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Journal of Molecular Catalysis B: Enzymatic 78 (2012) 111– 118

Contents lists available at SciVerse ScienceDirect

Journal of Molecular Catalysis B: Enzymatic

jo u rn al hom epa ge: www.elsev ier .com/ locate /molcatb

nfluence of different immobilization techniques for Candida cylindracea lipase onts stability and fish oil hydrolysis

arolina Pizarroa,∗, María C. Branesb, Alejandro Markovitsb, Gloria Fernández-Lorentec,osé Manuel Guisánd, Rolando Chamya, Lorena Wilsona

Escuela de Ingeniería Bioquímica, Facultad de Ingeniería, Pontificia Universidad Católica de Valparaíso, General Cruz 34, Valparaíso, ChileIndustria Química Härting S.A., Av. Presidente Frei Montalva 6000, Santiago, ChileInstituto de Fermentaciones Industriales, CSIC, Juan de la Cierva 3, 28006 Madrid, SpainDepartamento de Biocatálisis, Instituto de Catálisis – CSIC, Campus UAM, Cantoblanco, 28049 Madrid, Spain

r t i c l e i n f o

rticle history:eceived 2 December 2011eceived in revised form 29 February 2012ccepted 16 March 2012vailable online 27 March 2012

eywords:tabilityipasemmobilizationish oiliphasic reaction

a b s t r a c t

The type of enzyme immobilization has been associated with the degree of stabilization/modification ofthe protein structure, which ultimately determines its activity and stability under different conditions(temperature, solvent and substrate). The thermal stability and the stability in solvated reaction mediumof different derivatives of Candida cylindracea lipase (CCL) were evaluated for their hydrolysis activity ofp-nitrophenyl butyrate and in obtaining Omega-3 fatty acids EPA and DHA from fish oil triacylglycerols.CCL was immobilized by hydrophobic interactions and by covalent binding. In addition, hydrophobicderivatives were cross linked with functional polymers, such as dextran aldehyde and dextran sulfate,after chemical amination of its surface to prevent lipase desorption. Fish oil hydrolysis reactions werecarried out using 35 U of biocatalyst per gram of oil, at 37 ◦C and with a 1:1 aqueous/organic phaseratio. The aqueous phase was a 40% polyethylene glycol/phosphate buffer solution, pH 7, and the organicphase a 1:4 fish oil/hexane solution. According to its half-life time, the most thermostable derivative

corresponds to CCL immobilized in glyoxyl-agarose. Derivatives of CCL immobilized on octyl-agarose(O-CCL) and CCL immobilized on octyl-agarose cross linked with dextran sulfate (O-CCL-NH2-DxSO4)showed an activation effect of about 10 folds compared to its initial activity when they were incubatedin solvent and co-solvent biphasic reaction media. After 48 h of reaction, O-CCL and O-CCL-NH2-DxSO4

were those derivatives with highest yields of fish oil hydrolysis; its differential rate of hydrolysis detected acids

for polyunsaturated fatty

. Introduction

Omega-3 fatty acids, EPA and DHA, play a significant role inuman health such as the suitable development of the nervous sys-em and the prevention of cardiovascular diseases, among others1,2]. Thereby, it raises an important market for nutraceuticals andietary supplements that contain these polyunsaturated fatty acidsPUFAs). Their main source is marine food and enzymatic hydroly-is of fish oil constitutes an interesting biotechnological alternativeor its elaboration. Several studies report the use of different lipasesor this purpose [3–5].

The lipase from Candida cylindracea (CCL), also named Candidaugosa (CRL), is a 57 kDa glycosylated enzyme composed by 534

mino acids that does not require cofactors for its catalytic activ-ty [6]. This lipase has been reported as neither stereo-specificor a fatty acid specific lipase [7,8], making it one of the most

∗ Corresponding author. Tel.: +56 32 2273656; fax: +56 32 2273803.E-mail address: carolinapizarrotorres@gmail.com (C. Pizarro).

381-1177/$ – see front matter © 2012 Elsevier B.V. All rights reserved.oi:10.1016/j.molcatb.2012.03.012

make them a valuable tool for EPA/DHA glycerides concentration.© 2012 Elsevier B.V. All rights reserved.

interesting commercially available lipases for the hydrolysis of fishoil. Despite this no-specificity, it has been reported that CCL hasa marked lower hydrolysis rate for PUFAs. Precisely, this propertysupports the successful use of this enzyme for Omega-3 fatty acidsconcentration [9,10]. Enzyme immobilization is usually a preferredform for its industrial application due to the better enzyme stabil-ity achieved which allows biocatalyst reuse. One characteristic oflipases is its activation by hydrophobic interfaces, mechanisticallyexplained by the enzyme conformational change to an open, activestructure [11,12]. This interfacial activation allows its immobiliza-tion by adsorption in hydrophobic supports [13,14]. Immobilizationof different lipases by covalent attachment has also been ana-lyzed [15,16]. This immobilization involves different regions of thelipase surface, in which the equilibrium between an open and aclosed structure conformation depends on the reaction mediumhydrophobicity.

