Biochemical Engineering Journal 18 (2004) 6571

Activity and stability of a crude lipase from Penicillium aurantiogriseum in aqueous media and organic solvents
V.M.G. Lima a,b , N. Krieger a, , D.A. Mitchell b , J.D. Fontana c
Chemistry Department, Applied Chemistry Research Centre (CEPESQ), Federal University of Parana (UFPR), P.O. Box 19081, Centro Politcnico, Jardim das Amricas, Curitiba 81531-990 Parana, Brazil b Solid State Fermentation Laboratory, Biochemistry and Molecular Biology Department, UFPR, P.O. Box 19046, Centro Politcnico, Jardim das Amricas, Curitiba, 81531-990 Parana, Brazil c Biomass Chemo/Biotechnology Laboratory (LQBB), Biochemistry and Molecular Biology Department/Pharmacy Department UFPR, P.O. Box 19046, Centro Politcnico, Jardim das Amricas, Curitiba, 81531-990 Parana, Brazil Received 14 January 2003; accepted after revision 2 June 2003
a

Abstract The effects of various environmental conditions and chemical compounds on the activity and stability of the lipolytic preparation obtained from a wild strain of Penicillium aurantiogriseum were characterized during a preliminary evaluation of its potential for use in biocatalysis. In aqueous solution, the optimum pH for activity was 8.0 and the enzyme was stable between pH 6.0 and pH 9.0. In assays of 1 min duration carried out at pH 8.0, enzyme activities were quite high from 37 to 70 C, with a maximum at 60 C. However, thermal stability was rather low at temperatures higher than 28 C. Hydrolytic activity was enhanced by Mg2+ , Zn2+ , Co2+ and Mn2+ , but was inhibited by Cu2+ , Ba2+ and Hg2+ , while Ca2+ had no effect. Sodium azide activated the enzyme. Triton X-100 caused an inhibition of 52%, while Tween 80 and SDS had negligible effects on enzymatic activity. Despite the low ratio of the activity towards p-nitrophenyl palmitate (pNPP) in organic medium to that in aqueous medium (RO/A = 4.3 102 ), the enzyme showed a good stability in organic solvents with high log P values, the best result being in n-heptane (114% residual activity). These promising results with the crude preparation justify the undertaking of purication studies and the use of the pure enzyme in a more in-depth investigation for its potential in biocatalysis in organic solvents. 2003 Elsevier B.V. All rights reserved.
Keywords: Lipase; Penicillium aurantiogriseum; Enzyme activity; Enzyme biocatalysis; Filamentous fungi; Kinetic parameters

1. Introduction Lipases (E.C.3.1.1.3) have been dened as carboxylesterases that hydrolyse acylglycerides with acyl chains with more than 10 carbon atoms [1]. Industrial applications for these enzymes are mainly related to their use in detergents. Nevertheless, the broad potential for their application in different elds, from the chemical and pharmaceutical industries to the paper industry, is described in a large number of papers per year. This interest comes from the ability of these enzymes to catalyze not only hydrolytic reactions but also synthetic ones, the latter occurring in organic media when the water content is sufciently limited. Further, they can have a broad substrate specicity, but they also can be very selective in the reaction catalyzed, including a high enantioselectivity [1].
Corresponding author. E-mail address: nadiak@quimica.ufpr.br (N. Krieger).

A considerable number of bacterial and fungal lipases have been commercially produced, the latter being preferable because fungi generally produce extracellular enzymes, which facilitates recovery of the enzyme from the fermentation broth. Amongst fungi, the genus Penicillium contains many lipase producers [25]. Lipases of Penicillium aurantiogriseum (previously Penicillium cyclopium) have been extensively studied and nd industrial application in the production of monoacylglycerides [6]. Recently, we have isolated and optimized the production of a lipase from a strain of P. aurantiogriseum [7]. The strain produced the enzyme in an inorganic medium supplemented with olive oil and, after optimization of the culture conditions and medium composition, a volumetric activity of 24 U ml1 was achieved, with a very high specic activity (480 U mg per protein). These results suggest that it might be possible to reduce production costs, since current methods for production of lipase by strains of Penicillium use media with signicant concentrations of yeast extract

