enzyme assay (2) 06.01.2014

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 fluorogenic and chromogenic substrates, sensors, and

enzyme fingerprinting, which are our particular areas of interest.

1. Introduction

Enzyme assays are analytical tools to visualize enzyme acti-

vities. 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 produc-

tion 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 comple-

mentation of a biosynthetic pathway in an auxotrophic

bacterial or yeast strain. Both high-throughput screening

and genetic selection are viable protocols for discovering and

improving enzymes.3–12 Important developments of enzyme

assays are also constantly occurring in relation to drug

discovery efforts, medical diagnostics and in the area of

cellular and tissue imaging. Enzyme assays are also used in

enzyme model studies.13

The majority of enzyme assays are developed to test isolated

enzymes or enzyme containing samples such as culture suspen-

sions in 96-well microtiter plates or similar parallel liquid

phase systems. The simplest and most practical enzyme assays

are based on synthetic substrates that release a colored or

fluorescent product upon reaction or induce a directly detect-

able change in solution such as a precipitation. Many such

substrates are commercially available and often serve as

reference substrates to determine absolute activities of enzyme

samples in Units. Enzyme reactions may also be assayed using

indicators which respond indirectly to product formation or

substrate consumption. The indicator may be as simple as a

pH-indicator and as complex as a functionalized nanoparticle.

Many assays are also based on analytical instruments such

HPLC, GC, MS, NMR or IR spectrometers. These instru-

ments often allow access to reaction parameters not otherwise

accessible such as enantioselectivity and therefore play a

Department of Chemistry and Biochemistry, University of Berne,Freiestrasse 3, Berne, 3012, Switzerland.E-mail: [email protected]; Fax: +41 31 631 80 57;Tel: +41 31 631 43 25

Jean-Louis Reymond

Jean-Louis Reymond was bornin Switzerland in 1963. Hegraduated from the ETH inZurich in 1985 and receivedhis PhD from the Universityof Lausanne in 1989. In 1990he joined the Scripps ResearchInstitute first as a post-doctoralfellow with R. A. Lerner, thenas an assistant professor. In1997 he moved to the Univer-sity of Berne, Switzerland, asan associate professor, andbecame a full professor in1998. His research touches onthree areas of bioorganic chem-

istry: (1) high-throughput screening assays for biocatalysis; (2)artificial protein design with peptide dendrimers; and (3) smallmolecule drug discovery.

Viviana Fluxa

Viviana Fluxa was born inSantiago de Chile in 1980. Shereceived her MSc in chemistryfrom Fribourg University in2006. While at Fribourg, shestudied diethylstilbestrol deriva-tives in the laboratory of Chris-tian Bochet. In March 2006, shejoined the Reymond group atthe University of Berne in theDepartment of Chemistry andBiochemistry. Her current re-search interests include proteaseprofiling with FRET peptidesand exploring the biological ac-tivity of cyclic peptides libraries.

34 | Chem. Commun., 2009, 34–46 This journal is �c The Royal Society of Chemistry 2009

FEATURE ARTICLE www.rsc.org/chemcomm | ChemComm

critical role in biocatalysis for the discovery and optimization

of selective enzymes by directed evolution. Enzyme assays

have been the subject of a recent volume providing an over-

view in the perspective of biocatalysis.14 The present article

highlights the aspects of fluorogenic and chromogenic sub-

strates, sensors, and enzyme fingerprinting, which are our

particular areas of interest.

2. Chromogenic and fluorogenic substrates

Fluorogenic and chromogenic enzyme substrates form the

cornerstone of enzyme assay technology. They incorporate a

chromophore whose absorbency or fluorescence properties

change as a result of the enzyme reaction. The key advantage

of these substrates is that the assay is very simple and the

signal produced is directly related to the enzyme-catalyzed

reaction. If the colored or fluorescent product is soluble, the

assay is well-suited for microtiterplate based assays. On the

other hand, if the product is insoluble in the reaction media,

the substrate can be used for screening bacterial cultures on

agar plates. Almost all examples to date focus on a small

family of fluorophores and chromophores, in particular

umbelliferones, nitrophenols, fluoresceins, rhodamines and

BODIPY dyes, all of which are relatively large aromatic

groups which tend to influence both substrate binding

(stronger binding), catalytic turnover (reactivity may be

lowered or absent compared to non-labeled substrates), and

solubility. Encouraging recent reports by Nau and co-workers

suggest that new, non-aromatic fluorophores might provide an

alternative for FRET type assays.15,16 Alternatively various

chemistries can be implemented to separate the enzyme reac-

tive group from the fluorophore in a variety of reaction types,

as exemplified with the Clips-O method below.17 Further

developments in these directions are still possible and will

mark the future of this type of assays.

