Lipase assays for conventional and molecular screening: an overview

Biotechnol. Appl. Biochem. (2003) 37, 63–71 (Printed in Great Britain) 63

REVIEWLipase assays for conventional and molecular screening:an overview

Rani Gupta1, Pooja Rathi, Namita Gupta and Sapna Bradoo2

Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi 110 021, India

Lipases are versatile biocatalysts that can performinnumerable different reactions. Their enantio-,chemo- and stereo-selective nature makes them animportant tool in the area of organic synthesis. Unlikeother hydrolases that work in aqueous phase, lipasesare unique as they act at the oil/water interface. Besidesbeing lipolytic, lipases also possess esterolytic activityand thus have a wide substrate range. Hence, the lipaseassay protocols hold a significant position in the field oflipase research. Lipase activity can be estimated using awide range of assay protocols that differ in terms oftheir basic principle, substrate selectivity, sensitivityand applicability. As the value of these enzymes con-tinues to grow and new markets are exploited, de-velopment of new or improved enzymes will be a keyelement in the emerging realm of biotechnology.Hence, development of faster and simpler protocolsincorporating newer and more specific substrates is theneed of the hour. In this endeavour, methods that couldbe adopted for molecular screening occupy an im-portant position. Here, an overview of the lipase assayprotocols is presented with emphasis on the assays thatcan be adopted for the molecular screening of thesebiocatalysts.

Introduction

Lipases are an important group of biocatalysts with anunsurpassed role in swiftly growing biotechnology that isbased mainly on their remarkable ability to carry out novelreactions both in aqueous and non-aqueous media. Lipases(triacylglycerol acylhydrolase ; EC 3.1.1.3) catalyse the hydro-lysis of triacylglycerols to release free fatty acids andglycerol. This hydrolytic reaction is reversible, and inthe presence of decreased amounts of water, often in thepresence of organic solvents, the enzymes are effectivecatalysts for various inter-esterification and trans-esterifica-tion reactions [1–4]. Lipolytic reactions occur at the lipid}water interface where lipolytic substrates usually formequilibria between monomeric, micellar and emulsifiedstates. Further, lipases show extreme versatility regardingfatty-acyl-chain-length specificity, regiospecificity and chiral

selectivity [5]. Owing to their immense importance, thesemulti-faceted enzymes have tremendous potential in areassuch as food technology, detergents, the chemical industryand biomedical sciences. Hence reviews on lipases forseveral years have focused on their biotechnological impetus[6–9].

The latest trend in lipase research is towards thediscovery of novel enzymes and a greater understanding ofpreviously discovered enzymes and their functional signifi-cance using molecular tools such as recombinant DNAtechnology, random mutagenesis, site-directed mutagenesisand protein engineering [10]. Lately, directed evolution hasemerged as a key technology to generate enzymes with newand improved properties [11,12]. An emerging area ofinterest is the metagenome approach to tap the microbialsources of novel enzymes from the hitherto undiscoveredwealth of molecular diversity [13].

Thus with the advent of rapid methods for discoveringnovel enzymes or altering the properties of enzymes, thereis paramount interest in the development of screening toolsthat can be used to search for the best performance withrespect to specific property. An efficient screening orselection system is an absolute prerequisite to identifying thelipase-producing species}clones from a diverse populationas well as to pinpointing enzyme variants that display thedesired properties during molecular screening. With thisviewpoint, in the present review on lipase assays, besidesconventional procedures which have also been discussed byVerger’s group [5], equal emphasis has been placed onprocedures adopted for molecular screening of lipases.

Lipase assay procedures

Lipases can be viewed both as lipolytic and esterolyticenzymes, being catalysts for a large number of esters. Theactivity of lipases can thus be assayed by monitoring therelease of either fatty acids or glycerol from triacylglycerols

Key words : esterolytic, fatty acid, hydrolase, lipid, lipolytic.1 To whom correspondence should be addressed (e-mail

microzyme!123india.com).2 Present address : Biotechnology Department, Center for Chemistry and

Chemical Engineering, Lund University, Box 124, SE 22100, Lund, Sweden.