1369-703X/$ see front matter 2003 Elsevier B.V. All rights reserved. doi:10.1016/S1369-703X(03)00165-7

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V.M.G. Lima et al. / Biochemical Engineering Journal 18 (2004) 6571 Table 1 Extinction coefcient of pNP at different pH values Buffer Citratephosphate Citratephosphate Phosphate Phosphate TrisHCl TrisHCl GlycineNaOH GlycineNaOH pH 6.0 7.0 7.0 8.0 8.0 9.0 9.0 10.0 Extinction coefcient (103 ) (l mol1 cm1 ) 1.7 8.7 8.0 1.5 1.4 1.7 1.6 1.9

and peptone, which are not only relatively expensive but can also increase the cost of enzyme recovery after the fermentation [7]. In the present paper we characterize the activity and stability of the crude lipolytic extract from P. aurantiogriseum in various conditions of temperature and pH and in the presence of various organic solvents and metal ions, as part of ongoing studies to evaluate its potential for use in biocatalysis in organic media. 2. Materials and methods 2.1. Microorganism P. aurantiogriseum Dierckx was previously isolated in our laboratory as a contaminant of soybean oil and characterized by the Fungal Culture Collection, FIOCRUZ (Oswaldo Cruz Foundation, Rio de Janeiro, Brazil) and deposited as IOC 4212. It was maintained as a spore suspension in 50% (v/v) glycerol/water at 4 C. Before each experiment, it was subcultured onto Czapek agar with 1% (v/v) olive oil [7]. 2.2. Growth conditions The medium used was previously optimized by Lima et al. [7]. It contained, per liter, 2 g KNO3 , 0.5 g MgSO4 7H2 O, 1.0 g K2 HPO4 , 0.44 g ZnSO4 7H2 O, 1.12 g FeSO4 7H2 O, 0.203 g MnSO4 7H2 O, 12.50 g (NH4 )2 SO4 and 1% (v/v) olive oil. The inoculum was prepared in a 250 ml Erlenmeyer ask containing 50 ml of medium, while the fermentation was done in 500 ml Erlenmeyers containing 125 ml of medium. Cultures were started by the addition of 1 ml of a spore suspension (108 spores/ml) and were incubated at 29 C in a rotary shaker at 120 rpm. 2.3. Preparation of the crude extract After 72 h of culture, when the lipolytic activity was maximal [7], the contents of the ask were ltered through gauze to remove the mycelium. Ammonium sulphate was added to the supernatant to 80% of saturation, with mild agitation. The extract was then maintained under gentle stirring at 4 C for 12 h, after which it was centrifuged at 12 000g for 10 min. The supernatant was removed and the precipitate was re-suspended in a minimal volume (approximately 1 ml) of 50 mM phosphate buffer pH 7.0. To produce sufcient enzyme for the characterization studies, re-suspended precipitates from several asks, each treated as described above, were pooled. This suspension was dialyzed against the same buffer, at 4 C, with two exchanges. The molecular weight cut-off of the dialysis membrane was 14 000 Da. The dialyzed fraction retained within the bag was used for the activity and stability studies. This concentrated crude extract had a volume of 20 ml (after dialysis), a protein content of 0.3 mg ml1 and a volumetric activity of 39.3 U ml1 . It