2.1 Phenolate and aniline release

Indican is a chromogenic glycosidase substrate belonging to a

family of natural product glycosides found in plants such as

Isatis tinctoria and Polygonum tinctorum, the traditional source

to produce indigo is by fermentation.18 Cleavage of the glyco-

sidic 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 fluorescent 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 first 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 fluorescent umbelliferone anion 7.23,24 The

signal is only visible above pH 7 where the product exists as a

fluorescent anion. The same principle allowed substrates for

aldolase catalytic antibodies25–27 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,33–35 as wells as a fluorogenic substrate for

Baeyer–Villiger 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

Fig. 1 Chromogenic enzyme substrates releasing nitrophenolate.

Noelie Maillard, born in

Delemont (Switzerland) in

1981, studied chemistry at the

University of Neuchatel, and

complete her diploma thesis

in inorganic chemistry at the

Institute of Chemistry, Univer-

sity of Neuchatel with Prof.

Dr Thomas R. Ward. She

started her PhD in 2006 under

the supervision of professor

Jean-Louis Reymond in the

Department of Chemistry and

Biochemistry at the University

of Bern (Switzerland). Her current research interests include

the development of new methods for the screening of peptide

dendrimer libraries and its application in different reactions types.

Noelie Maillard

This journal is �c The Royal Society of Chemistry 2009 Chem. Commun., 2009, 34–46 | 35

conditions used to assay BVMO. Substrate 8 and further analogs

are currently the only available fluorogenic substrates for

BVMO and they are readily obtained by alkylation of various

chloroketones with umbelliferone.

The ketone or aldehyde leading to b-elimination may also be

formed by chemical oxidation of a primary 1,2-diol or

1,2-aminoalcohol reaction product in the so-called Clips-Ot

substrates (Fig. 3).17 A typical example is epoxide 10, which

provides a highly reliable probe of epoxide hydrolase activity for

screening in microbial cultures.37 The substrate is hydrolyzed by

epoxide hydrolases to form the 1,2-diol 11. In the presence of

sodium periodate (NaIO4), the diol is rapidly and quantitatively

oxidized to form aldehyde 12, which undergoes a b-elimination

to form umbelliferone 7 as above. Further examples include

thermally stable lipase, amidase and phosphatase substrates,38–41

an HIV-protease substrate,42 and a ceramidase substrate.43

Further strategies for indirect phenolate release include intra-

molecular carbamate or carbonate cyclization. The aldolase

Fig. 3 The Clips-Ot epoxide hydrolase substrate.

Fig. 4 A chromogenic substrate for aldolase catalytic antibodies.

Fig. 5 Enzyme assays with direct and indirect release of a fluorescent

aniline.

Fig. 2 Fluorogenic enzyme substrates with indirect release of

umbelliferone.


antibody substrate 13 features an interesting recent example of

this approach (Fig. 4).44 This substrate undergoes sequential

retroaldolization, b-elimination and intramolecular carbamate

cyclization to release a catechol which can be made visible by

formation of the insoluble black precipitate 14 in the presence of

Fe(III). Related carbonate and carbamate cyclization are also

used in substrate for epoxide hydrolases45 and acylases.46,47

Anilines such as nitrophenyl aniline and rhodamine are

frequently converted to amides to form chromogenic or

fluorogenic amidase and protease substrates, as in the protease

probe 15 designed for caspases (Fig. 5).48 The aniline can also

be released indirectly, for example via a ‘‘self-immolative’’

quinone methide mechanism as in the fluorogenic peptide

substrate 16 for the prostate specific antigen (PSA),49 or by

spontaneous hydrolysis of a urea following oxidation of a

phenol to a catechol and subsequently an orthoquinone as in

the recently reported tyrosinase substrate 17.50

2.2 FRET

Many enzyme assays are based on FRET (‘‘Forster or Fluores-

cence Resonance Energy Transfer’’) as detection principle, for

instance to measure hydrolytic reactions separating fluorophore

from quencher in the case of proteases,15,16,51–56 cellulases,57 and

lipases,58–60 and in the synthetic direction for fucosyl trans-

ferases.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 effect.62–64 Fluorescence of a label can

also be modulated by proximity effects such as medium effects.