# 2003 Portland Press Ltd

64 R. Gupta and others

or fatty acid esters. Further, since lipases act at the oil}waterinterface, change in the properties of the interface is animportant criterion for measuring lipolysis.

Estimation of fatty acids releasedThe fatty acids released by lipase-mediated hydrolysis canbe determined qualitatively by gel-diffusion assay andquantitatively using (i) titrimetry, (ii) colorimetric assays,(iii) fluorescence, (iv) chromatographic procedures (TLC}GC}HPLC) and (v) immunological methods.

Qualitative estimation : plate assay Lipase-producing strainsfrom a wide population are conventionally screened ontributyrin (tributyrylglycerol) agar plates. A zone of tributyrinhydrolysis is indicative of either esterase or lipase activity.More recently, agar plates supplemented with olive oil havealso been employed for screening lipase-positive colonies[14–16].

Gel-diffusion assay has been widely used to screenlipases in culture supernatants of various microbes as, onthe lipolysis of triacylglycerol, a clear zone is produced[17–19]. Alternatively, an indicator is added to the agar thatin turn forms complexes with the acids, and a coloured zoneis produced. The indicators used are Nile Blue Sulphate [20]and Victoria Blue [17]. These tests are very convenient forfast screening of lipolytic microbes growing on agar plates.However, some false-positive results can result from acid-ification of the medium due to the generation of acidicmetabolites other than free fatty acids that are released bymicrobial lipases.

To overcome these limitations, Kouker and Jaeger [21]employed the fluorescence dye Rhodamine B to observe thezone of lipolysis as an orange fluorescence under UV light at350 nm. Rhodamine forms a fluorescent complex with freefatty acids. Thus the lipase-producing colonies give aflourescent halo that is visible under UV light. In place oftriolein (trioleoylglycerol), olive oil has also been used inthe agar medium. This process has been used recently byJette and Ziomek [22] and Jarvis and Thiele [23]. Thisfluorescent dye method allows quantification of very minutelevels of lipase activity.

Quantitative estimation : titrimetry This is one of the oldestand most widely used quantitative assays, on account of itssimplicity, accuracy and reproducibility. This is a reliabletechnique for characterizing lipase action and specificity, aswell as the interfacial activation phenomenon [24]. Triolein(or, as a cheap alternative, olive oil, which contains 70%triolein) is a generally used, internationally accepted sub-strate [25]. Morever, tributyrin, triacetin (triacetylglycerol)and tripropionin (tripropionylglycerol) can also be used assubstrates for the estimation of enzyme activity [26,27].However, lipases have a higher rate of hydrolysis on longer-

chain triacylglycerols such as triolein as compared withshorter triacylglycerols. The pH-stat method is a highlysensitive as well as a quantitative method that can measurethe release of even 1 µmol of released fatty acid}min [8].However, at a pH value of less than 7, where free fatty acidsare not fully ionized, pH-stat titration is either inaccurate orimpossible to perform, even after introducing a correctionfactor [28]. Here the lipase activity is determined by simpletitration of the assay mixture to pH 9.0}10.0 after stoppingthe reaction by ethanol [29–32] or by adopting the AmericanOil Chemists Society acid value method. Here, a sample isdrawn and mixed with propan-2-ol and toluene and titratedto a phenolphthalein end point. This method is often used tomeasure free fatty acids in crude and refined fats and oils[33]. One unit of lipase activity is defined as the amount ofenzyme that catalyses the release of 1 µmol of fatty acid fromolive oil}min per ml under the standard assay conditions[34]. However, titrimetry is extremely laborious and time-consuming. Colorimetric and fluorimetric assays providethe option of simpler and more rapid assays, although thesubstrates are expensive and therefore not used as widelyas the standard assay protocols.

Quantitative estimation : spectrophotometric assay p-Nitro-phenyl esters of various-chain-length fatty acids are generallyused as substrates and release of p-nitrophenol is measuredspectrophotometrically at 410 nm [35,36]. Short-chainesters are water-soluble and therefore their hydrolysisprovides a measure of esterase, rather than lipase, activity.However, p-nitrophenyl palmitate is used to measure lipaseactivity. A major limitation of this assay is that the enzymicreactions cannot be performed at acidic pH owing to thelack of absorbance of p-nitrophenol at acidic pH [37]. Thusenzyme activity at only neutral and alkaline pH values can beascertained by this procedure. For acidic enzymes, this assayhas been used by raising the pH of the reaction mixture afterculmination of the reaction. Another important consider-ation while performing this assay at different pH values isthat p-nitrophenol has different absorption coefficients atdifferent pH values. Therefore standards at the appropriatepH value should be incorporated into the assay protocol.