was stored at 4 C, with the addition of 0.02% (w/v) sodium azide. For the experiments of activity in organic solvents, 10 ml of the crude extract was lyophilized for 12 h at 45 C in a Jouan LP3 lyophilizer. 2.4. Lipase assay in aqueous solution and protein determination The p-nitrophenyl palmitate (pNPP) method was used for most of the activity determinations [3,8]. Activity tests were also done with other esters of p-nitrophenol (pNP), namely, p-nitrophenyl acetate (pNPA), p-nitrophenyl butyrate (pNPB), p-nitrophenyl caproate (pNPC) and p-nitrophenyl caprate (pNPCA). The coefcient of extinction of pNP at 410 nm was determined as a function of pH (Table 1). One unit was dened as the amount of enzyme liberating 1 mmol of pNP per minute at 37 C, under the conditions of the assay. The titrimetric method was also used to conrm the lipolytic activity, using olive oil and tributyrin (Sigma) as substrate [9]. An aqueous emulsion of each substrate was prepared with 20% (w/v) of substrate, 6% (w/v) of triethanolamine and 74% (v/v) of 50 mM phosphate buffer pH 8.0, which was strongly agitated for 30 min. One milliliter of enzyme sample was added to 5 ml of this emulsion and then incubated at 37 C for 20 min under mild agitation (300 rpm) with a magnetic stirrer. Sixteen milliliter of a 1:1 (v/v) solution of ethanol and acetone was then added, and this solution titrated with 50 mM NaOH. For this method, one unit of lipolytic activity was the amount of the enzyme that produced 1 mol of free fatty acids per minute, at 37 C. Protein was determined by the method of Bradford [10]. 2.5. Lipase assay in organic media The activity of the lipase crude extract, in the presence of organic media, was determined according to Pencreach and Baratti [11], with modications. The assay was done in twelve identical 25 ml Erlemeyers asks by adding 5 ml of a 10 mM pNPP solution in n-heptane to 1 mg of the crude lyophilized extract (14.7 g of protein) in each ask. The

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mixture was incubated in a shaker at 160 rpm and 37 C. At 5 min intervals two asks were removed and 9.5 ml 0.1 M NaOH was added to each. The mixture in each ask was agitated for 20 s to extract the pNP to the aqueous phase. The phases were separated and the aqueous phase containing the pNP was read at 410 nm in a Shimadzu spectrophotometer. The molar extinction coefcient of the pNP under these conditions (3.53 103 l mol1 cm1 ) was estimated by using standard solutions of pNP in n-heptane and applying the extraction procedure as described above. One unit of activity is dened as the amount of enzyme that liberates 1 mmol of pNP per minute under the assay conditions. 2.6. Effect of temperature on activity and stability Activity assays were done using the pNPP method with 50 mM phosphate buffer pH 8.0, at temperatures between 10 and 70 C. The reaction was followed during 5 min and activities were calculated on the basis of the product concentration at 1 min. Stability assays were done by incubating concentrated crude extract at 28, 37, 45 and 50 C, in the absence of substrate. Samples were removed and assayed for lipolytic activity by the pNPP standard method, at 37 C. 2.7. Effect of pH on activity and stability Activity assays were done using the pNPP method at 37 C in assay mixtures buffered to various pH values. The following buffers were used: citrate-phosphate pH 6.0 and 7.0; phosphate pH 7.0 and 8.0; TrisHCl pH 8.0 and 9.0 and glycineNaOH pH 9.0 and 10, all at 50 mM. Stability assays were done by incubating crude extract at 28 C for 1 h in buffers of different pH values (glycineHCl pH 2.2 and 3.0; citrate pH 3.06.0; citratephosphate pH 3.07.0; phosphate pH 7.0 and 8.0; TrisHCl pH 8.0 and 9.0 and glycineNaOH pH 9.0 and 10, all at 50 mM). In each assay 10 l of crude extract was added to 2.69 ml buffer solution. Lipase activity of each sample was then measured at 37 C, using 50 mM phosphate buffer, pH 8.0, by the pNPP method. 2.8. Effect of various compounds on enzyme stability The enzyme was incubated in the presence of various salts and detergents at 28 C for 1 h, at specied concentrations. The residual activity was then measured using the standard pNPP assay system. 2.9. Stability in organic solvents Ten microliter of crude extract was adsorbed onto a disc of lter paper of 3 mm diameter and incubated at 28 C in the presence of 1.4 ml of one of various water-immiscible

organic solvents, as described by Sztajer et al. [12]. After 1 h, the lters were taken out, the solvents were evaporated and phosphate buffer 50 mM pH 8.0 was added. For water-soluble solvents, the enzyme was incubated directly (10 l of crude extract in 1.4 ml of solvent). The residual activity was measured using the standard pNPP assay system.