The use of organic solvents in biphasic reaction media not onlyinfluences the mass transfer in the reaction system but also hasgreat effect on the enzyme structure [17]. Using molecular dynam-ics simulations, the effect of organic solvents on the structure and

1 Cataly

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12 C. Pizarro et al. / Journal of Molecular

ynamics of CRL has been previously evaluated [18,19]. Severaltrategies have been assessed in order to improve lipase proper-ies, such as the co-immobilization with poly-ionic polymers thatncrease its stability in the presence of organic solvents [20,21].

The structural changes produced by lipase immobilization andhe presence of solvents in the reaction could influence its cat-lytic ability and/or modulate its selectivity [22–24]. Therefore,he objective of this study is to assess the enzyme stability inolvents/co-solvents and its thermal stability, using derivativesf CCL prepared under different immobilization protocols, for itspplication in fish oil triacylglycerol hydrolysis.

. Experimental

.1. Materials

CCL, 153.7 mg of protein content/g of powder and with 46 U/gf protein, was supplied by Lyven (Colombelles, France). Dex-ran was from Fluka (Buchs, Switzerland), sulfate-dextran and-nitrophenyl butyrate (p-NPB) were from SIGMA Chemical Co. (St.ouis, MO). Octyl-sepharose CL-4B and cyanogen bromide activatedepharose 4B (CNBr) were purchased from GE Healthcare. Cross-inked agarose 10 BCL was from Hispanagar S.A. (Burgos, Spain).olyethylene glycol (PEG) 4000 and ethylene glycol were purchasedrom Merck (Darmstadt, Germany). Other reagents and solventssed were analytical or HPLC grade. Fish oil (mackerel oil fromouth Pacific Korp S.A. Chile) was provided by Härting SA (Santiago,hile).

.2. Methods

.2.1. Determination of enzyme activityThe standard activity assay was performed as previously

escribed [25]. Briefly, the absorbance increase at 348 nm pro-uced by the p-nitrophenol release in the hydrolysis of 0.4 mM-nitrophenyl butyrate in 25 mM sodium phosphate buffer at pH

at 30 ◦C was measured with the aid of a thermostatized spec-rophotomer. A unit of pNPB activity (U) is defined as the amountf enzyme necessary to hydrolyze 1 �mol of pNPB/min under theescribed conditions. The massic activity of each derivatives corre-ponded to the measurement of the derivative activity per gram ofupport.

.2.2. Immobilization of CCL in different supportsCCL was immobilized using different protocols of immobiliza-

ion. In all cases the immobilization yield was calculated fromhe quotient between the activity of the immobilized enzyme andctivity of the suspension at the end of immobilization.

i. Immobilization of CCL on octyl-agaroseCCL was immobilized on the hydrophobic support octyl-

agarose (O-CCL) at low ionic strength as previously described[13]. Octyl-agarose was added to a buffered lipase solution ina ratio 1/10 (g/mL). Lipase solution was in a concentration of76.9 mg protein/L in 25 mM sodium phosphate buffer at pH 7.Immobilization was performed at room temperature with mildstirring. After 1 h, derivatives were filtered and washed withdistilled water.

i. Immobilization of CCL on octyl-agarose and cross-linking withpolyfunctional polymers(a) Chemical amination of the lipase surface

This was accomplished by incubating octyl derivatives in

ethylenediamine (EDA) 1 mM pH 4.75 in a ratio of 1/10(g/mL), which forms the linkage between the EDA aminogroup and the surface enzyme carboxylic groups (termi-nal carboxylic, Asp and Glu), as previously described [26].

sis B: Enzymatic 78 (2012) 111– 118

The reaction is catalyzed by the addition of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) 10 mM. After1.5 h under mild stirring at room temperature, the aminatedderivatives are filtered and washed with abundant distilledwater.

