Substrate Arrays for Fluorescence-Based

Enzyme Fingerprinting
and High-Throughput Screening
JEAN-LOUIS REYMOND
Department of Chemistry and Biochemistry, University of Berne, Berne, Switzerland

Indirect release of fluorogenic phenols such as umbelliferone was used as a chemical principle
to prepare fluorogenic substrates for a variety of enzymes in which the enzyme-reactive group
is separated from the fluorescent reporter group, allowing structural and functional diversifica-
tion. Fluorescent probes were obtained for enzymes useful for asymmetric synthesis including
aldolase catalytic antibodies, transaldolases, alcohol dehydrogenases, lipases, epoxide hydrolases,
proteases, and Bayer-Villiger monoxygenases. Arrays of structurally related substrates were pre-
pared and used to record the activity of enzymes on multiple substrates simultaneously in parallel
formats such as microtiter plates, microarrays, and substrate cocktails, leading to enzyme-specific
activity patterns. Arrays of nonlabeled substrates were assayed using indirect product sensors such
as the adrenaline-periodate back-titration assay for 1,2-diols or the copper–calcein assay for amino
acids. Many of the fluorogenic substrates and sensors described here are either commercially
available or accessible in one or two simple synthetic steps and can be used for high-throughput
screening of enzyme activities.

Key words: catalytic antibodies; enzyme assays; fluorogenic substrates; chemosensors; activity
fingerprinting; profiling; proteases; lipases; epoxide hydrolases; alcohol deydrogenases; Bayer-
Villiger monoxygenases; microarrays; fluorescence; high-throughput screening; acridone; umbel-
liferone; adrenaline; fluorescein.

Introduction which catalyzes an enantioselective protonation reac-

One of the main activities in biocatalysis consists
in developing enzymes for industrial applications, par-
ticularly in the areas of fine chemical synthesis, food
processing, and polymers. Enzymes are discovered and
improved by screening various microbial collections
and mutant libraries for the desired activity. Enzyme
assays, which make up analytical methods for the mea-
surement of enzyme activity, play a critical role in this
process, especially assays suitable for high-throughput
screening in microtiter plate or in agar plates.1
The facilitating role of fluorescence assays for high-
throughput screening during enzyme discovery can be
illustrated by two examples in the area of catalytic
antibodies. Antibodies with catalytic activities can be
discovered by screening series of monoclonal antibod-
ies binding specifically to small molecules mimick-
ing the reaction’s transition state.2 Antibody 14D9,
Address for correspondence: Jean-Louis Reymond, Department of
Chemistry and Biochemistry, University of Berne, Freiestrasse 3, 3012
Berne, Switzerland. Voice: +41 31 631 43 25; fax: +41 31 631 80 57.
jean-louis.reymond@ioc.unibe.ch
Ann. N.Y. Acad. Sci. 1130: 12–20 (2008).