3. Results and discussion 3.1. Effect of pH on lipolytic activity and pH stability Lipolytic activity (36 U ml1 ) was maximal at pH 8.0 (Fig. 1). The activity depended not only on the pH, but also on the type of buffer used. Higher activities were observed with phosphate buffer than with citratephosphate buffer or TrisHCl. This activation of lipases from the genus Penicillium by phosphate buffer has already been described in Ref. [13]. Lipases produced by the genus Penicillium have been reported as having optimal activity at neutral or slightly basic pH values [4,13], and the current lipolytic extract ts into this pattern. The lipolytic activity of the crude extract remained stable in the pH range of 5.09.0 (Fig. 2) during pre-incubation for 1 h at 28 C, with the residual activity remaining above 80%. In fact, incubation at pH values in the range of pH 6.08.0 caused an increase in the measured activity (20%), compared to the activity of the crude extract without incubation (see Fig. 2). The pH stability of the lipolytic extract differs from that reported in the literature for related enzymes. Fungal lipases are, in general, quite stable in the pH range from 4 to 7 and unstable in alkaline pH values, as has been observed for the lipases of P. citrinum [5], Penicillium roqueforti [13] and Mucor sp [14], as well as for lipases A and B of P. restrictum [15]. However, a deeper comparison among the pH stability results is not possible because of the different methods,
40 35

Activity (U mL-1)

30 25 20 15 10 5 0 5 6 7 8 9 10 11

pH
Fig. 1. Effect of pH on the lipolytic activity of the crude extract. Assay conditions: 37 C, the buffers used were citratephosphate pH 6.0 and 7.0 ( ), phosphate pH 7.0 and 8.0 ( ), TrisHCl pH 8.0 and 9.0 ( ) and glycineNaOH pH 9.0 and 10.0 ( ), all at 50 mM, protein concentration 2.96 g/ml, and 7.9 mM of pNPP.

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140 120 100 80 60 40 20 0 0 2 4 6 8 10 12

120

Residual activity (%)

Residual activity (%)

100 80 60 40 20 0 20 40 60 80 100 120 140

pH
Fig. 2. pH stability of the lipolytic crude extract. Residual activities after 1 h incubation at 28 C in the presence of different buffers: glycineHCl pH 2.2 and 3 ( ), citrate pH 36 ( ), citratephosphate pH 3.07.0 ( ), phosphate pH 6.08.0 ( ), TrisHCl pH 8.0 and 9.0 ( ) and glycineNaOH pH 9.0 and 10.0 ( ), all buffers at 50 mM concentration. Assay conditions: 37 C, phosphate buffer pH 8.0, 50 mM, protein concentration: 2.96 g/ml, 7.9 mM pNPP. The activities were compared to the activity determined in phosphate buffer, pH 7.0, 50 mM without pre-incubation.

Time (min)
Fig. 4. Temperature stability of the lipolytic crude extract. The extract was incubated at 28 C ( ), 37 C ( ), 45 C ( ) and 50 C ( ). Assay conditions: 37 C, phosphate buffer pH 8.0, 50 mM, protein concentration: 2.96 g/ml, 7.9 mM pNPP. The activities were calculated on the basis of product concentrations after 1 min.

substrates, and degrees of purication of each enzyme in the various reports. 3.2. Effect of temperature on lipolytic activity and stability of the crude extract The lipolytic activity against pNPP, measured in terms of the amount of product produced during the rst minute of incubation, increased with temperature from 30 C up to 60 C (Fig. 3). The highest activity was observed at 60 C (41.0 U ml1 ), but high activities were also found in the range from 50 (38.4 U ml1 ) to 70 C (39.6 U ml1 ). Activity determinations beyond 70 C were not done because of the difculties in rate estimation caused by the spontaneous hydrolysis of pNPP at temperatures above 70 C.
45

Activity (U mL-1)