For example, in an aminonitrobenzofurazane-labeled g-cyclo-dextrin reported as an a-amylase fluorogenic substrate, cleavage

of the cyclodextrin ring by the amylase exposes the fluorophore

to water and reduces fluorescence intensity.65 Fluorescent sub-

strates 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

response to kinase activity, the probe provides a strong lumines-

cence enhancement, resulting from the increased ability of the

probe to bind and sensitize Tb3+ and Eu3+ ions upon phos-

phorylation.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-pyrenyl-

butanoyl)glycerol in the presence of lipoproteins and albumin.68

Ester hydrolysis releases the pyrene carboxylate, which then binds

to albumin, resulting in a fluorescence increase. The commercially

available FITC-conjugates of casein are used as protease sub-

strates, whereby proteolysis removes the autoquenching and leads

to stronger fluorescence of the fluorescein chromophores.69

Recently reported protease-sensitive nanofibers consisting of

aggregated b-sheet forming peptides also rely on dilution-induced

release of autoquenching upon proteolytic cleavage.70

2.3 Other approaches

The enzyme reaction may also directly modify the chromophore

itself as a detection mechanism. For example microbial growth

can be monitored by following the activity of nitroreductases

that reduce the nitro group of various 7-nitrocoumarins such as

20 to form the corresponding 7-aminocoumarins 21 as a

fluorescent products (Fig. 7).71 Peroxidases react with a variety

of aromatic compounds to form colored products, and indoles

are substrates for cytochromes, forming indigo upon secondary

oxidation of the hydroxyindole primary product.72 Alcohol

dehydrogenase and aldolases can be screened using 6-methoxy-

naphthaldehyde and related substrates.27 The primary amine in

substrate 22 is oxidized by monoamine oxidase for form an

aldehyde, ultimately forming the fluorescent indole 23 by con-

densation of with the aniline amino group.73 The recent report

of an assay for fatty acid dehydrogenases based on chromo-

phore modification by the enzyme is also of interest.74

Luminescent products such as luciferase substrates (biolumines-

cence)75 or 1,2-dioxetanes (chemiluminescence)76 released from

enzyme reactions are also of interest.

Substrate dissolution or product precipitation or crystalliza-

tion may sometimes form the visible signal, without formation

of a colored product. Classically, microbial cultures producing

active lipases will form a clearing zone on agar plates prepared

with tributyrin. Certain polymer degrading enzymes can be

screened by recording dissolution of an insoluble substrate, in

Fig. 7 Fluorogenic substrates with direct modification of the

chromophore upon reaction.

Fig. 6 A FRET probe for following nucleophilic addition reactions

to aldehydes.