Colorimetric assays are based on hydrolysis of thecolourless ester β-naphthyl caprylate (octanoate) to yieldcoloured β-napthol, which is measured spectrophotome-trically at 560 nm, [27,38–40]. α-Naphthyl esters such asα-naphthyl acetate, naphthyl propionate and naphthylbutyrate are also used as substrates.

Other assays based on the formation of a copper soapof the fatty acid in the presence of a dye indicator [41,42] andbased on the metachromatic properties of the cationic dyeSafranine [43] have also been used by some researchers.However, such assays are not routinely used in laboratories.


Review: lipase assays for conventional and molecular screening 65

Besides measurements of the coloured product, spec-troscopic assays are also based on precipitation of fatty acidswith calcium or copper. von Tigerstrom and Stelmaschuk[44] have described a method that involves precipitation offatty acids released by Tween 20 hydrolysis, with CaCl2, andthe increase in absorbance is measured at 500 nm. Theenzyme reactions were linear with time, at least up to an A500

value of 1.2, and were proportional to the enzyme con-centration. This turbidimetric method is much simpler and atleast 36 times more sensitive than the titrimetric assay withTween 20, and at least four times more sensitive than aspectrophotometric assay with p-nitrophenyl palmitate.

Quantitative estimation : fluorescence assay Fluorescent com-pounds have also been used for lipase assay. The methodinvolves measurement of the fluorescent fatty acid releasedbecause of lipase activity [45,46]. In this method described byWilton [45,46], quantities as low as 20 pg of purified porcinepancreatic lipase could be detected. It is also possible to usetriacylglycerols having one of the alkyl groups substitutedwith a fluorescent group such as pyrenyl [47,48]. Duque etal. [49] developed an assay using fluorogenic and isomericallypure 1-(3)-o-alkyl-2,3-(3,2)-diacylglycerols as substrates.This lipase assay is continuous and does not requireseparation of substrate and reaction products.

A non-fluorescent substrate 4-methylumbelliferyloleate has also been employed to release highly fluorescent4-methylumbelliferone after lipase action [50]. Roberts [51]developed a rapid and highly sensitive fluorescence-basedassay using 4-methylumbelliferyl butyrate. This methodcould detect low lipolytic activity (10 nmol}h per ml) andwas 6000-fold more sensitive than conventional titration,which measures activity in the range of µmol}min per ml.

A quantitative fluorescence lipase assay based on theinteraction of Rhodamine B with fatty acids released duringthe enzymic hydrolysis of triacylglycerols has been describedby Jette and Ziomek [22]. The assay is linear over the rangeof 0.5–2 mM oleic acid and 0.05–1 µg of pure lipase. Themethod allows flexibility in the choice of substrate. A largenumber of samples can be assayed simultaneously, making itpractical for assaying lipase activity in column fractionsduring purification. This method is rapid and can beautomated further.

Quantitative estimation : chromatographic procedures Chro-matography is a conclusive method for direct determinationof the release of fatty acids following enzyme-catalysedhydrolysis of a lipid substrate.

TLC: a quantitative analysis of the released free fattyacids from triacylglycerols can be carried out using densi-tometric or autoradiographic methods with radiolabelledtriacylglycerols. These methods are very sensitive and candetect fatty acid to a few picomol [52]. The main disadvantage

is that these procedures are very time-consuming anddiscontinuous.

GC: in the official American Oil Chemists Societymethod, the fatty acids are converted into their methylesters and subjected to quantification by GC. Recently,Bereuter and Lorbeer [53] have developed a temperature-based GC procedure to assay the activity and selectivity oflipases, and to test various reaction conditions.

HPLC: the product of lipolysis can also be easilyidentified using HPLC and has been used for lipases fromPseudomonas sp., Candida rugosa, Rhizopus arrhizus, Geo-trichum candidum [54] and Penicillium sp. [55].

