An easy and fast gel diffusion assay for detecting and monitoring lipase activity by quantification of fluorescein is described. By measuring the intensity of fluorescein, it is possible to obtain a calibration curve with a regression coefficient better than by using the radius of fluorescent haloes. Through the quantification of fluorescence intensity of fluorescein released after the hydrolysis of a fluorescent ester, fluorescein dibutyrate, used as substrate in agar plates, commercial and skimmed milk lipase activity were studied. Moreover, with this method, lipase activity can be monitored in reaction medium that contains compounds which are affected by turbidity or cause measurement interference for UV-spectrophotometer and fluorimeter. In this experiment, boiled skimmed milk was dispersed in the agar gel with fluorescein dibutyrate, and it was used as a reaction medium to mimic natural conditions. The development of such an assay has a potential for applications in industries ranging from pharmaceuticals to food production and monitoring.U radu je opisana jednostavna i brza metoda određivanja i praćenja aktivnosti lipaze pomoću fluoresceina. Mjerenjem intenziteta fluorescencije fluoresceina može se dobiti kalibracijska krivulja s koeficijentom regresije boljim od onog dobivenog mjerenjem promjera fluorescentne zone. Kvantitativnim određivanjem intenziteta fluorescencije fluoresceina, oslobođenog hidrolizom estera fluorescein dibutirata na podlozi s agarom, ispitana je aktivnost komercijalne lipaze, te one izolirane iz obranog mlijeka. Ovom se metodom može pratiti aktivnost lipaze u reakcijskoj smjesi koja sadržava spojeve što uzrokuju zamućenje ili interferiraju s uzorkom pri mjerenju UV-spektrofotometrom i fluorimetrom. U istraživanju je upotrijebljena reakcijska smjesa od prokuhanog obranog mlijeka raspršenog na agar gelu s fluorescein dibutiratom. Daljnim bi se razvojem ova metoda mogla primijeniti u farmaceutskoj i prehrambenoj industriji, zaštiti okoliša i sl

Anna Lante

Giovanna Di Pierro

Giovanna Lomolino

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ISSN 1330-9862 scientific note(FTB-2708)A Quantitative Fluorescence-Based Lipase AssayGiovanna Lomolino, Giovanna Di Pierro and Anna Lante*Department of Agronomy, Food Natural Resources, Animals and Environment, Agripolis, University ofPadova, Viale dell’Università 16, IT-35020 Legnaro, PD, ItalyReceived: January 13, 2011Accepted: February 9, 2012SummaryAn easy and fast gel diffusion assay for detecting and monitoring lipase activity byquantification of fluorescein is described. By measuring the intensity of fluorescein, it ispossible to obtain a calibration curve with a regression coefficient better than by using theradius of fluorescent haloes. Through the quantification of fluorescence intensity of fluores-cein released after the hydrolysis of a fluorescent ester, fluorescein dibutyrate, used as sub-strate in agar plates, commercial and skimmed milk lipase activity were studied. More-over, with this method, lipase activity can be monitored in reaction medium that containscompounds which are affected by turbidity or cause measurement interference for UV--spectrophotometer and fluorimeter. In this experiment, boiled skimmed milk was dispersedin the agar gel with fluorescein dibutyrate, and it was used as a reaction medium to mimicnatural conditions. The development of such an assay has a potential for applications inindustries ranging from pharmaceuticals to food production and monitoring.Key words: agar plate assay, hydrolytic activity, lipase, milkIntroductionIn the search for novel enzymes, it is necessary toapply functional tests during high-throughput screeningsthat are capable of detecting enzyme activities with highselectivity and sensitivity (1). In this regard, several meth-ods have been developed for the measurement of lipa-ses, an important group of ubiquitous biocatalysts whichhave relevant applications in the food, detergent and phar-maceutical industries (2). As reported by Josef et al. (3),lipases not only catalyze the hydrolysis of triglyceridesbut also perform