C 2008 New York Academy of Sciences.
doi: 10.1196/annals.1430.000 12
tion,3 was discovered by screening approximately 60
different monoclonal antibody–producing hybridoma
cell lines using a high-performance liquid chromatog-
raphy (HPLC)-based assay.4 The project required the
tedious and expensive subcloning of a large number of
hybridoma cell lines to enable the activity screening.
By contrast, antibody 10F11, which catalyzes a very
unusual retro-Diels-Alder reaction,5 was discovered
by direct high-throughput screening of approximately
15,000 samples of initial, unstabilized hybridoma cell
lines, using the fact that the reaction produces a blue
fluorescent product.6 In this experiment cell culture
resources were concentrated on catalytically active hy-
bridoma from the very beginning. Eventually, the ex-
periment resulted in over 10 different catalytic anti-
bodies for the reaction using only a fraction of the
resources used previously for cell culture.
The advantage of fluorescence-based screening for
biocatalyst discovery led us to attempt to expand the
range of reactions available to fluorescence screening.
Initial experiments used acridone-labeled substrates
that were separated and identified at very low concen-
tration by thin-layer chromatography, however high-
throughput screening was still quite difficult to carry
Reymond: Fluorescence-Based Enzyme Screening
FIGURE 1. Fluorogenic enzyme substrates with β-elimination of umbelliferone. The assay is run in
microtiter plate wells in 100 µL of aqueous buffer (20 mM borate pH 8.8) with 100 µM substrate, 2 mg/ml
bovine serum albumin (BSA), and the biocatalyst (5–50 µg/ml).
out in this format.7 We next investigated antibody-
based fluorogenic sensors to selectively reveal product
formation.8 This sensor format was compatible with
high-throughput liquid handling in microtiter plates
but required expensive reagents, rendering it unprac-
tical. We therefore turned our attention to simple
fluorogenic substrates and sensors. At the time, clas-
sical fluorogenic enzyme substrates were almost en-
tirely confined to hydrolytic reactions involving bond-
cleaving reactions releasing a fluorescent of colored
phenols, such as the hydrolyis of 4-methyl-umbelliferyl-
β-D-galactoside. We explored the possibility of expand-
ing such fluorogenic processes to include nonhydrolytic
reactions with chiral functional groups.9
The structural diversity of fluorogenic substrates
made available by these developments led us to pro-
pose the principle of enzyme activity fingerprinting as
a new analytical tool for characterizing enzyme activ-
ities. Enzyme fingerprinting consists of recording the
activity of a given enzyme with an array of structurally
related but different substrates to characterize the ac-
tivity by an enzyme-specific reactivity pattern.10 This
review summarizes key discoveries in the area of fluo-
rogenic substrates and sensors for biotransformations
and their use to form substrate arrays suitable for en-
zyme activity fingerprinting. The issue of delivering
13
user-friendly screening assays is also discussed by high-
lighting our most favorable assays for practical use.
Separating the Enzyme Reactive
Group from the Fluorophore
Fluorophores are typically conjugated polycyclic
aromatics with a defined substitution pattern. In classi-
cal fluorogenic enzyme substrates the enzyme reactive
group is directly bound to the fluorophore, implying
that almost no structural and functional diversity is
allowed in the vicinity of the enzyme reactive group
which makes direct contact to the enzyme. We rea-
soned that it might be possible to expand the chemical
and structural diversity of fluorogenic substrates by
separating the fluorophore from the functional group
undergoing a biotransformation by triggering the flu-
orophore activation indirectly. This would also make
it possible to diminish or even suppress the influence
of the fluorophore on enzyme binding and reactivity,
which is generally not desirable in biocatalysis screen-
ing.
This principle was first realized in a chiral
fluorogenic alcohol dehydrogenase substrate (1)11
(FIG. 1). Oxidation of the secondary alcohol to
14 Annals of the New York Academy of Sciences

FIGURE 2. Periodate activated fluorogenic substrates. (5) substrate for epoxide hydrolases. (8) sub-
strate for lipases and esterases. (9) reference commercial lipase substrate. Assays are operated under the
conditions described in the legend of FIGURE 1, in the presence of 1 mM NaIO 4 as additional reagent.