Quantitative estimation : immunological methods Immuno-logical methods or ELISA-based procedures are highlyspecific and sensitive and can be adopted easily. Indeed, theyhave been used for detection of lipases in Staphylococcusaureus [56]. However, they could not be adopted as routinelaboratory assays, since a purified enzyme sample as well asthe raising of poly- or mono-clonal antibodies are pre-requisites. Several ELISAs have been developed specificallyto quantify amounts of pure lipases, e.g. human pancreaticlipase, human gastric lipase, lipoprotein lipase and hepaticlipase [57,58]. Extending the conventional ELISA techniqueto specific lipases, Aoubala et al. [59] developed twosandwich ELISA techniques for evaluating the interfacialbinding of human gastric lipase to lipid monolayers. Thisassay made it possible for the first time to measure theenzymic activity of the lipase on dicaprin (didecanoylglycerol)monolayers as well as to determine the correspondinginterfacial access of the enzyme.

Lipase assays based on the properties of the interfaceLipases act at the oil}water interface and hence the interfacialsurface pressure is an important criterion for measuringlipase activity. Activity at the interface is solely the propertyof lipases and thus is indicative of true lipases.

Monomolecular film technique This technique has been usedprimarily to study lipolysis at the lipid}water interface andspecifically for a quantitative investigation of interfacialregulation [8,60]. This method is highly sensitive, and verylow lipid amounts are required to perform reliable kineticmeasurements. However, in terms of the amounts of lipaseused, the monomolecular film technique requires as muchlipase as the pH-stat method (0.1–1 µg}assay). Moreover,this technique is not widely used mainly because elaborateand extensive set-ups are required for accurate estimationof lipolytic activity.

Oil-drop method The change in surface tension of a lipidmonolayer caused by lipase-mediated hydrolysis can bemonitored by the oil-drop method [61]. This methodconsists of forming an oil drop in a syringe containing the oil.



The shape of the oil is directly correlated to the interfacialtension of oil}water. When no detergent or fatty acid ispresent in the medium, the drop is shaped like an apple.Lipase added to the water phase binds to the oil}waterinterface and hydrolyses the substrate. The released prod-ucts remain in the interface and consequently the interfacialtension decreases. The shape of the drop changes to a pearform and at a certain point leaves the support. A computer-controlled device has been developed for measurement inthis type of lipase assay [62].

As compared with the other interfacial techniques, theoil-drop tensiometer presents the unique advantage of beingable to monitor lipase activities on natural long-chaintriacylglycerols at a closely controlled oil}water interface[63,64]. However, the oil-drop methodology requires theoil to be freed carefully from any natural tensioactivecompounds such as free fatty acids and di- and mono-acylglycerols because of the amphipathic character of thesecontaminants, which might decrease the initial interfacialtension. Clean materials and equipments are also a strictrequirement for the oil-drop methodology to give reliableresults.

Atomic force microscopy Atomic force microscopy has beenused for studying the enzymic hydrolysis of mixed bilayers ofacylglycerols}phospholipids by Humicola lanuginosa lipase.Mica-supported lipid bilayers are hydrolysed by H. lanuginosalipase and, as the products dissolve in buffer, regions of thebilayer with deep defects are detected by the atomic-force-microscopic tip. Increases in the areas of the holes in the lipidbilayer are recorded as a function of time and specific activityof the enzyme is thus estimated, assuming one molecule oflipase to be acting in each hole. These data provide the firstnanoscale picture of the kinetics of lipid degradation bylipases [5].

IR spectroscopy A continuous assay for measuring lipase-catalysed hydrolysis of triacylglycerols in reverse micellesusing Fourier-transform IR spectroscopy was developedby Walde and Luisi [65]. Lipolysis can be monitored byrecording the Fourier-transform IR spectrum of the entirereaction mixture. Fatty acid esters and free fatty acids (peakmaximum at 1751 and 1715 cm−1 respectively) can bequantificated on the basis of their molar absorption coef-ficients and Beer’s law. This method was applied to measurethe lipolysis of various substrates (e.g. trioctanoylglyceroland vegetable oils).