esterification, interesterefication, acido-lysis, alcoholysis, and aminolysis. Currently, the basic char-acteristics driving the optimization of any procedure thatmeasures enzymatic activity are that the method is simple,direct, specific, and ready for automation. Agar platemethods, which are useful for studying and quantifyingenzymes present in biological extracts (4), may providean alternative to other methods and an interesting start-ing point from which to develop new assays. Visuali-zation of lipase on gel media can be accomplished byusing dyes because the drop in pH due to released fattyacids can be monitored by colour changes of a pH indi-cator dye (2–5). Since there is a linear relationship be-tween the diameter of the fatty acid diffusion spot andthe logarithm of the enzyme concentration, the techniqueis very convenient for detecting lipolytic organisms, eventhough metabolic acidification of the medium caused byfactors other than free fatty acids released by enzymaticactivity can give false results. Fluorescence assays canmeasure the reaction products that become fluorescentupon hydrolysis and can be continuously monitored (6).Moreover, these methods are very sensitive and less sus-ceptible to compounds that cause background signals(7). Sandoval and Marty (8) reported the use of a gelassay that relied on a fluorescent indicator to measure invivo synthetic activity of lipases derived from microor-ganisms. This protocol is based on the esterification of along-chain alcohol and different fatty acids contained ina rhodamine B agar plate. It is very sensitive, but it isnot suitable for bacterial strains which metabolize fattyacids. To overcome the problems described above, wemeasured the hydrolytic activity of a commercial prepa-479G. LOMOLINO et al.: Fluorescence-Based Lipase Assay, Food Technol. Biotechnol. 50 (4) 479–482 (2012)*Corresponding author; Phone: ++39 049 827 2920; Fax: ++39 049 827 2929; E-mail: anna.lante@unipd.itration of lipase and of fresh skimmed milk through quan-tification of fluorescein, a fluorescent molecule attachableto many substrates (9), which is released after enzymecatalysis. Moreover, to avoid complications when scal-ing up from the laboratory to industrial applications, theenzyme activity of fresh skimmed milk was tested notonly in a TBS agar (synthetic buffer), but also using boiledskimmed milk which was dispersed in agar gel to mimicnatural conditions.Materials and MethodsPlates (15 cm in diameter) were filled with 2 % (bymass per volume) agarose (A5093-500G; Sigma-Aldrich,St. Louis, MO, USA) dissolved in microwave oven in TBS(Tris-buffered saline) at pH=7. The solution was cooledto 70 °C, and 50 mM of fluorescein dibutyrate (FDB;Sigma-Aldrich) and 0.2 % Triton X-100 (BDH Chemicals,Poole, UK) were added. To disperse fluorescein dibuty-rate, we used Triton even though it is a UV-absorbingdetergent and the fluorescent measurement was madeunder UV excitation.A volume of 1 mL of 50 mM FDB and 50 mL of gelsolution were transferred into plates and cooled at roomtemperature. The thickness of the resulting gel was 3mm. The volume and thus the depth of the agar in theplate is an important parameter as it influences signalstrength (data not shown).Wells (3 mm in diameter) were obtained using a corkborer with a volume of about 50 mL. To obtain a cali-bration curve, increasing amounts of fluorescein (Sigma--Aldrich) (0.3, 0.6, 0.9, 1.2, 1.5, 1.8 and 2.1 mg) dissolvedin TBS were loaded into each well. Additionally, increas-ing quantities (3, 6, 9 and 12 mg of protein) of a com-mercial lipase from hog pancreas (Fluka, Sigma-Aldrich,30.1 U/mg) dissolved in TBS and fresh skimmed milk(FSM) (10, 15, 20 and 25 mg of protein) were loaded intoeach well. Protein quantification was carried out by theBradford Coomassie method using Pierce Coomassieprotein assay kit (Thermo Scientific Pierce, Rockford, IL,USA). The calibration curve and enzymatic test of thecommercial lipase and of FSM were carried out at thesame time and under the same conditions at 39 °C.After an hour, fluorescent haloes were visible under UVlight. To monitor the time evolution of the radius