particular cellulases.77 More sophisticated systems were recently

investigated, such as the formation of hydrogels78,79 or

dipeptide nanotubes.80 In a recent example, incorporating an

ester-containing substrate in a liquid crystal allowed to assay a

lipase through its action on the alignment layer.81

2.4 Fluorescence activated cell sorting (FACS)

Fluorescent and/or fluorogenic substrates have been used to

directly identify cells expressing active enzymes in liquid culture

based on fluorescence-activated cell sorting (FACS), which

provides a particularly meaningful application of such sub-

strates in high-throughput screening.82,83 For example mutants

of the protease OmpA were displayed on the surface of E. Coli

cells and a cell-surface adherent fluorogenic substrate for

protease was added to the culture.84,85 Cells expressing an active

protease became fluorescent and could be sorted out by FACS,

which allowed discovering mutants with a 30-fold improved

activity. The same technique has been used for sorting micro-

emulsion droplets where an enzyme gene and expression

machinery are compartmentalized together with a fluorogenic

substrate system.86–88 Both methods allow to screen very large

numbers of variants (4107). Glycosides labeled with Bodipy

dyes were used to screen sialyl transferases in living cells, relying

on the fact that this substrate but not its sialylated product is

cell permeable. Thus, after washing unreacted substrate, cells

containing an active enzyme retained the fluorescent product

and were separated by fluorescence activated cell sorting.89 A

high-throughput screening technique for the identification and

isolation of enantioselective enzymes based on FACS has been

developed on the idea of labeling each of the two enantiomers

with a different fluorescent dye. This method allows to evaluate

108 cells (and number of clones generated) within a few hours.90

3. Indicator assays

A variety of relatively simple chemosensor systems based on

chromogenic or fluorogenic reagents can convert a chemical

transformations into a detectable signal, often through a func-

tional group specific reaction or by a separation effect. Bio-

sensors binding to either substrate or product may be used

similarly. Such indicator assays can be used to assay reactions

of specific, unlabeled substrates, which is necessary whenever a

very specific 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 measure-

ment 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 enzyme-

catalyzed reaction detectable consists in further converting the

reaction product by a second enzyme to form a second product

and so on, until one of these follow-up reactions produces a

detectable signal. The vast majority of enzyme-coupled assays

involve an oxido-reductase, usually an alcohol dehydrogease

(ADH), using NAD or NADP as cofactors, as exemplified

recently in the selection of enantioselective aldolase mutants.91,92

Recent interesting examples include the determination of the

enantioselectivity of lipases and esterases using acetate ester

substrates on the basis of an acetate detection kit.93 The assay

was used recently to select a double mutant of Bacillus Subtilisis

esterase with inverted enantioselectivity towards acetylated

tertiary alcohols.94 The enantioselectivity of ADH enzymes has

also been used to determine the enantioselectivity of alcohols

formed by the catalyzed addition of diethyl zinc to aldehydes,95

for a transition metal catalyzed epoxide opening reactions96 and

the conversion of benzaldehyde and acetyl cyanide to the corres-

ponding acetylated cyanohydrins.97,98 A pair of enzymes was

described that are able to differentiate the 1,2-hexanediol anti-

podes Lactobacillus kefir alcohol dehydrogenase, highly S selective,

and horse liver alcohol dehydrogenase, modestly S selective. This

allows one to obtain simultaneous enantioselectivity readouts on

two distinct substrates for the Co(III)-salen-mediated hydrolytic

kinetic resolution of epoxides.99 It has been shown that only

partially enantioselective dehydrogenases are generally sufficient

for the ee-determination of chiral alcohols.100

The reduction of hydrogen peroxide to water catalyzed by

peroxidases occurs with oxidation of various chromogenic

dyes, such as ABTS.101 Horseradish peroxidase (HRP) and

H2O2 as oxidant was used to detect the naphthol product

formed by hydroxylation of naphthalene by a P450cammonoxygenase.102,103 Turner and co-workers have developed

the reaction to follow the activity of an enantioselective

microbial monoamine oxidase (MAO-N) on amines. The

MAO-N produces hydrogen peroxide as a byproduct, which

is revealed by a peroxidase and 3,30-diaminobenzidineas a

chromogenic reagent.104,105 In a related experiment, the same

group has recently reported a novel high-throughput screening

method to determine both the rate and the enantioselectivity

of asymmetric ketone reduction by ketoreductases (KRED) in

the presence of an R-selective alcohol oxidase, and optionally

horseradish peroxidase (HRP) and ABTS (Fig. 8).106 An

R-selective KRED induces multiple turnovers producing

Fig. 8 Dual-wavelength spectrophotometric tracking of two chromo-

gens (NADP+ and ABTS) for the determination of ketoreductase

(KRED) activity and enantioselectivity.


many equivalents of NADP+, or ABTS+ if HRP is added to

the assay. By contrast, an S-selective KRED results in only

one turnover of NADPH since no R-alcohol is produced as an

oxidase substrate.