There exist other methods, such as NMR, for quan-tifying lipase activity in biphasic microemulsions [66], or IRspectroscopy, for measuring lipase-catalysed hydrolysis oftriacylglycerols in reverse micelles [65]. A conductometricmethod has been described using a short-chain substratetriacetin [67]. Amaya et al. [68] have reported the use of

lipase-catalysed synthesis of octyl linolenate in a hexane-microaqueous reaction system as a new assay method forlipase activity in organic solvents.

From the foregoing description it can be concludedthat there are several methods available for lipase assay(Table 1). However, the method to be selected dependsupon the user’s requirements and available resources. Ofall, titrimetry is the most reliable and widely used pro-cedure involving hydrolysis of fats and oils. Besides this,the p-nitrophenyl palmitate assay is important when hand-ling large numbers of samples.

Once a lipase has been identified, its biocatalytic utilitydepends upon its substrate specificity, positional specificityand enantioselectivity. The major usage of lipases is expectedto be in the pharmaceutical sector due to their chiralselectivity, which makes them indispensable biocatalysts.However, the methods available for testing the enantio-selectivity of lipases are generally expensive as well asneedful of sophisticated instruments like HPLC. Therefore,the future goals of lipase research are to develop inexpensiveand quick assays for testing the enantioselectivity of theseenzymes. Further, a more sensitive method needs to bedeveloped to detect small amounts of enantioselectiveactivity [10]. Selection of relatively cheap and pure enanto-meric compounds coupled with an efficient screening systemshould form the basis of selection of a highly efficientenantioselective catalyst.

Qualitative testing for enantioselectivityThis is used where lipase-catalysed resolution of racemicesters for the production of optically pure carboxylic acidsor alcohols is the desired transformation. For this, sus-pensions of the enantiomeric esters in an agar medium areplaced in simple Petri plates. Solutions of the enzymesare added and clearing of the treated areas is observedvisually. If the pure enantiomeric ester is hydrolysed, theproducts of hydrolysis (that is, a fatty acid and an alcohol) aresoluble in the medium, causing the treated area to clear. If noreaction takes place, the area remains translucent [4,69].

Quantitative testing for enantioselectivityAder et al. [69] have developed a facile screening systembased on the enantioselective separation of reaction pro-ducts by HPLC on chiral supports. Using a commerciallyavailable column (Chiralcel OB) all products resulting fromthe enantioselective enzymic hydrolysis were separatedsimultaneously. A direct determination of the achievedconversions, as well as configurations and enantiomericpurities of both substrates and products, can be achieved inone single experiment. The obtained data allow rapiddetermination of the enantioselectivities [70] displayed bythe employed biocatalysts, and thus a rapid evaluation oftheir synthetic usefulness.



Tab

le1

Ass

ays

for

the

dete

rmin

atio

nof

lipas

eac

tivity

Ass

ayan

dsu

bstr

ate

Prod

uct

anal

ysed

Prin

cipl

ein

volv

edR

emar

ksR

efer

ence

(s)

Plat

eas

says

Trib

utyr

in,a

cylg

lyce

rols

and

este

rsof

long

-cha

infa

tty

acid

sSh

ort-

chai

nfa

tty

acid

sH

alo-

base

dor

colo

urch

ange

ofPh

enol

Red

/Vic

toria

Blue

/Nile

Blue

Sulp

hate

,or

mea

sure

men

tof

fluor

esce

nce

afte

rco

mpl

exat

ion

offa

tty

acid

with

fluor

esce

ntdy

eR

hoda

min

eB.

Con

veni

ent

for

rapi

dsc

reen

ing.

[14–

23]

Titr

imet

ryFa

tsan

doi

ls,tr

iacy

lgly

cero

ls,m

ethy

lest

ers

Fatt

yac

ids

Neu

tral

izat

ion

reac

tion

eith

erdi

rect

lyby

pH-s

tat

orby

pHin

dica

tor.

Mos

tre

liabl

ean

dco

mm

only

used

proc

edur

e.[2

4,33

]

Spec

trop

hoto

met

ryFa

tty

acid

conj

ugat

esof

β-n

apht

hol

β-N

apht

hol

Estim

atio

nof

β-n

apht

holb

yco

mpl

exat

ion

with

Fast

Blue

BB.