andfluorescence, measurements were taken every hour for 4h. To reproduce the natural turbid conditions of milk,the enzyme activity of FSM was then tested in boiledskimmed milk incorporated into the agarose gel (BSM),heated at 100 °C for 30 min in order to denature consti-tutive enzymes, especially lipase, and then supplement-ed with FDB. Images of enzymatic activity were acquiredby a digital camera and analyzed with the Gel Doc 1000system (Bio-Rad, Hercules, CA, USA). Radius and fluor-escence measurements were taken using Molecular Ana-lyst® software (Bio-Rad).The standard curves were obtained from (i) a log trans-formation of increasing fluorescein quantities and theradius of the haloes, and from (ii) the increasing quanti-ties of fluorescein and the intensity of fluorescence con-verted to counts (fluorescence units, FU) by the Mole-cular Analyst® software. For the regression analysis andt-test (p<0.05), Excel and CoHort software (Monterey,CA, USA) were used, respectively.Results and DiscussionFig. 1 shows the quantification of lipase activity usingeither the measurement of the radius of the fluorescenthalo created by fluorescein, or the intensity of fluores-cence. The obtained images were enhanced by Molecu-lar Analyst® and converted into peaks and related mea-surements. Fig. 1a shows the fluorescence intensity peakscreated by increasing doses of fluorescein. In order to480 G. LOMOLINO et al.: Fluorescence-Based Lipase Assay, Food Technol. Biotechnol. 50 (4) 479–482 (2012)050100150200250024681012140.9 1.2 1.5 1.8m(fluorescein)/μgy=89.524x+10.857 R2=0.99650 0.5 1.0 1.5 2.0 2.5m(fluorescein)/μgy=6.0893x–7.7961 R2=0.947852.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4log( (fluorescein)/μg)mr/mm50100150Fluorescenceintensity/FUa)b)c)Fluorescenceintensity/FUFig. 1. Quantification of fluorescein: a) an example of densito-metric analysis of increasing doses of fluorescein. Image anal-ysis was performed by avoiding the black internal hole of thefluorescent halo. Intensity of fluorescence is converted to counts(fluorescence units, FU) by Molecular Analyst® software. A num-ber of 255 counts represents the value of fluorescence satura-tion; b) linear regression curve between the intensity of fluores-cence, expressed by the measure of the height of the peaks andthe fluorescein mass; c) linear regression curve between the loga-rithm of fluorescein mass and the radius of stained zone formedon agarose plate. All values are means of three trials. Coeffi-cient of variation (CV) is less than 1 %verify the relationship between the intensity of fluores-cence, as measured by the height of the peaks, and thequantity of fluorescein, a model of regression was stud-ied (Fig. 1b). The regression analysis reveals a lineartrend. The correlation coefficient, R2, was found to be0.9965, which is better than the acceptance criteria of0.9900. This method of fluorescein quantification wascompared with methods proposed by other authors (4–9) that consider only the measurement of the diameteror the radius of the haloes. However, as shown in Fig.1a, the halo borders are not clear. The diffusion of fluor-escence does not allow easy measurement of the radiusof the halo. The linear relationship (Fig. 1c) between theradius and the log of fluorescein quantity produces a re-gression coefficient of 0.9478. The value is lower thanthat obtained using peak intensity and is outside the ac-ceptance criteria. This method is also slower because itrequires a log transformation.The linear range of Fig. 1 corresponds to a very lowquantities of fluorescein (0.3–2.1 mg), but due to the con-trasting effect of white fluorochrome signal and the blackbackground under UV light, enhanced by the MolecularAnalyst software, it is possible to load even lower con-centrations of fluorescein, increasing the sensitivity of themethod (data not shown). Besides, the calibration curveof Fig. 1b, encompassing a low change in concentration,shows how small quantities of fluorescein produce greatvariations of intensity, further demonstrating the high sen-sitivity of the method. Sensitivity of an assay is essentialin most