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ANALYTICAL

BIOCHEMISTRY

Analytical Biochemistry 363 (2007) 297–299

Notes & Tips

A modified para-nitrophenyl palmitate assay for lipase syntheticactivity determination in organic solvent

Yun Teng, Yan Xu *

Laboratory of Brewing Microbiology and Applied Enzymology, School of Biotechnology, Key Laboratory of Industrial Biotechnology,

Ministry of Education, Southern Yangtze University, Wuxi, Jiangsu 214036, People’s Republic of China

Received 19 November 2006Available online 26 January 2007

Due to the diversity and increasingly new applicationsof various enzymes, many enzymatic activity assay meth-ods are developed every year. The same situation couldalso be applied to the case of lipases (EC 3.1.1.3). As a toolfor organic syntheses, more and more attention has beenpaid to the lipase-catalyzed reactions in nonaqueous mediaduring recent years [1]. Esterification and transesterifica-tion are two typical reactions catalyzed by this enzyme[2], and numerous important products, such as flavor esters[3], monoacylglycerols, optically pure building blocks [4],and biodiesel [5], could be produced by those reactions.However, not all of the lipase can catalyze the above reac-tions in organic solvent, so a suitable enzyme must be cho-sen for the proposed procedure.

Enzyme selection depends on the activity determination;therefore, many different lipase assay methods have beendeveloped for various purposes [6–8]. Most of the methodsare based on hydrolytic assays. However, a problem occurswhen the synthetic activities of the enzymes in organic sol-vents do not correspond with the hydrolytic activities inaqueous solutions [9,10]. Enzymes identified by the hydro-lytic methods are not necessarily suitable for the desiredsynthetic reactions. As a result, several methods based onesterification and transesterification activities have beenproposed [11–13]. Most of these methods are based ongas chromatography (GC),1 high-performance liquid chro-

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doi:10.1016/j.ab.2007.01.026

* Corresponding author. Fax: +86 510 85864112.E-mail address: yxu@sytu.edu.cn (Y. Xu).

1 Abbreviations used: GC, gas chromatography; HPLC, high-perfor-mance liquid chromatography; HTS, high-throughput screening; pNPP,para-nitrophenyl palmitate; pNP, para-nitrophenol; CAL, Candida antarc-

tica lipase; BCL, Burkholderia sp. lipase; PCL, Penicillium sp. lipase; CRL-1,Candida rugosa lipase; PFL, Pseudomonas fluorescens lipase; CRL-2,Candida rugosa lipase, type VII; PPL, porcine pancreas lipase, type II;RCL, Rhizopus chinensis lipase; GC–FID, gas chromatography–flameionization detector.

matography (HPLC), or titrimetric determinations In thecase of screening and investigating large numbers of en-zymes, those methods are extremely time-consuming andexpensive. Recently, a high-throughput screening (HTS)method for hydrolase synthetic activity assay based onfluorescence detection was developed to avoid the time-consuming problem [14]. Based on the same mechanism,another fluorometric assay method for protease-catalyzedtransesterification was also developed [15]. However, thefluorometric assay is not always available in ordinary bio-chemistry laboratories; furthermore, the fluorescence sub-strate is expensive and high-quality reaction solvents arerequired.

Selection of suitable reaction and substrate is crucial foran activity assay. In the case of lipase activity assay, thereaction should be catalyzed by lipase in organic solvent,and the substrate should be detected by a simple method.Unfortunately, because most of the substrates for lipase as-say could exhibit absorbance characteristics only in a cer-tain aqueous phase after hydrolysis by lipase, selection ofa proper substrate for determining the lipase syntheticactivity in organic solvent became a key issue. Pencreachand Baratti determined lipase hydrolytic activity in organicsolvent using para-nitrophenyl palmitate (pNPP) as thesubstrate by an extracting procedure [16]. In the currentwork, based on Pencreach and Baratti’s description, wepropose a modified method to determine the syntheticactivity in the organic solvents. The method is based onlipase-catalyzed transesterification reactions betweenpara-nitrophenyl esters and ethanol. The yield of para-ni-trophenol (pNP) was detected by a simple spectrophoto-metric method and then used as the indicator forsynthetic activity determination.

pNPP, a well-known substrate for lipase hydrolysisactivity, was selected as the substrate in this work. Thetransesterification between pNPP and ethanol catalyzed