Hydrolytic enzymes such as glycosidases have been used as

secondary enzymes to follow the production of chromogenic

substrates from non-reactive precursors through a primary

enzyme such as a glycosidase, glycosyl transferase, glyco-

synthases.107–110 Similarly, proteases have been used to track

prolyl cis–trans isomerases,111–113 kinases,114,115 and peptide

deformylase.116 Luciferases produce light by oxidation under

consumption of ATP, oxygen and an oxidizable substrate such

as luciferin or an aldehyde and reduced flavin. A number of

assays have been reported that use a luciferase as secondary

enzyme to screen a reaction producing one of the luciferase

substrates as a product. The oldest method to quantify

ATP relies on firefly luciferase and luciferin.117 Luminescent

bacteria have been used to monitor the activity of aldolase

catalytic antibodies releasing nonanal.118 The same pheno-

typic screen was recently used to discover new oxido-reductase

enzymes, whereby the substrate spectrum of the bacterial

luciferase was explored and extended.119 Firefly luciferase

was recently used as the substrate undergoing refolding by

the chaperone Hsp90.120 An assay for monoamine oxidases A

and B was recently reported where the phenol-type substrate

for the luciferase is released by b-elimination of a primary

aldehyde oxidation product. For inhibitor assay, it should be

noted that typically 1–3% of compound libraries inhibit

luciferase activities.121

3.2 Functional group selective reagents

Kazlauskas and co-workers have exploited the well-known

fact that ester hydrolysis lowers the pH of the reaction medium

to develop a colorimetric assay to screen lipases and esterases

for enantioselectivity.122,123 pH-indicators have also been used

in the context of sol–gel encapsulated enzymes.124 Recently a

pH-indicator assay was reported for screening glycosyl trans-

ferases based on the acidification induced by glycosyl transfer

from UDP-GalNAc,125 and from glycosyl fluorides.126 Func-

tional group selective chromogenic or fluorogenic reagents

have been used to detect enzyme activities, including reagents

for amines formed by amidases,127,128 ammonia from nitrile

hydrolysis,129 aldehydes from vinyl ester cleavage130,131 and

from periodate cleavage of epoxide hydrolysis products,132,133

epoxides,134 thiols from thiolactones,135,136 phosphorylated

peptides from kinase reactions,137–139 UDP from glycosyl

transferases,140 dimedone from lipase reactions,141 and amino

acids from amidases.142 For example, amino acids can be

detected by fluorescence in real-time using the non-fluorescent

Cu(II) complex of calcein 24. The amino acid displaces Cu(II)

from calcein, whereby calcein regains its green fluorescence.

This simple assay is suitable to screen acylases, amidases and

proteases, as illustrated for the case of aminopeptidase, for

which no other fluorescence assay is known (Fig. 9).143,144

Adrenaline serves as the reporter for detecting for 1,2-diols

(28) and 1,2-aminoalcohols by back-titration of sodium

periodate (Fig. 10). Adrenaline (29) reacts quantitatively

and rapidly with sodium periodate to form the deep red

Fig. 9 A fluorescence assay for aminopeptidase using a calcein

sensor.

Fig. 10 The adrenaline test for enzymes.


adrenochrome (30), a reaction which is does not take place if a

1,2-diol or 1,2-aminoalcohol has already consumed the perio-

date oxidizing agent. This provides a practical end-point assay

for a variety of hydrolytic enzymes.145,146 The adrenaline assay

can be used to screen the enzymatic hydrolysis of epoxides (25)

by epoxide hydrolases, triglycerides such as tributyrin 26 or

various acetate esters147 by lipases and the dephosphorylation

of phytic acid (27) by phytases. The adrenaline test for epoxide

hydrolase was recently adapted to an automated format for cell

culture.148 Sodium periodate also decolorizes certain chromo-

phores and the assay was used to screen epoxide hydrolases

using fluorescein as periodate reporter.149

In an unusual yet very practical example of indirect sensing

of an enzyme reaction, epoxide hydrolase activity on butane-

oxide was detected in E. coli cultures on agar-plate using

Safranin O.150 Oxidation of the 1,2-diol product by E. Coli

modified the membrane potential and lead to accumulation of

the red dye in the colonies producing active enzyme, allowing

for direct selection.