(b) Sulfate dextran cross-linkingAs previously described [27], the cationic interaction cross-

linking was performed by the addition of 0.5 mg of DxSO4(100,000 Da) per gram of protein contained in the octyl-amino derivative to a suspension of the derivative in 100 mMphosphate buffer at pH 7 in a ratio of 1/10 (g/mL). After 2.5 hof mild stirring at room temperature, the cross linked aminoderivatives (O-CCL-NH2-DxSO4) were filtered and washedwith abundant distilled water.

(c) Aldehyde dextran cross-linkingDextran polymer was activated as previously described

[28]. The covalent cross-linking with the polymer was per-formed adding 8 g of sodium periodate to 3.33 g of dextran(25,000 Da) previously dissolved in 100 mL of distilled water.After 3 h of reaction at room temperature, the solution wasdialyzed for approximately 16 h in 50 volumes of distilledwater in order to eliminate the formaldehyde produced dur-ing the oxidation procedure. The derivative cross-linking wascarried out by the addition of 10 mg of the above describedpolymer per gram of protein contained in the octyl-aminoderivative suspended in 100 mM phosphate buffer at pH 7 ina ratio of 1/10 (g/mL). After 16 h of reaction time, sodiumborohydride (1 mg/mL) is added to reduce the remainingaldehyde groups and the amino aldehyde bonds. The solutionwas mildly stirred for 1 h at room temperature, and finallythe cross-linked amino derivatives were filtered and washedwith abundant distilled water.

iii. Immobilization of CCL on CNBr-activated agaroseCNBr-activated support was added to a buffered lipase

solution in a ratio 1/10 (g/mL) as previously has beendescribed [29,30]. Lipase solution was in a concentrationof 76.9 mg protein/L of 25 mM sodium phosphate buffer atpH 7. Immobilization was performed at low temperature(4 ◦C) with mild stirring for 15 min. The immobilization pro-cedure was stopped by blocking the amine groups with 1 Methanolamine (pH 8) for 1.5 h under mild stirring and roomtemperature. Finally, the covalent CNBr-CCL derivative waswashed with abundant distilled water.

iv. Immobilization of CCL on glyoxyl-agaroseThe glyoxyl-agarose support is obtained from the activa-

tion of agarose with glyoxyl groups as described previously[31]. The immobilization of CCL in glyoxyl-agarose (Gx-CCL)was performed by adding this activated support to a bufferedsolution of lipase in a ratio 1/10 (g/mL) at low temperature(4 ◦C) with gentle stirring for 30 min. The concentration ofenzyme solution was 76.9 mg protein/L of 25 mM sodiumphosphate buffer, which was previously adjusted to pH 10with a saturated solution of sodium carbonate. This pro-cedure preferently immobilizes lipase surface regions withhigher density of amino groups with pK close to 10 [29].The immobilization is completed by a reduction step withsodium borohydride 1 mg/mL. After 30 min the derivativewas washed with distilled water.

2.2.3. Thermal stability studiesThermal stability studies were carried out incubating the

enzyme derivatives (1 g in 10 mL of 25 mM sodium phosphate,

pH 7) and soluble enzyme (76.9 mg protein/L of 25 mM sodiumphosphate buffer, pH 7) at 30 ◦C, 40 ◦C, 50 ◦C and 60 ◦C. Sam-ples were periodically withdrawn and their activity was measuredusing the pNPB assay described in Section 2.2.1. The residual

Catalysis B: Enzymatic 78 (2012) 111– 118 113

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Table 1Immobilization yield and activity of different derivatives of CCL.

Derivative Immobilizationyield [%]

Specific activity (p-NPB)[U/g support]

O-CCL 97.1 101.2O-CCL-NH2-DxSO4 93.8 40.4

C. Pizarro et al. / Journal of Molecular

ctivity was calculated as the ratio between the activity at a givenime and the activity at time zero of incubation. The calcula-ion of half-life time and kinetic parameters was obtained fromorrelating experimental data with different expressions of ther-al inactivation previously proposed [32] by nonlinear regression

Levenberg–Marquardt algorithm) using the statistical softwarePSS (IBM).

.2.4. Stability in solvents and co-solvent studiesThe stability of soluble CCL and its derivatives was evaluated

y incubation at 37 ◦C in non-reactive conditions using cyclohex-ne and hexane as solvents, and propanol, ethylene glycol andolyethylene glycol as co-solvents. The incubation in solvents waserformed at a 1:1 ratio of each solvent with a solution of 25 mModium phosphate pH 7. The incubation in co-solvents was per-ormed by adding the appropriate amount of co-solvent to a 25 mModium phosphate solution, pH 7, to equal the polarity of a 50%ropanol solution used as a reference (calculated using dielectriconstant of the solution, εm = 46). Samples were taken periodicallyith a pipette with cut-tip and their activity was measured asescribed in Section 2.2.1. The residual activity is calculated as theatio between the activity measured at a given time and activity atime zero of incubation.