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Fig. 1. Comparison of the various methods applied for the determinationof the synthetic activities of different lipases. In the titrimetric method foresterification (black bars), experiments were conducted in a 50-mlstoppered conical flask containing 5 ml stock solution (0.6 M palmiticacid and 0.72 M ethanol in n-heptane) and 50 mg lipase. The flask wasshaken at 200 rpm for 15 min at 40 �C. After adding 10 ml N,N-dimethylformamide to stop the reaction, the mixture was titrated withKOH methanol solution (0.2 M) using thymol blue (0.3% [w/v] inmethanol) as the indicator. In the colorimetric method for transesterifi-cation (medium gray bars), Experiments were carried out in a volume of5 ml containing 10 mM pNPP and 1 M ethanol in n-heptane, and then25 ll samples were extracted by the aqueous alkaline phase and detected at410 nm. In the GC method for transesterification (light gray bars),experiments were carried out in a volume of 5 ml containing 10 mM pNPPand 1 M ethanol in n-heptane, and then 150 ll samples were extracted bythe aqueous alkaline phase. Then 80 ll supernatant was taken, mixed with20 ll 2-hexanol (2.5 g/L, internal standard), and analyzed by GC. Dataare means of at least three determinations.

298 Notes & Tips / Anal. Biochem. 363 (2007) 297–299

by lipases was carried out in n-heptane, and the system wasused as a model reaction. Liberation of pNP was detectedusing a common spectrophotometer (UV-2102PC, Unico[Shanghai] Instruments, China) after extracting aliquotsof sample from the reaction medium with 1 ml of 0.1 MNaOH solution [16]. To validate the effectiveness of themethodology, it was necessary to verify or demonstratethe following: (i) the capability of transesterification be-tween pNPP and ethanol, which could be considered asthe lipase’s catalytic ability or synthetic activity in organicsolvent, and (ii) the ability to distinguish various lipaseswith different synthetic activities.

Seven common commercial lipases were used in thisstudy. Candida antarctica lipase (CAL, Novo435) was a giftfrom Novo Nordisk (Denmark); Burkholderia sp. lipase(BCL, Amano PS-C), Penicillium sp. lipase (PCL, AmanoG), Candida rugosa lipase (CRL-1, Amano AY), and Pseu-

domonas fluorescens lipase (PFL, Amano AK) were pur-chased from Amano Pharmaceutical (Japan); andCandida rugosa lipase (CRL-2, lipase type VII) and porcinepancreas lipase (PPL, type II) were purchased from Sigma(Germany). Rhizopus chinensis lipase (RCL, whole-cell li-pase prepared in our laboratory [3]) was selected to inves-tigate the efficiency of the colorimetric method and themodel reaction. The colorimetric assays were carried outin 2-ml Eppendorf tubes. Lipase of 0.01 to 0.3 U wasweighed out corresponding to weights of 1 to 20 mg andthen was mixed with 0.5 ml stock solution (10 mM pNPP[Sigma] in n-heptane). To start the reaction, 30 ll ethanol(1 M) was added to the reaction mixture. The mixturewas incubated at 40 �C with a shaking speed of 200 rpmfor 5 to 30 min. After settling the lipase in the reaction mix-ture for 30 s, 25 ll of the clear supernatant was taken andthen immediately mixed with 1 ml of 0.1 M NaOH in a1 ml cuvette. The pNP liberated was extracted by the aque-ous alkaline phase, and then the extraction was detected at410 nm against a blank without enzyme using a UV–visiblespectrophotometer. When necessary, either the organicsample was diluted with n-heptane or less volume of samplewas taken to ensure measurement within its linear rangeThe molar extinction coefficient of pNP was estimated at1.27 · 10[3] M[–1] cm[–1] by using standard solutions ofpNP in n-heptane and extraction described above. Toavoid undesired hydrolysis due to the presence of water(especially in the transesterification reaction), all reactantsand solvents were dried by molecular sieves before use.