The

este

ris

not

stab

leat

extr

eme

pH.

[38–

40]

p-N

itrop

heny

lest

ers

p-N

itrop

heno

lC

olou

red

prod

uct

mea

sure

dat

410

nm.

Con

veni

ent

met

hod.

Pref

erre

ddu

ring

purif

icat

ion

proc

edur

es.D

isadv

anta

geof

unde

rgoi

ngsp

onta

neou

shy

drol

ysis.

[35,

36]

Tw

eens

Fatt

yac

idPr

ecip

itatio

nof

fatt

yac

idw

ithca

lciu

mor

copp

eran

dm

easu

rem

ent

oftu

rbid

ity.

Sim

ple,

repr

oduc

ible

and

sens

itive

;can

beus

edfo

rqu

antit

ativ

eas

says

but

ofte

nus

edfo

rpl

ate

assa

ys.

[44]

Fluo

resc

ence

assa

yT

riacy

lgly

cero

lsw

ithal

kylg

roup

subs

titut

edw

itha

fluor

esce

ntgr

oup,

e.g.

conj

ugat

edpy

reny

lgro

upFl

oure

scen

tfr

eepr

enyl

grou

psSh

iftin

fluor

esce

nce

wav

elen

gth

afte

rtr

iacy

lgly

cero

lhyd

roly

sis.

Rap

idas

say,

but

expe

nsiv

esu

bstr

ate

limits

itsus

age.

[48,

48]

Non

-fluo

resc

ent

4-m

ethy

lum

belli

fery

lole

ate

Fluo

resc

ent

4-m

ethy

lum

belli

fero

nePr

oduc

tis

anal

ysed

,as

itis

fluor

esce

nt.

[50,

51]

Chr

omat

ogra

phic

proc

edur

es(T

LC/G

C/H

PLC

)T

riacy

lgly

cero

ls,fa

tsan

doi

lsFa

tty

acid

sA

naly

sisan

dqu

antif

icat

ion

ofth

epr

oduc

tor

resid

uals

ubst

rate

thro

ugh

spec

ific

colu

mns

.U

sede

pend

sup

onav

aila

bilit

yof

the

inst

rum

ent.

Tim

e-co

nsum

ing

for

rout

ine

anal

ysis,

but

ofte

nre

com

men

ded

for

subs

trat

e-sp

ecifi

city

dete

rmin

atio

n.

[53–

55]

Inte

rfac

ialp

ress

ure

:mon

olay

erm

etho

dLi

pid

Fatt

yac

ids

Cha

nge

insu

rfac

epr

essu

redu

eto

brea

kdow

nof

tria

cylg

lyce

rol.

Hig

hly

sens

itive

.Ela

bora

tean

dex

tens

ive

set-

ups

requ

ired

for

accu

rate

estim

atio

n.[8

,60]

Inte

rfac

ialp

ress

ure

:oil-

drop

met

hod

Lipi

dFa

tty

acid

sO

il-dr

opsh

ape

ism

onito

red

;cha

nges

from

appl

eto

pear

shap

eup

onhy

drol

ysis.

Exte

nsiv

ese

t-up

sar

ere

quire

d.[6

1–64

]

Inte

rfac

ialp

ress

ure

:ato

mic

forc

em

icro

scop

yLi

pid

bila

yers

Fatt

yac

ids

Reg

ions

ofbi

laye

rshy

drol

ysed

bylip

ases

show

ing

deep

defe

cts

are

dete

cted

byth

eat

omic

-forc

e-m

icro

scop

ytip

.

Prov

ided

the

first

nano

scal

epi

ctur

eof

kine

tics

oflip

idde

grad

atio

nby

lipas

es.S

ophi

stic

ated

inst

rum

ents

invo

lved

.

[5]

IRsp

ectr

osco

pyV

eget

able

oils,

trio

ctan

oylg

lyce

rol

Fatt

yac

ides

ters

and

free

fatt

yac

ids

Lipo

lysis

mon

itore

dby

reco

rdin

gth

eFo

urie

r-tr

ansf

orm

IRsp

ectr

umof

the

entir

ere

actio

nm

ixtu

re.