applications where the aim is the identificationof dilution of an enzyme, and therefore weak enzymaticactivity (6). The linear trend of the calibration curve isobserved until the maximum value of 255 counts (FU)that represents, for the Molecular Analyst software, thesaturation limit of fluorescence. The main advantage offluorimetric assays besides their sensitivity is the pos-sibility to continuously follow the reaction kinetics (2).There are many synthetic enzymatic substrates thatrelease fluorescein upon hydrolysis. In this experiment,a synthetic ester, FDB, was chosen to study lipase activ-ity. The lipolytic activity of increasing doses of lipasesof different origin was studied by using agar plates inwhich FDB, detergent and TBS were present. Table 1shows the lipolytic activity of the commercial enzymeand of FSM during 4 h of incubation (T1, T2, T3 andT4). As shown in Table 1, the lipase activities as esti-mated by the intensity of fluorescein released after thehydrolysis and as predicted by the radius of the halo arenot statistically significantly different as determined bythe t-test (p<0.05). Both methods show an increase in thelipolytic activity of both the commercial lipase and theFSM over time. The coefficient of variation is higher if theenzyme activity is estimated as a function of the radiusof the halo. This is due to the diffusion of fluorescencearound the border that makes it difficult to measure, asshown in Fig. 2a, which shows the haloes of fluoresceinreleased by increasing amounts of FSM-derived lipasesover time. It is likely that some FDB is incorporated intomicelles and that the lipase molecules that diffuse out ofthe well sample may hydrolyze FDB in the micelles.Fluorescence assays have not only got the advantageto be very sensitive but also they are less susceptible tointerference by compounds causing background signals.For instance, enzymatic activity can be measured in crudelysates and even in turbid solutions (8,10).It is known that synthetic esters, such as FDB, areinsoluble in aqueous buffers and in the presence of sur-factants. Turbidity caused by such synthetic esters hasbeen considered an interfering factor in techniques suchas spectrophotometric reading (11). Thus, the screeningmethod presented here, which is in gel media, allowsthe calculation of enzyme activity even if insoluble com-pounds that produce turbidity or interference are pre-sent. This method is specific for the quantification of theanalyte without interference, because the estimation of481G. LOMOLINO et al.: Fluorescence-Based Lipase Assay, Food Technol. Biotechnol. 50 (4) 479–482 (2012)Table 1. Comparison of lipase activities using TBS and BSM as reaction mediaBuffer Sample TimeFluorescence activityFUCV%r(fluorescein halo)mmCV%TBS lipaseT1 0.86±0.084 9.7 0.978±0.322 32.9T2 1.212±0.141 11.6 1.001±0.309 30.8T3 2.034±0.155 7.6 1.89±0.76 40.2T4 2.548±0.392 15.4 2.321±0.89 38.34TBS FSMT1 0.07±0.011 15.0 0.051±0.014 27.5T2 0.092±0.004 3.9 0.079±0.021 26.5T3 0.164±0.25 15.0 0.178±0.078 43.8T4 0.350±0.018 5.2 0.321±0.112 34.9BSM FSMT1 0.03±0.005 16.8 0.025±0.011 44.0T2 0.054±0.008 14.8 0.044±0.018 40.9T3 0.064±0.013 20.2 0.057±0.025 43.9T4 0.081±0.015 18.5 0.078±0.028 35.9Evolution of radius of fluorescein halo and fluorescence activity was monitored every hour for 4 h (T1–T4). To check the precision ofthe method, the corresponding coefficients of variation (CV) were calculated at the fluorescein mass of the used range. All valuesare means of three determinationsthe fluorescence intensity and haloes of fluorescein arenot affected by turbidity.Since this method monitors only fluorescence, the li-polytic activity of FSM was studied when boiled skimmedmilk was incorporated into the agarose gel (Fig. 2b). Thisallowed the study of the lipolytic activity under chemi-cal and physical conditions similar to natural turbid con-ditions of milk. As shown in Fig. 2b, the intensity andradius of haloes of fluorescein increased as a function oftime and quantity of the loaded sample, although theuse of BSM in the reaction medium causes slight back-ground noise. These findings are quantified