40 35 30 25 20 30

The high activities herein obtained between 50 and 70 C for the lipase of P. aurantiogriseum are not reported for lipases produced by other fungi of the genus Penicillium, for which the highest activities occurred between 25 and 45 C [4,5,12,13,15,16]. For example, a temperature of 40 C for maximal activity was reported for P. cyclopium lipases (II and III) [2,17], using dicaprylin (Lipase I) and p-nitrophenyl laurate (Lipase III) as substrates. The lipolytic extract from P. aurantiogriseum therefore presents the highest temperature for maximum activity so far reported for Penicillium lipases. However, the optimal temperature is not constant for an enzyme and can vary according to the purity of the substrate, the presence of inhibitors and with the method used in the analysis. The enzyme is not particularly stable at elevated temperatures (Fig. 4). After 30 min of incubation of the extract at various temperatures, there was only 32% residual activity after incubation at 50 C, 45% after incubation at 45 C and 77% after incubation at 37 C. Loss of activity did not follow simple rst order decay kinetics. The two-step model of Henley and Sadana [18] tted well to the data, yielding the results in Table 2. These results suggest that the rst step
Table 2 Parameters of the Henley and Sadana [18] two-step denaturation modela at the various temperatures where denaturation was observed Parameter First order rate constant for the 1st step of the denaturation reaction First order rate constant for the 2nd step of the denaturation reaction Activity of the intermediate enzyme form relative to the native enzyme Activity of the nal denatured enzyme relative to the native enzyme 37 C 0.39 0.0044 0.80 0 45 C 0.37 0.0058 0.50 0 50 C 0.41 0.4154 0.66 0.30

40

50

60

70

80

Temperature (oC)
Fig. 3. Effect of temperature on the lipolytic activity of the crude extract. Assay conditions: 3070 C, phosphate buffer pH 8.0, 50 mM, protein concentration: 2.96 g/ml, 7.9 mM pNPP. The activities were calculated on the basis of product concentrations after 1 min.

a The Henley and Sadana model assumes two sequential rst-order denaturation steps: E E1 and E1 E2 .

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of the denaturation process is little affected by temperature, and yields an enzyme form with signicant residual activity. The rst order rate constant for the second step of the denaturation process increases signicantly between 45 and 50 C, and yields a denatured form with little or no residual activity. In general, fungal lipases are not stable at temperatures above 40 C. However, there have been few reports of moderately thermostable lipases, such as that of P. wortmanii [4], which retained 90 and 55% of the initial activity after 60 min of incubation at temperatures of 45 and 50 C, respectively. The lipase I from P. cyclopium was stable at 30 C for 1 h and lost 10% of its activity after 1 h of incubation at 35 C [19]. The lipase II was stable at 40 C for 15 min, being quickly deactivated at temperatures above this point [2]. 3.3. Stability in organic solvents The stability in organic solvents is an important characteristic of lipases. It can determine whether the enzyme can be used to catalyze synthetic reactions and also to predict which solvent would be better to perform the reaction. The effect of the organic solvent depends on the nature of both enzyme and solvent [20]. Hydrophilic solvents (2.5 < log P < 0), such as acetone and ethers, are usually incompatible with enzyme activity; whereas water-immiscible solvents (2 < log P < 4), such as alkanes or haloalkanes retain the catalytic activity due to the fact that they do not strip off the crucial bound water from the enzyme surface [12,20,21]. It is believed that water-miscible organic solvents strip water from the enzymes, leading to the unfolding of the molecule with exposure of the inner hydrophobic residues and that this denaturation occurs at a much faster rate than in a pure aqueous system [22]. Therefore, the stability of the crude extract produced by P. aurantiogriseum against several hydrophobic solvents was studied. As shown in Table 3, the enzyme had a good stability in water-immiscible organic solvents with a residual activity of 113.6% for n-heptane, 92.3% for hexane, 91.0% for isooctane and 82.3% for toluene, these results being in agreement with Zaks and Klibanov [21]. It was proposed that residues
Table 3 Stability of the lipolytic crude extract from P. aurantiogriseum in organic solvents Organic solvent Phosphate buffer pH 8.0 Methanol Iso-propanol Ethanol Acetone Butanol Toluene Hexane n-Heptane Isooctane log P 0.76 0.28 0.24 0.23 0.8 2.5 3.5 4.0 4.51 Residual activity (%) 100 0 1.9 0 0 6.4 82.3 92.3 113.6 91.0