the corresponding ketone allowed subsequent β-
elimination of umbelliferone as a fluorogenic process,
an assay which was also coupled to ester hydrolysis to
enable the detection of enantioselective lipases.12 The
principle was exploited for aldol type substrates for
aldolase catalytic antibodies (2)13 and transaldolases
(3)14 More recently, we showed that the simple probe
(4),15 which undergoes enolization followed by elimi-
nation of the fluorescent product umbelliferone, allows
one to detect aldolase-active small molecule and den-
dritic catalysts.16 The same principle can be extended
for indirect probes for lipases and acylases by releasing
(4) as a reaction product.17
The β-elimination for fluorophore release also al-
lowed screening of epoxide hydrolases using epox-
ide (5), whose hydrolysis produces the 1,2-diol (6)
(FIG. 2).18 Screening is carried out in the presence of
sodium periodate, which does not react with the epox-
ide but rapidly cleaves the 1,2-diol product of epoxide
hydrolysis to yield the unstable aldehyde intermediate
(7), from which β-elimination of umbelliferone fol-
lows. The assay is compatible with whole-cell micro-
bial screening.19 The release of the 1,2-diol (6) was
also exploited to form remarkably active fluorescence
probes for lipases such as (8).20 In this case, substrate
(8) showed negligible reaction in the absence of en-
zyme and was much highly reactive toward lipases.
By contrast the commercially available lipase probe
(9) showed much lower specific enzyme reactivity and
underwent spontaneous cleavage in buffer.
Enzyme Activity Fingerprinting
This selectivity toward specific enzyme reactions
combined with the possibility of altering the enzyme re-
active head of the substrate by functional group varia-
tions led us to formulate the concept of enzyme activity
fingerprinting, which consists in recording the activity
pattern of a given enzyme-containing sample against
a series of structurally distinct substrates.21 This activ-
ity pattern, which could be represented as an array of
colored pixels to facilitate data visualization, might be
considered as a “fingerprint” in the sense of an identifi-
cation tool if the measurements would be practical and
reproducible and would formally render an image of
the functional potential of the enzyme (FIG. 3).22 These
features could be ensured by the absence of nonspecific
Reymond: Fluorescence-Based Enzyme Screening
FIGURE 3. Principle of enzyme activity fingerprinting.
An enzyme-containing test sample is subjected to a series of
enzyme reactions in parallel (such as microtiter plate assays)
or in a combined way (substrate cocktail, microarray). The
pattern of individual reactivities obtained provides a multidi-
mensional set of information forming the activity fingerprint,
conveniently displayed as a grayscale bitmap as shown.
reactivity in our periodate-triggered substrates. The
fingerprint concept was related to the multisubstrate-
based identification of microbial strains using the API
ZYM system,23 which is used routinely in clinical mi-
crobiology.24 However, the API ZYM associated each
substrate with a different enzyme and produced a pat-
tern representing a panel of enzyme activities. In con-
trast, our fingerprinting arrays would produce a pat-
tern allowing us to distinguish two related enzymes of
the same type from one another.
We first demonstrated the enzyme fingerprinting
concept using an array of periodate-activated epoxides,
amides, and esters analogs of (5) and (8) featuring
various functional groups.21 To our delight, testing a
series of hydrolytic enzymes including various lipases,
esterases, acylases, and epoxide hydrolases showed that
each different enzyme indeed produced a specific and
readily reproducible reactivity pattern, which could be
conveniently represented in a two-color reactivity scale
indicating activity and stereoselectivity (FIG. 4).
The patterns observed with our first array were spe-
cific but could not be interpreted easily aside from the
simple distinction between functional group types, such
as epoxide hydrolysis versus ester, amide, or carbonate
hydrolysis. We next investigated lipases and esterases
using a series of periodate-activated monoester sub-
strates (10a-h) (FIG. 5) in both enantiomeric forms.25
In this case the reactivity patterns observed indicated
the chain-length selectivity of the enzymes. As ex-
pected, lipases were more active on long-chain sub-
strates, while esterase preferentially cleaved short-
chain substrates. Surprisingly, a selectivity for inter-
mediate chain-length substrates was apparent as the
second principal component of the observed diversity.
The experiment was later repeated with a related se-
ries of water soluble esters (11a-e) and (12a/b) de-
15
rived from fluorescein.26 These substrates allowed the
investigation of the reactivity of lipases and esterases
in pure aqueous environment, in contrast to classi-
cal fluorogenic probes for these enzymes requiring
cosolvents. The fluorescein-derived substrates showed
very high and specific enzyme reactivity, undergoing
cleavage within seconds. Substrates (11a-e) further-
more showed reduced nonspecific cleavage due to the
oxymethyl ether–type linkage between the enzyme re-
active ester and the fluorophore.27 In this case esterases
again stood out for their reactivity against short chain
substrates, although their reactivity against intermedi-
ate chain length (C8 and C10) was also remarkable.
Interestingly, lipases and esterases were clearly distin-
guished from one another by their different reactivity
in pure aqueous buffer versus buffer containing 20%
dimethyl sulfoxide as cosolvent, whereby esterases were
more active in aqueous environment while lipases re-
quired the cosolvent for highest activity, as shown by
the color-coded pattern (FIG. 5).
We next explored technical improvements to facil-
itate enzyme activity fingerprinting. On the enzyme
assay side of the experiment, we developed an in-
direct sensor system for product formation based on
back-titration of sodium periodate using adrenaline.28
This colorimetric assay is suitable for a broad range
of hydrolytic processes. We used it to carry out fin-
gerprinting with commercially available epoxides for
epoxide hydrolases, and polyol acetate for lipases and
esterases.29 However this assay format still required
microtiter plate handling, which we perceived as an
important limitation for fingerprinting because it re-
quired large volumes of enzyme solution and multiple
parallel pipetting operations, which were sources of
error and made high-throughput activity fingerprint-
ing difficult. We therefore investigated other assay for-
mats for fingerprinting. One of them used substrate
microarrays. Substrate arrays have been reported for
proteases and certain hydrolases,30 however attempts
to analyze lipase reactivity using microarrays had not
been reported. We developed a periodate-coupled tag-
ging method for microarray assay using an acyl-chain
length series of 12 substrates (13a-f) (6 pairs of enan-
tiomers), which allowed us to record the activity of
several lipases on microarrays (FIG. 6).31 Although the
system is elegant, the reactivity on the glass surface in
this system is limited to a handful of lipases despite
extensive optimization of surface attachment chem-
istry and assay conditions. Furthermore, the assay does
not reproduce the activity of the enzymes in solu-
tion, suggesting that other phenomena than substrate
binding and turnover determine the outcome of the
assay.
16 Annals of the New York Academy of Sciences