in Table 1.The coefficient of variation (CV) is higher when BSM isused in the reaction medium and when the halo radiusis considered. However, there is not statistically signi-ficant difference between the two methods of quantifica-tion (t-test, p<0.05), and the interference caused by BSMin the reaction medium is very low and does not pre-vent the estimation of lipolitic activity of FSM. The li-pase activity of FSM, calculated with BSM in the reactionmedium, was lower than that detected in TBS, probablydue to the competition of two substrates, FDB and en-dogenous lipids in boiled skimmed milk (12).ConclusionThe method proposed here is a simple and fast as-say for measuring lipase and esterase activities. To ourknowledge, this is the first report on the use of gel diffu-sion assay for determining these enzymes by using fluo-rescein dibutyrate, fluorescent substrate with ester link-age, based on the activity quantification by the measureof fluorescence intensity of the halo. It is useful for mea-suring a large number of samples simultaneously, allow-ing for easy automation. This is an alternative to high--throughput methods such as microplate-based fluorescentlipase assay that have many wells per plate, and has theadvantage of not requiring a plate reader or fluorometer,which may not be broadly available in many laborato-ries.Another advantage is that it allows the determina-tion of enzyme activity even in the presence of insolublecompounds that produce turbidity or cause measurementinterference for UV-spectrophotometer and fluorimeter.In our laboratory this method allowed us to compare thelipolytic activity of milk of different mammals. Moreover,it should be possible to extend this method to other en-zyme assays based on fluorescent substrates. The develop-ment of an assay such as this holds great promise forapplications in industries ranging from pharmaceuticalsto food production and monitoring.AcknowledgementsThe authors gratefully acknowledge Prof Paolo Spet-toli for critical reading of the manuscript and Dr Fede-rico Zocca for technical assistance.References1. J. Grognux, D. Walher, E. Nyfeler, J.L. Reymond, Universalchromogenic substrates for lipases and esterases, Tetrahe-dron Asymmetry, 15 (2004) 2981–2989.2. F. Hasan, A.A. Shah, A. Hameed, Methods for detectionand characterization of lipases: A comprehensive review,Biotechnol. Adv. 27 (2009) 782–798.3. B. Josef, P.W. Ramteke, G.O. Thomas, Cold active micro-bial lipases: Some hot issues and recent developments, Bio-technol. Adv. 26 (2008) 457–470.4. F. Zocca, G. Lomolino, A. Lante, 3,4-Dihydroxyphenylala-nine gel diffusion assay for polyphenol oxidase quantifi-cation, Anal. Biochem. 383 (2008) 335–336.5. H. Scholze, H. Stütz, F. Paltauf, A. Hermetter, Fluorescentinhibitors for the qualitative and quantitative analysis oflipolytic enzymes, Anal. Biochem. 276 (1999) 72–80.6. J.P. Goddard, J.L. Reymond, Recent advances in enzymeassays, Trends Biotechnol. 22 (2004) 363–370.7. D. Gilham, R. Lehner, Techniques to measure lipase andesterase activity in vitro, Methods, 36 (2005) 139–147.8. G. Sandoval, A. Marty, Screening methods for syntheticactivity of lipases, Enzyme Microb. Technol. 40 (2007) 390–393.9. M. Schmidt, U.T. Bornscheuer, High-throughput assays forlipases and esterases, Biomol. Eng. 22 (2005) 51–56.10. L.F. Hernández, J.C. Espinosa, M. Fernández Gonzáles, A.Briones, â-Glucosidase activity in a Saccharomyces cerevisiaewine strain, Int. J. Food Microbiol. 80 (2003) 171–176.11. P. Jain, S. Jain, M.N. Gupta, A microwave-assisted micro-assay for lipases, Anal. Bioanal. Chem. 381 (2005) 1480–1482.12. H.C. Deeth, Lipoprotein lipase and lipolysis in milk, Int.Dairy J. 16 (2006) 555–562.482 G. LOMOLINO et al.: Fluorescence-Based Lipase Assay, Food Technol. Biotechnol. 50 (4) 479–482 (2012)Fig. 2. Lipolytic activity of increasing quantities of fresh skim-med milk over time, as measured on plates in the presence of:a) TBS and b) BSM as reaction media. See Table 1 for defini-tions of T1, T2, T3 and T4

Kvantitativno određivanje lipaze pomoću fluoresceina

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