3.3 Bio- and nano-sensors

While the above example apply simple chemical reactivity

principles in the context of enzyme assays, some sensor

systems rely on more sophisticated detection schemes with

biosensors, vesicles and gold nanoparticles, a type of assay

which may be assigned to the ‘‘nano’’ world. The first notable

example concerns antibodies for the so-called cat-ELISA

assay developed in the context of catalytic antibody

research.151–156 Further examples ADP selective aptamers

for kinase sensing,157 or lectins for testing glycosyl transferases

on microarray displayed substrates.158–161 Vesicles containing

synthetic multifunctional pores (SMPs) have been used for

enzyme assays.162–166 The SMPs are incorporated in the vesicle

membrane and serve as channels for the escape of fluorescein,

which results in a fluorescence increase since dilution removes

autoquenching. Substrate/product ratios in an enzymatic

reaction can be monitored whenever substrate and product

differentially modulate the flow of fluorescein through the

SMPs. Gold nanoparticles have been used for a variety of

enzyme assays. Solutions of Au(III) (HAuCl4) are reduced by

NAD(P)H, catechols, or thiols, to form colored suspensions of

gold nanoparticles, allowing a colorimetric assay for enzymes

such as lactate dehydrogenase,167 acetyl choline esterase168

and tyrosinase.169 Aggregation of gold nanoparticles in

suspension induces a color change from red to purple grey,

allowing assays for kinases mediated by a biotin–avidin

aggregation trigger,170,171 for proteases using a synthetic

peptide with a protease-specific sequence flanked by a pair

of S-acetyl cysteine residues,172 for alkaline phosphatase173

and ATP sensing174 using charge induced aggregation of

nanoparticles, and for endonucleases using two sets of gold

nanoparticles coated with complementary single-strand DNA

substrates.175

4. Fingerprinting

The information content of screening assays can be increased

by analyzing multiple substrates simultaneously. The first

multi-substrate analysis method was the APIZYM system in

the 1960s.176–180 In this method a set of 19 or 32 different

enzyme substrates, including chromogenic substrates for

lipases and esterases, aminopeptidases, chymotrypsin, trypsin,

phosphatases, sulfatases and b-galactosidases, is used in a

multi-well format to analyze microbial cultures. The analysis

produces a reactivity pattern indicating which enzymes are

produced by the microorganism. This information serves to

identify the microorganism, and forms the basis of microbial

strain identification in hospitals. Enzyme fingerprinting

proposes to focus the analysis on a single enzyme using a

series of structurally related substrates to characterize its

selectivity.181 The data may be used to identify reactive

substrates, or for functional classification of the enzymes.40

Generally one analyzes reactivity fingerprints across a series of

similar substrates, such as series of peptides, however it should

be mentioned that certain enzymes may also catalyze reactions

of different functional groups, which is called substrate

promiscuity.182,183 Fingerprinting with multiple substrates

should be distinguished from the so-called activity-based

protein profiling, which is based on labeled suicide enzyme

inhibitors to identify reactive enzymes in protein

mixtures.184–192 The critical problem of enzyme fingerprinting

is to provide a reliable method for producing the data,

and most research efforts are currently still focusing on

this problem. The true potential of fingerprinting will be

realized when reagents will be available for measuring multi-

dimensional datasets reporting enzyme selectivities and

substrate preferences as readily as what is currently possible

with single substrates.

4.1 Parallel assays in microtiter plates

Assays with multiple substrates in microtiterplates have been

mostly used for hydrolase profiling.147,193–195 For example,

fingerprinting lipases and esterases with 16 periodate-activated

fatty acid ester substrates in both enantiomeric forms showed,

as expected, that lipases were more active on long-chain

substrates, while esterases preferentially cleaved short-chain

substrates. More surprisingly, a selectivity for intermediate

chain length substrates was apparent as the second principal

component of the observed diversity, an information not

otherwise accessible (Fig. 11).40 Using a related series of

water-soluble esters 31a–e and 32a/b derived from fluorescein,

fingerprinting showed that lipases and esterases differ from

one another by their reactivity in pure aqueous buffer vs.

buffer containing 20% dimethyl sulfoxide as co-solvent.