of the hydrophobic solvent, which may be transferred to the assay system with the enzyme after the pre-incubation step, keeps it in the open conformation and therefore the lid of the enzyme does not block the active site crevice, keeping a exible conformation and therefore increasing the activity [23]. The same behavior was found for the lipases from P. simplicissimum [12], from Mucor sp. [14], from Mucor hiemalis f. hiemalis [24] and from Rhizopus oryzae [25]. 3.4. Effect of various compounds on the stability of lipolytic activity The effect of salts on the stability was examined by adding different compounds to the standard reaction mixture (Table 4). The activity was enhanced by chlorides of Mg2+ , Zn2+ , Co2+ and Mn2+ but was inhibited by Cu2+ and Ba2+ , as was observed for the lipase of M. hiemalis f. hiemalis [24]. The enzyme was strongly inhibited by HgCl2 , as observed for lipases from Mucor sp. [14], P. expansum [16] and A. oryzae [26], which could suggest the existence of SH-groups [16]. The ion Ca2+ is known to promote the hydrolytic activity of some kinds of lipases [12,16,25], but the lipase from P. aurantiogriseum, as well as the lipase from A. oryzae [26], are not activated by addition of this ion. The azide assay was done in order to verify its interference with the enzymatic activity, since it is regularly used to maintain crude extracts free of contamination, as was done in the current work. The results of this assay showed that incubation with azide enhanced the enzyme activity by 30%. In fact, this compound did not show any inhibitory effect on the activity of other lipases, such as lipases from P. simplicissimum [12], A. oryzae [26] and P. expansum [16].
Table 4 Stability of the lipolytic crude extract from P. aurantiogriseum in the presence of salts and detergentsa Substance Controlb BaCl2 MgCl2 CaCl2 ZnCl2 CoCl2 KCN CuSO4 MnCl2 HgCl2 EDTA NaN3 Triton X-100 (HLEc 13.5) Tween 80 (HLEc 15) SDS (HLEc 40) 1 mM 1 mM 1 mM 1 mM 1 mM 1 mM 1 mM 1 mM 1 mM 1 mM 10 mM 1 mM 0.01% 0.01% 0.01% Concentration Residual activity (%) 100 70 113 79 112 105 38 69 128 0 118 109 130 52 109 100

a The enzyme was incubated in the presence of various compounds at 28 C for 1 h. b Control, without the addition of any substance. c HLE, hydrophilic/lipophylic equilibrium. The higher the value of the HLE, the more hydrophilic the detergent [33].

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Surfactants are frequently used in the preparation of the emulsion in lipolytic assays and also in the purication and characterization of lipases. They can potentially cause denaturation of the enzyme by disrupting its tertiary structure. On the other hand, they can prevent aggregation of the enzyme and, consequently, their addition may increase the lipase activity. Therefore, the choice of the surfactant either in the preparation of the emulsion or in the purication steps is crucial. For instance, the lipase of P. expansum, when stored at pH 6.09.0, was stable only in presence of 0.1% Tween 20, and the detergent prevented aggregation of enzyme [16]. Our enzyme was stable in 0.01% Tween 80 (109% residual activity) and 0.01% SDS (100% residual activity), after being incubated for 60 min at 28 C (Table 4). However, incubation with Triton X-100 caused a decrease of almost 50% in the enzyme activity. This last result is important because the emulsion used in the pNPP assay contains 0.01% of Triton X-100 and therefore could inhibit the enzyme. Further studies are currently being done to determine the inuence of the detergent during the assay and the need to substitute it. 3.5. Lipolytic activity with different substrates The denition of which reactions a lipase catalyses is not an easy subject, and it has been discussed for more than 10 years. Recently, lipases have been dened as carboxylesterases that hydrolyze acylglycerides, with acyl chains with more than 10 carbon atoms [1]. Our enzyme does this, hydrolyzing triolein (Table 5), and therefore under this denition it would be classied as a lipase. Even though a greater activity was obtained with tributyrin than with triolein (Table 5), it is not possible to afrm that the lipase of P. aurantiogriseum has a greater specicity for short-chained triacylglycerols, because the assays were not done in such a manner to guarantee the same interfacial area, this being a factor that can determine the velocity of the reaction [27]. The interfacial area depends on the type of substrate, because this determines the characteristics of the emulsion. Due to the complexity
Table 5 Comparative study of lipolytic activity of the crude extract against various different substratesa Substrate pNPA (C2) pNPB (C4) pNPC (C6) pNPCA (C10) pNPP (C16) Triolein (C18) Tributirin (C4) Method Spectrophotometric Spectrophotometric Spectrophotometric Spectrophotometric Spectrophotometric Titrimetric Titrimetric Activity(U mg1 ) 0 47.1 73.9 94.0 87.8 3.2 17.8