FIGURE 4. Enzyme fingerprints from a periodate activated substrate array. The substrates are derivatives of (5) and
(8) (FIG. 2) with different substitution patterns and enzyme reactive functional groups as shown in the array reference at
lower right (cpds numbers from original Ref. 22). The relative activity is color-coded in each array position according to
the relative reaction rate (color intensity) and enantioselectivity (blue to green ratio) according to the reference scale at
lower left. Below each array the enzyme abbreviation is given in three-letter code (see Ref. 22), and the number indicates
the maximum reaction rate of the most reactive substrate in the array in pM/sec.

The most practical format we identified for enzyme
fingerprinting involved formulating substrate cocktails
analyzable by HPLC.32 In this technique, the enzyme
is exposed to a well-defined mixture, or cocktail, of sev-
eral substrates selected for reactivity type and optimal
product separation. Fingerprinting is carried out in a
single, one-vial operation during which the enzyme
reaction with the cocktail is allowed to proceed for a
measured amount of time, and the product pattern
is determined by HPLC analysis. Each substrate and
product is labeled with a strong chromophore facili-
tating selective identification at a specific wavelength.
This labeling allows one to operate at low substrate
concentration, thus avoiding inhibition effects between
different substrates. We have reported cocktails to an-
alyze lipases and esterases and proteases.33 We also
demonstrated cocktail profiling of microorganisms in
a setup resembling the API ZYM series, but allowing
the analysis of thermophilic microorganisms.34 Sub-
strate cocktails currently under development in our
laboratory have proven useful in the characterization
of novel enzymes from the metagenome.35
User-Friendly Enzyme Assays
Enzyme assays are applied in biocatalysis research
during high-throughput screening as well as during
Reymond: Fluorescence-Based Enzyme Screening 17

FIGURE 5. Fingerprint analysis of chain length and cosolvent selectivity of lipases and esterases. All assays were run
in microtiter plates according to the conditions described in FIGURE 1 (substrates 11–12) or FIGURE 2 (substrates 10a-h).
The array represent the activity data with substrates 11a-h and 12a-b on a series of lipases and esterases (code at each
column heading) according to the reference color-scale shown below. Color intensity represents the relative reaction rate
(value of the maximum observed reaction rate is given below each column) and the blue-to-green ratio indicates the relative
reactivity in aqueous buffer vs. buffer + cosolvent. (Data from Ref. 26.)

