In developing this assay using the cleavage of immobilised substrate
peptides, the authors point out a previously neglected feature of such
reaction systems. The enzyme is effectively in excess, while a significant
fraction of the substrate is present as the complex with the enzyme.
Hence the kinetic expression resembles the Michaelis–Menten equation,
but has the enzyme concentration in place of the substrate concentration.
Use of this expression allows a better quantitation of enzyme activity and
estimation of inhibitor potency.
42. Gutierrez OA, Chavez M, Lissi E: A theoretical approach to some
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The authors develop the kinetic model previously introduced [41]. In
particular, they show the consequences for optimal assay sensitivity of
choices of enzyme concentration, reaction time and data analysis
method. They also consider inhibitor effects and the optimal conditions
for quantitation of these.
43. Kim JH, Hong JA, Yoon M, Yoon MY, Jeong HS, Hwang HJ: Solid-
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46. Houseman BT, Mrksich M: The role of ligand density in the
enzymatic glycosylation of carbohydrates presented on
self-assembled monolayers of alkanethiolates on gold.
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