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Enzyme assays
Jean-Louis Reymond,* Viviana S. Fluxa` and Noelie Maillard
Received (in Cambridge, UK) 6th August 2008, Accepted 4th September 2008
First published as an Advance Article on the web 17th October 2008
DOI: 10.1039/b813732c
Enzyme assays are analytical tools to visualize enzyme activities. In recent years a large variety of
enzyme assays have been developed to assist the discovery and optimization of industrial
enzymes, in particular for white biotechnology where selective enzymes are used with great
success for economically viable, mild and environmentally benign production processes. The
present article highlights the aspects of uorogenic and chromogenic substrates, sensors, and
enzyme ngerprinting, which are our particular areas of interest.
1. Introduction
Enzyme assays are analytical tools to visualize enzyme activities. In recent years a large variety of enzyme assays have
been developed to assist the discovery and optimization of
industrial enzymes, in particular for white biotechnology
where selective enzymes are used with great success for
economically viable, mild and environmentally benign production processes.1,2 In this context the enzyme assays serve to
screen collections of enzymes available from strain collections,
metagenomic libraries and libraries of mutant enzymes
obtained by random or directed mutagenesis from known
enzymes. This type of screening must be distinguished
from genetic selection experiments, in which a mutation/
selection protocol is set up to allow for the survival of
microorganisms producing an active enzyme, e.g. by complementation of a biosynthetic pathway in an auxotrophic
bacterial or yeast strain. Both high-throughput screening
Viviana Fluxa
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to produce indigo is by fermentation.18 Cleavage of the glycosidic bond forms an unstable hydroxyindole intermediate,
which dimerizes oxidatively at air to form indigo as a blue
precipitate. A number of enzyme substrates have been designed
following this natural product example. For instance, numerous
glycosides of uorescent or colored phenols are used to test
glycosidases, e.g. nitrophenyl b-galactoside (1) for detection
of b-galactosidase activity, p-nitrophenyl caproate (2) as a
chromogenic lipase substrate, and the nitrophenyl octyl ether
3 to detect cytochrome P450 activity19,20 (Fig. 1). Note that the
yellow color of nitrophenolate is only visible above pH 7.
Naphthols can be detected indirectly by a secondary reaction
with diazonium salts to form azo-dyes, a principle used in
cytochemistry to test esterase activities in tissue samples with
naphthyl acetate,21 and recently adapted to assay aldolase
antibodies in agar plates.22
The range of phenol release substrates can be extended using
indirect release mechanisms, as we rst demonstrated with the
alcohol dehydrogenase enzyme substrate 5 (Fig. 2). This chiral
secondary alcohol is oxidized by the enzyme to form the
corresponding ketone 6, which is unstable and undergoes a
b-elimination reaction catalyzed by bovine serum albumin to
produce the blue uorescent umbelliferone anion 7.23,24 The
signal is only visible above pH 7 where the product exists as a
uorescent anion. The same principle allowed substrates for
aldolase catalytic antibodies2527 and proline-type catalysts,28,29
transaldolases,30 transketolases,31 and lipases.32 A related scheme
involving an intermediate hemiacetal provides various lipase and
esterase substrates,3335 as wells as a uorogenic substrate for
BaeyerVilliger monoxygenases (BVMO) in the form of the
2-aryloxyketone 8,36 via the intermediate lactone 9 which may
be considered as a lactonase-type probe, although it is quite
unstable and spontaneously hydrolyzes in the whole cell
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Fig. 1
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response to kinase activity, the probe provides a strong luminescence enhancement, resulting from the increased ability of the
probe to bind and sensitize Tb3+ and Eu3+ ions upon phosphorylation.67 Fluorescence modulations by aggregation,
dilution or phase change are also related to FRET substrates.
Thus, lipases can be assayed with 1,3-dioleoyl-2-(4-pyrenylbutanoyl)glycerol in the presence of lipoproteins and albumin.68
Ester hydrolysis releases the pyrene carboxylate, which then binds
to albumin, resulting in a uorescence increase. The commercially
available FITC-conjugates of casein are used as protease substrates, whereby proteolysis removes the autoquenching and leads
to stronger uorescence of the uorescein chromophores.69
Recently reported protease-sensitive nanobers consisting of
aggregated b-sheet forming peptides also rely on dilution-induced
release of autoquenching upon proteolytic cleavage.70
FRET
Many enzyme assays are based on FRET (Forster or Fluorescence Resonance Energy Transfer) as detection principle, for
instance to measure hydrolytic reactions separating uorophore
from quencher in the case of proteases,15,16,5156 cellulases,57 and
lipases,5860 and in the synthetic direction for fucosyl transferases.61 Aldehyde 18 features an interesting use of FRET, in
which addition of a nucleophile to the aldehyde such as an
antibody catalyzed aldol addition to form 19 (Fig. 6) removes the
intramolecular quenching eect.6264 Fluorescence of a label can
also be modulated by proximity eects such as medium eects.