Expe

nsiv

ean

dso

phist

icat

edin

stru

men

tsin

volv

ed.

[65]



Molecular screening of lipases

An emerging area of research in the field of enzymology is todevelop radically different and novel biocatalysts throughvarious molecular approaches such as recombinant DNAtechnology, protein engineering, directed evolution and themetagenomic approach. The success of the molecularapproach involves the proper combination of molecular bio-logical techniques coupled with efficient high-throughputassays for lipases with newer and improved properties [12].Efficient screening procedures have to handle every singlemember of a given library of potential candidates and identifythe biocatalyst that possesses the desired properties. By andlarge, this employs a number of enzyme assays describedearlier. However, the prerequisite of such assays is simplicity,rapidity and sensitivity to be able to distinguish a positiveclone}protein variant from a massive pool of negative ones.Various high-throughput screening systems for assayingenantioselective catalysts and enzymes have been dealt within detail by Reetz [12]. A brief description of the assaysystems adopted in molecular screening of lipases is pre-sented here.

Assays for screening enzymic activityThe simplest method to distinguish variants of a colony isbased on their enzymic activity on the specific substrates. Inthis category are placed the widely used agar-plate assay,which involves the appearance of a zone of hydrolysisaround the positive colony, or the filter assay. Here, theselection criterion is the enzymic activity of the samples ona specific substrate. These assays are conclusive with respectto the catalytic potential of a clone or protein variant.

Agar-plate assays The most commonly employed assayprocedure for molecular screening of lipases is the agar-plateassay, which involves the appearance of a clearance haloaround the lipase-producing colony}clone. Depending onthe cloning vector used, the agar medium may or may notcontain an inducer, e.g. isopropyl β-D-thiogalactoside isincorporated into the medium when the cloning vectorincludes the β-galactosidase operon.

The tributyrin diffusion agar method is the method ofchoice for researchers the world over [71–78]. In additionto tributyrin, other esterolytic substrates are also employed,such as Tween 80 alone or in combination with Nile Blueor Neat’s foot oil with Cu2+ salts [21,79]. However, asmentioned previously these substrates are easily hydrolysedby esterases and are not indicators of true lipase. Therefore,the clones selected based on these protocols have to beconfirmed by some other true lipase assay.

A conclusive lipase plate assay is the trioleoylglycerol}Rhodamine B agar plate assay described by Kouker andJaeger [21]. This assay procedure has been used widely to

screen lipase-positive clones [14–16,78,80,81]. However,different researchers have used different concentrations ofRhodamine B in the medium, such as 0.001% (w}v)[14–16,81] and 0.0002% (w}v) [73], which could be becauselower concentrations of Rhodamine B do not always lead toa clear fluorescent halo. Another drawback of this protocolis that a clear zone is visible under UV light only when a highenzyme titre is obtained. Also, sometimes a non-fluorescenthalo due to the hydrolysis of olive oil is visible under visiblelight only.

Filter assays This is another way to conclusively selectpositive clones. Colonies on an agar plate are overlaid withfilter papers soaked in lipase substrate solutions. Positiveclones are detected by development of colour due to thehydrolysis of the substrate by the enzyme. Reiter et al. [82]used Whatman filter papers soaked with β-naphthyl-2-chloro-propionate and Fast Blue B dye to screen for esteraseactive colonies. Prim et al. [73] confirmed the lipolytic ac-tivity of positive clones of a genomic library by using filterpaper containing 4-methylumbelliferone oleate as the sub-strate. A similar method was used by Tan and Miller [76] toconfirm lipase activity in a broth culture of recombinantEscherichia coli.

Assays for screening large numbers of samplesMolecular techniques such as protein engineering anddirected evolution are based on screening of a massivenumber of samples. Thus it is imperative to have assayprotocols that can rapidly screen a large library and selectthe desired variants.

Titre plate assays have to a large extent enhanced theprobability of discovering desired variants by increasingscreening throughput of genetic diversity libraries. Reactionsthat take place in titre plates allowing for spectro-photometric detection of the products are the method ofchoice in the development of high-throughput screeningmethods [83].