The transesterifications were also monitored by gaschromatography–flame ionization detector (GC–FID, Agi-lent 6820, Agilent Technologies, Shanghai, China). Thereaction was carried out exactly the same as the proceduredescribed above. After settling the lipase in the reactionmixture for 30 s, 150 ll of the clear supernatant was takenand then immediately mixed with 3 ml of 0.1 M NaOH toremove the pNP. The mixture was thoroughly mixed andcentrifuged at 8000 rpm for 10 min, and then 80 llsupernatant was taken and mixed with 20 ll 2-hexanol(2.5 g/L, internal standard). The sample was analyzed by

GC–FID equipped with a polyethylene glycol column(AC20, 30 m · 0.22 mm · 0.25 lm, SGE International,Australia). Fig. 1 shows that the catalytic conversion ratedetermined by both methods (colorimetric assay andGC–FID) agreed very well. The figure also shows thatthe transesterification reaction between ethanol and pNPPcould be catalyzed by several lipases and that the catalyticeffect could be determined by measuring the release of theamount of pNP that could be detected by the simple color-imetric method. Because transesterification and esterifica-tion are two typical reactions catalyzed by lipases, mostof the synthetic activity definitions are based on the tworeactions. To find out whether the two reactions correlatedwell with ethyl palmitate synthesis, the esterification be-tween palmitic acid and ethanol catalyzed by the same en-zymes was carried out. For each lipase, the experiment wasconducted in a 50 ml stoppered conical flask containing5 ml stock solution (0.6 M palmitic acid and 0.72 M etha-nol in n-heptane) and 50 mg lipase. The flask was shakenat 200 rpm for 15 min at 40 �C. After adding 10 ml N,N-di-methylformamide to stop the reaction, the mixture wastitrated with KOH methanol solution (0.2 M) using thymolblue (0.3% [w/v] in methanol) as the indicator. Fig. 1 showsthat the results determined by the two methods correlatedwell except for CAL and PFL. One possible reason for thisphenomenon could be that different lipases have differentsubstrate specificities [17]. In general, the higher the esteri-fication activity, the higher the transesterification activity

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Fig. 2. Kinetics of transesterification of pNPP with ethanol with differentamounts of BCL in n-heptane: ¤, 2.5 mg/ml BCL; h, 5 mg/ml BCL; D,10 mg/ml BCL; e, 15 mg/ml BCL; *, 25 mg/ml BCL Assays were carriedout in a volume of 5 ml containing 10 mM pNPP and 1 M ethanol in n-heptane, and then 25 ll samples were extracted by the aqueous alkalinephase and detected at 410 nm at regular time intervals. Data are means ofat least two determinations.

Notes & Tips / Anal. Biochem. 363 (2007) 297–299 299

for the same lipase. Therefore, it could be concluded thatthe new colorimetric method based on transesterificationreaction measurement could be used to evaluate the lipasesynthetic activity in organic solvent.

Fig. 2 depicts the kinetics curve of the transesterificationreaction between pNPP and ethanol catalyzed by BCL withdifferent concentrations. As expected, the initial reactionrate increased proportionally to the lipase concentrationThis result confirmed the possibility of monitoring thetransesterification by the proposed colorimetric method.To evaluate whether this method could be used for lipasescreening, the synthetic activity of mycelium-bound lipasefrom 30 mutant strains of R. chinensis was assayed by thecolorimetric and titrimetric methods. The results showthe good consistency of the two measurement methods(see supplementary figure).

A simple modified colorimetric method for screening lip-ases with synthetic activity in organic solvent has been pro-posed and validated. The greatest improvement of thismethod compared with the Pencreach and Baratti methodis that lipase synthetic activity is detected in organic solventbase on a transesterification reaction [16]. With this meth-od, the substrate used was less expensive and was commer-cially obtainable, and the measurement time could beshortened greatly. Despite some drawbacks on dealing withsubstrate specificities and measurement accuracy stillremaining, the proposed method potentially provides anefficient and rapid way to screen synthetic activity of vari-ous lipases in organic solvent. Because only a small amountof substrate and enzyme is required, the method could alsobe useful in HTS formats to deal with a large amount ofenzyme screening.

Acknowledgments

This work was financially supported by the NationalNatural Science Foundation of China (3047006), the Pro-gram for Changjiang Scholars and Innovative Research

Team in University (IRT0532) and the Ministry of Educa-tion. PR China under Program for New Century ExcellentTalents in University (NCET).

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at doi:10.1016/j.ab.2007.01.026.

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