enzyme purification and handling. Ideally the assay
uses the authentic substrate for the targeted biotrans-
formation, but a fluorogenic derivative might be ac-
ceptable if it is easily prepared and its reactivity is highly
specific. Assay reagents should be commercially avail-
able or synthesizable in one step from cheap reagents
using inexpensive and high-yielding synthetic proce-
dures. These constraints do not leave much room for
even relatively simple synthetic sequences, including
the preparation of the epoxide hydrolase substrate (5)
and its analog (8) (See FIG. 2) for lipases, which we
have prepared and made available to others upon re-
quest. One recent success story in our laboratory con-
cerns 2-aryloxyketones such as (14), which serve as
fluorogenic probes for Bayer-Villiger monoxygenases
(BVMO), a class of enzyme of current interest in bio-
catalysis.36 These substrates are easily prepared in one
step and offer a very direct measurement of BVMO
activity for whole-cell assays. Similarly, the FRET-type
lipase probe (15) is accessible in two steps and of-
fers an unprecedented possibility to assay lipases under
strongly alkaline conditions (pH 11), which are relevant
to the industrial application of these enzymes.37 The
commercially available and inexpensive fluorescein-
derived calcein (16) is also noteworthy. This fluores-
cent ligand strongly binds to Cu(II) ions to form a
18 Annals of the New York Academy of Sciences

FIGURE 6. A substrate microarray for fingerprinting lipase activities. Aminofunctionalized glass slides
were coated covalently with bovine serum albumin and the substrate using appropriate spacers based on
active ester chemistry and reductive alkylation. The assay includes stepwise incubation and glass-washing
cycles following the indicated sequence. The array is then recorded using a microarray scanner. Each
spot is present in quadruplicate on the array. See Ref. 31 for details.

nonfluorescent complex, which can be used to moni-
tor various amino acid–releasing enzyme reactions.38
Indeed amino acids strongly complex Cu(II) ions, such
that their formation from nonchelating precursors in-
duces a fluorescence increase in solutions by displace-
ment of the quenching Cu(II) to form free calcein. This
sensor system allows one to follow the reaction of the
enzyme amnopeptidase by fluorescence (FIG. 7).39
We have recently reported a practical method to
miniaturize microtiter plate operation to the microliter
level to facilitate high-throughput screening with fluo-
rogenic substrates based on silica gel plates.40 Silica gel
plates are soaked in a solution of the fluorogenic sub-
strate in an organic solvent such as dichloromethane
and dried, whereby the substrate is adsorbed on the
silica gel particles. The substrate-impregnated plates
can be stored without degradation of the substrate
and serve to test small volume of enzyme-containing
solution for activity. Typically, one microliter of the
enzyme-containing test solution is deposited on the
plate manually or using a robotic arm, and the evolu-
tion of fluorescence is followed visually. The assay was
demonstrated for testing lipases and glycosidases using
the corresponding umbelliferyl esters and glycosides.
We also recently found that soaking synthesis support
beads makes it possible to carry out high-throughput
screening of synthetic catalysts produced by split-and-
mix combinatorial synthesis, a method which we have
used to discover catalytically active dendritic enzyme
models.41
Conclusion
Fluorescence assay for enzyme activity screening
and fingerprinting is key to facilitate discovery and rou-
tine activity checking in biocatalysis research. Simple
chemical principles such as β-elimination of umbellif-
erone allowed us to expand the scope of fluorogenic
substrates beyond classical hydrolytic processes to al-
dol condensation, epoxide hydrolysis, or Bayer-Villiger
oxidation. The structural diversification of fluorogenic
enzyme substrates allowed us to explore activity finger-
printing as a means to characterize enzyme function.
The continuous development of assay chemistry and
format is essential to render high-throughput screening
Reymond: Fluorescence-Based Enzyme Screening 19

FIGURE 7. User-friendly fluorescence enzyme assays. Substrate 14 is a convenient fluorogenic substrate for Bayer-
Villiger monoxygenases (BVMOs, see Ref. 36). Substrate 15 is a FRET-type probe for lipases that is stable to alkaline pH
and allows screening at pH 11, see Ref. 37. Calcein (16) is a commercial fluorescein derivative that can be combined with
Cu(II) to form a nonfluorescent complex. The complex acts as a sensor for free amino acids in aqueous buffer, allowing one
to follow enzyme reactions such as the hydrolysis of leucinamide by aminopeptidase under the conditions shown. Details
in Ref. 39.

available to microbiologists as a tool for the discovery
and improvement of functional enzymes.
Acknowledgment
This work was supported by the University of Berne,
the Swiss National Science Foundation, and Protéus
SA, Nı̂mes, France.
Conflict of Interest
The author declares no conflicts of interest.
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