of catalysis in heterogeneous systems, the mechanisms of action of lipases, as well as their kinetics, are still not yet well elucidated, with lipolytic activity frequently being related simply to qualitative characteristics of the interface [28]. The above denition hides the fact that these enzymes can hydrolyze a variety of compounds that contain carboxylic ester groups and not only acylglycerols [1]. The pNPP method has been used by several authors to characterize lipolytic activity, since this method is more reproducible, sensitive and rapid than titrimetric methods [3,29]. In fact, some recent works have suggested the use of synthetic long-chained substrates, such as pNPP, to characterize the activity of true lipases, and synthetic short-chained substrates, such as p-nitrophenylacetate (pNPA), to characterize esterases; the two activities being differentiated, therefore, only by the length of the fatty-acid chain [30,31]. Under this denition, the crude extract from P. aurantiogriseum would be said to contain true lipases since, in a test of activity against various pNP esters, all substrates except pNPA were hydrolyzed. Activity with these esters increased as the length of the acyl chain increased, with the highest activities being obtained with pNPCA (C:10), 94 U mg1 , and with pNPP (C:16), 87 U mg1 . However, the use of pNPP as a substrate for the determination of the activity of lipases has been questioned by other authors [1], because it is not necessarily hydrolyzed specically by lipases, being also hydrolyzed by other esterases. Therefore, it is advisable to use the pNPP method as a measure of lipolytic activity only once the enzyme has been shown to hydrolyze triacylglicerides. 3.6. Activity towards p-NPP in organic solvent Pencreach and Barati [32] proposed a method to compare the activity of lipases towards pNPP in aqueous medium and in n-heptane, using the ratio RO/A , which is the ratio of the activity in the organic solvent to the activity in aqueous medium. We used this method to compare the activities of P. aurantiogriseum lipase in the two media. The activity of the lipase towards pNPP in the organic medium was 13.6 U mg1 and for the aqueous medium, using the best conditions as determined before, was approximately 23 times higher (312.3 U mg1 ), giving a value of RO/A of 4.3 102 . Pencreach and Baratti [32] determined the RO/A for 36 lipases from different sources and found 19 values of RO/A lower than one. Activities in organic media related in the literature are usually lower than those observed in aqueous media, which has been ascribed to the low amount of available water, to the lower structural exibility of the enzymes in organic media and also due to diffusional effects, since it is a heterogeneous catalysis system. In any case, this is an initial study for the lipase of P. aurantiogriseum and further experiments are currently being done to optimize the conditions for the application of the enzyme in organic solvents.

a Assay conditions for the spectrophotometric method37 C, phosphate buffer pH 8.0, 50 mM, 3 mg/ml of pNP ester, 2.96 g protein/ml; titrimetric method37 C, 20 min incubation, 2.96 g protein/ml, 0.2 g/ml of triolein or tributyrin.

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4. Conclusion We have characterized the lipase produced by P. aurantiogriseum Dierckx IOC 4212 in an inorganic medium supplemented with olive oil. The good stability of the enzyme with organic solvents justies the search for potential applications of the enzyme in biocatalysis in such media. These further studies should be done with a puried preparation of the enzyme.

Acknowledgements This work was partially funded by the Brazilian Agencies Fundao Araucria and CAPES. Valeria M.G. Lima, David Mitchell and Nadia Krieger thank the Brazilian National Council for Scientic and Technological Development (CNPq, Conselho Nacional de Desenvolvimento Cientco e Tecnolgico) for research scholarships. References
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