Reetz and Jaeger [10] have described a screening systemusing p-nitrophenol esters as substrates. The 96-well micro-titre plates were loaded with the culture supernatants ofmutants followed by the addition of enantiomerically pure (R)and (S) substrates. In this way, up to 48 mutants per plate canbe screened. The enzyme-catalysed hydrolysis of each (R)}(S)hydrolysis pair was monitored by detecting the generation ofp-nitrophenol by spectroscopy. Using this system about500–600 mutants could be screened per day by one person.In such systems, fluorogenic substrates can also be applied,the advantage being that only small amounts of the substrateare needed [10].

Liebeton et al. [84] have used microtitre plates toscreen for a mutant producing an enantioselective lipasefrom a library. Each recombinant colony was resuspended in



a well of a microtitre plate filled with the growth medium andthe lipase inducer. Following growth, the culture broth wasadded to two wells of a second microtitre plate containing an(R)- or (S)-enantiomer and the reaction was monitoredusing a photometer.

Enantioselective reactions can also be monitored bydetecting the black-body radiation produced by the reactionof (R)- and (S)-configured enantiomers of a chiral alcoholusing an IR-thermographic camera [85]. Reetz and Jaeger[83] detected enantioselective lipases by carrying outacylation of the substrate in a microtitre plate and monitoringthe reaction in a time-resolved manner using an IR camera.

The company Biotrove used a 2500-channel nanotitreplate to perform over 30000 screens in less than 2 days todiscover a mutant lipase whose activity was 20-fold higherthan the wild-type. The clones from a mutant lipase librarywere added to the nanotitre plate and the lipase activity ineach channel of the plate was inferred by measuring the rateof fluorescence increase following addition of coumarinoleate.

The above assays are useful in systems showing highenzyme expression. However, these protocols cannot beapplied to all screening systems because of non-expressionof target lipase genes or extremely low and thereforeundetectable expression levels. In such cases, selection isbased on other protocols such as the detection of enzymeactivity in culture broth using assays such as titrimetry orcolorimetric assays, HPLC or MS. In other cases, detectionis based on consensus or specific DNA sequences such asthe N-terminal amino acid sequence or the substrate-bindingsequence of the target enzyme using protocols such ascolony, Southern or Western hybridization. However, allthese protocols have the disadvantage of being extremelytime-consuming and laborious. Thus there is a need forassays that are rapid and sensitive enough to be able todetect extremely low expression.

Conclusions

Lipases are the most versatile biocatalysts and bring about arange of bioconversion reactions using a variety of sub-strates, ranging from lipids to a wide array of esters. Inaddition, lipases have the unique property of working at thelipid}water interface. Owing to the wide substrate specificityof lipases, a number of assay protocols are employed.However, today the most widely used lipase assay protocolis the titrimetry assay using olive oil as a substrate.Determination of lipase activity at the lipid}water interface isalso indicative of a true lipase. However, recent structuralstudies on several lipases point towards the fact that anumber of these differ with respect to their properties.A variety of esters also serve as substrates for lipases. The

p-nitrophenyl palmitate assay is the method of choice forestimating the esterolytic activity. However, there is a needto distinguish esterolytic activity of lipases from esterases.Therefore, a number of assay protocols should be simul-taneously employed for an enzyme to be conclusivelyassigned as a true lipase. To make this endeavour possible,there is an urgent requirement for assays that are simpler,more rapid, more specific and which incorporate a widerrange of substrates than are currently used.

The most important property of lipases is theirenantioselective nature. However, the exploitation of thisproperty is restricted, owing to expensive and inaccessiblesubstrates. Thus the need of the hour is to develop moresensitive protocols involving cheaper substrates so thatscreening of enantioselective enzymes can be adopted as aroutine procedure in lipase research.

The development of enzymes with novel properties bysearching the present biological diversity or by creatingnewer enzyme variants hold a pivotal position in futureenzyme technology. However, before the discovery orcreation of enzyme variants can be put into practice, moreefficient screening methods have to be developed because,until recently, existing screening systems allowed for only afew dozen determinations or selectivity factors per day.Thus all efforts in this field of endeavour depend upon thedevelopment of further high-throughput screening systemsand the use of different substrates.

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Lipase assays for conventional and molecular screening: an overview

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