For example, in an aminonitrobenzofurazane-labeled g-cyclodextrin reported as an a-amylase uorogenic substrate, cleavage
of the cyclodextrin ring by the amylase exposes the uorophore
to water and reduces uorescence intensity.65 Fluorescent substrates for kinase substrates have been recently reviewed.66 In a
recent example, a luminogenic probe was developed for tyrosine
phosphorylation based on a short peptide sequence containing
an iminodiacetate moiety near the site of phosphorylation. In
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3. Indicator assays
A variety of relatively simple chemosensor systems based on
chromogenic or uorogenic reagents can convert a chemical
transformations into a detectable signal, often through a functional group specic reaction or by a separation eect. Biosensors binding to either substrate or product may be used
similarly. Such indicator assays can be used to assay reactions
of specic, unlabeled substrates, which is necessary whenever a
very specic reaction is being optimized. The main drawback of
indicator assays is that they are often sensitive to interferences
(e.g. other events than catalytic turnover may produce a signal,
or turnover may be masked) and may require narrow assay
conditions that render them incompatible with certain enzymes.
In addition the reporter chemistry may be rate limiting, which
prevents their use for kinetic studies. These potential limitations
may be overcome for a high-throughput screening application
by using proper controls and relying on an endpoint measurement rather than on measuring a reaction rate. As long as they
can test authentic substrates, indicator assays will remain one of
the best option for enzyme assays in the future.
3.1
Enzyme-coupled assays
One of the most straightforward methods to render an enzymecatalyzed reaction detectable consists in further converting the
reaction product by a second enzyme to form a second product
38 | Chem. Commun., 2009, 3446
Fig. 8 Dual-wavelength spectrophotometric tracking of two chromogens (NADP+ and ABTS) for the determination of ketoreductase
(KRED) activity and enantioselectivity.
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4. Fingerprinting
Meldal and co-workers reported the use of synthetic combinatorial libraries of millions of synthetic peptides as FRET
substrates to analyze protease reactivity on solid
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recently reported an assay for on-bead proteolysis of a solidsupported combinatorial library of N-acetylated non-tagged
octapeptides AcL (Fig. 13).214 After proteolysis, the free
N-termini are simply stained by reductive alkylation with a
tagged aldehyde, and the stained beads are analyzed.
4.3 Cocktail ngerprinting
Fig. 11 Fingerprint analysis of chain length selectivity of lipases and
esterases.
support,
a very practical method still under further
development today.212,213 One of the diculties in the analysis
is the necessity to introduce a uorescent label on the cleaved
peptide, which may reduce the reactivity of the protease. We
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containing three uorogenic protease substrates on a microarray on which spots of enzyme had been previously printed,
allowing high-throughput screening of enzyme inhibitors.232
Depositing uorogenic substrates on poly-lysine-coated glass
slides also allows ecient assays of various enzymes in nanodroplets.233 Nanodroplets for enzyme assays can be moved
using thermal gradients for mixing.234 Fluorogenic substrates
have also been arrayed with covalent attachment to the surface
of glass slides to allow activity proling experiments with
proteases and other hydrolytic enzymes using combinatorial
series of uorogenic coumarin-derived substrates35,235,236 and
with lipases using substrate of varying acyl chain length 35af,
relying on chemoselective oxidation of the 1,2-diol product
with sodium periodate followed by reaction with rhodamine
sulfohydrazide to detect conversion (Fig. 14).237
Microarrays have also been used for an elegant protease
proling method based on combinatorial libraries of
PNA-encoded dipeptidyl-rhodamine substrates.238241 An
important improvement in the study of the biology of phosphatases was developed in a microarray. Glass slides having
multiple peptide substrates of Ser/Thr phosphatases
immobilized on them, can be used to simultaneously determine
the preference of the enzymes for the dierent substrates.242
Protease proling was also recently reported based on a
multiplexed solution phase assay on microarrays.243 Microarrays were also reported for enantioselectivity, for example to
estimate the optical purity of amino acids after covalent
attachment by reaction with a pseudo-enantiomeric pair of
labels bearing two dierent uorophores based on Horeaus
method.244246 Mass spectrometry was applied for the creation
of biochips loaded with label-free oligosaccharide arrays, in
order to study glycosyltransferases activities.247
It should be mentioned that it is possible to handle lowvolume enzyme assays at the scale of one microliter per
assay using silicagel plates pre-impregnated with a uorogenic
substrate as the reaction medium, which provides a practical
42 | Chem. Commun., 2009, 3446
solution for miniaturization that is much simpler than microarrays.248 A robotic arm is used to dispense the enzyme
containing test solutions in a volume of one microliter per
assay, which results in a homogeneously dispersed spot on the
silicagel surface on which the enzyme reacts evenly with the
substrate (Fig. 15).
5. Conclusion
In recent years enzyme assays have greatly advanced in their
scope and in the diversity of detection principles employed. In
the 1990s high-throughput screening of enzyme activity was
perceived as a critical bottleneck in enzyme engineering due to
the advent of random mutagenesis methods for directed evolution, which multiplied demands for screening by orders of
magnitude. Developments of new screening methods based on
chemistry, biology and instrumentation have followed to rise to
this challenge, in part by reviving and rening older methods.
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Acknowledgements
This work was supported by the Swiss National Science
Foundation and Proteus SA, N mes, France.
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