Esterases were more active in aqueous environment, while

lipases required the cosolvent for highest activity, as shown by

the color coded pattern (Fig. 12).35

Microtiter-plate based assays have also been used to analyze

proteases using multiple peptide substrates to determine the

optimal substrate,196–199 in particular with positional scanning

libraries of millions of fluorogenic peptidyl coumarinamides as

series of 20 or 400 different substrate mixtures.200–208

4.2 On-bead assays

Meldal and co-workers reported the use of synthetic combi-

natorial libraries of millions of synthetic peptides as FRET

substrates to analyze protease reactivity on solid


support,209–211 a very practical method still under further

development today.212,213 One of the difficulties in the analysis

is the necessity to introduce a fluorescent label on the cleaved

peptide, which may reduce the reactivity of the protease. We

recently reported an assay for on-bead proteolysis of a solid-

supported 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 fingerprinting

Due to its separating power, chromatographic screening offers

the possibility to assay several different substrates simulta-

neously. A single HPLC-analysis returns the activity finger-

print, in which the relative amounts of product formation

defining the reactivity profile or fingerprint, can be precisely

reproduced if the cocktail composition is controlled. We have

demonstrated this principle for a fingerprint analysis of lipases

and esterases using a cocktail of 20 monoacyl-glycerol

analogs,215 and for proteases using a cocktail of five hexa-

peptides.216 Such characterization tools may prove useful to

identify novel enzymes with unusual selectivities, as well as in

the area of diagnostics. Similarly, the classical APIZYM

substrate palette for microbial characterization can be formu-

lated as a cocktail reagent, allowing 16 different enzyme

reactivities to be determined in a single analysis.217 A cocktail

of fluorescent umbelliferyl glycosides was recently used to

characterize various glycosidases using an HPLC-based

assay.218 Substrate cocktails can also be analyzed by mass

spectrometry in a variety of setting including enantioselectivity

determination using a mixture of two isotopically labeled

pseudo-enantiomers,219–225 for glycosyl transferase reac-

tion using substrate mixtures226–230 or for protease activity

determination using a mixture of peptide substrates.231

4.4 Microarrays

Miniaturization of fingerprinting (and screening) down to

the scale of a few nanoliters per datapoint is possible with

microarrays printed on glass slides. A nanospray system was

used to homogeneously distribute nanodroplets of a solution

Fig. 11 Fingerprint analysis of chain length selectivity of lipases and

esterases.

Fig. 12 Fingerprint analysis of cosolvent selectivity of lipases and

esterases.

Fig. 13 On-bead protease profiling on TentaGel beads.


containing three fluorogenic protease substrates on a micro-

array on which spots of enzyme had been previously printed,

allowing high-throughput screening of enzyme inhibitors.232

Depositing fluorogenic substrates on poly-lysine-coated glass

slides also allows efficient assays of various enzymes in nano-

droplets.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 profiling experiments with

proteases and other hydrolytic enzymes using combinatorial

series of fluorogenic coumarin-derived substrates35,235,236 and

with lipases using substrate of varying acyl chain length 35a–f,

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

profiling method based on combinatorial libraries of

PNA-encoded dipeptidyl-rhodamine substrates.238–241 An

important improvement in the study of the biology of phos-

phatases 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 different substrates.242

Protease profiling was also recently reported based on a

multiplexed solution phase assay on microarrays.243 Micro-

arrays 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 different fluorophores based on Horeau’s

method.244–246 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 low-

volume enzyme assays at the scale of one microliter per

assay using silicagel plates pre-impregnated with a fluorogenic

substrate as the reaction medium, which provides a practical

solution for miniaturization that is much simpler than micro-

arrays.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 evolu-

tion, 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 refining older methods.

Fig. 15 High-throughput screening with microtiter reaction on

silicagel plates.

Fig. 14 Periodate-coupled lipase assay on microarrays.


For what concerns future developments, the demand for

new enzyme assays remains high in the context of high-

throughput screening in enzyme engineering, in particular

for sensing regio-, stereo- and enantioselectivity. Remarkably,

most of these problems have been in principle solved by

instrument-based assays such as NMR, MS, HPLC and MS,

not reviewed here, but the methods are often complicated and

expensive to implement. Therefore, most application examples

in enzyme engineering continue to use fluorogenic and

chromogenic substrates and indicator assays as the main

screening tool, simply because when available such assays

are simple to use and inexpensive. Despite of their apparent

drawbacks in terms of possible artefacts, the most useful

assays seem to be indicator assays that are compatible with

a range of different substrates. In particular enzyme-coupled

assays will probably remain high on the list for many years to

come, with assays producing a colored precipitate being the

most useful for high-throughput screening as they can be

applied on agar plates, on paper, or in microtiter plates.

Improvement in fingerprinting reagents should also be

considered in the future to enable high-throughput screening

with a diverse set of substrates simultaneously, which would

allow a much more productive use of enzyme libraries.

Acknowledgements

This work was supported by the Swiss National Science

Foundation and Proteus SA, Nımes, France.

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