.2.5. Hydrolysis of fish oil triacylglycerolsHydrolysis reactions were carried out using 35 U of biocatalyst

er gram of fish oil, at 37 ◦C and with a 1:1 aqueous/organic phaseatio. The aqueous phase was a 40% (v/v) PEG/phosphate bufferH 7 solution and the organic phase a 1:4 fish oil/hexane solu-ion; considered as the best conditions for reaction medium in thistudy (Section 3). The reaction was kept under constant agitationo achieve a homogeneous dispersion of phases. Dispersion sam-les were periodically taken with a pipette with the tip cut off and,fter separation by centrifugation, the organic phase was analyzedy GC chromatography (see Section 2.2.6). The percentage of fattycid hydrolysis was obtained by the ratio between the initial fattycid concentration in the triacylglyceride and the concentration ofatty acids released after hydrolysis reaction. The specific rate ofydrolysis of each fatty acid was calculated from the percentage ofydrolysis per gram of derivative in time at initial reaction times.

.2.6. Quantification of fish oil fatty acids released by thenzymatic hydrolysis reaction

The determination of the fatty acid profile was performedy gas chromatography (GC). The samples were analyzed in

gas chromatograph HP6890 Series II (Hewlett-Packard, USA)quipped with a flame ionization detector (FID) and a split/splitlessnjector. The injection volume was 0.5 �L at a split ratiof 1:30. The injector and FID temperatures were 300 ◦C and20 ◦C, respectively. A methylilpheenylpolysiloxane HP-5 column30 m × 0.32 mm × 0.25 �m) and helium gas carrier at a constantow of 1 mL/min were used. After derivatization with N,O-is(trimethylsilyl)acetamide, fatty acids were quantified using an

nternal standard of �-sitostanol (64%).

. Results and discussion

.1. Characterization of CCL derivatives

Specific activity and immobilization yields obtained for eachCL derivative are presented in Table 1. All derivatives prepared onydrophobic supports showed a very high yield of immobilization,

ontrary to that observed with those formed by covalent bindingetween enzyme and support (CNBr-CCL and Gx-CCL). The highernzyme activity per gram of support was obtained with O-CCLerivative whose union is achieved by hydrophobic interactions.

O-CCL-NH2-DxCHO 93.8 47.6CNBr-CCL 62.7 47.8Gx-CCL 33.6 45.3

Probably this interaction fixes the open state of the lid, which hidthe active site, providing a more active enzyme structure. This effecthas been previously reported for other lipases [33,34].

The specific activity obtained with cross linked derivatives (O-CCL-NH2-DxSO4 and O-CCL-NH2-DxCHO) was lower than theircounterpart without cross linkage (O-CCL), probably because thepolymer layer over the lipase surface could cause a stiffness effectof its structure and reduce substrate access to catalytic site and/orits conversion to product.

3.2. Thermal stability study of CCL and its derivatives

Fig. 1 shows the time course of residual activity of CCL andits derivatives at different temperatures. As may be corroboratedwith half-life times and kinetic parameters for all biocatalysts pre-sented in Table 2, enzyme immobilization strongly increased itsthermal stability compared to that of the soluble enzyme (CCL).Even at the lowest temperature tested (30 ◦C), CCL decreased itsactivity by 60% compared to the CCL derivatives that remainedalmost completely active. While CCL became almost inactive at40 ◦C, all derivatives remained similarly active (over 80% activity)within a long period of time (Fig. 1b). Increasing temperature to50 ◦C (Fig. 1c) clearly unraveled the difference in the stability ofthe biocatalysts. While Gx-CCL and O-CCL-NH2-DxSO4 showed asimilar behavior and decreased its activity only in 25%, CNBr-CCLpresented the highest instability at short time courses compared toother derivatives. This might be explained because the immobiliza-tion on CNBr occurs through a single covalent bond remaining verysimilar to the structure of the native enzyme form without immo-bilization. An intermediate behavior was observed with the O-CCLderivative, consistent with the concept that the immobilization bya hydrophobic surface, clearly involves structural modifications ofthe protein contributing to maintaining the open configuration ofthe lipase at higher temperatures compared to the one-point cova-lent immobilization produced by CNBr-CCL.

The increase of enzyme stability has been associated with therigidization of its structure [35], preventing unfolding states whichlead to an inactive conformation of the protein. This phenomenon isevident in the least inactivated multipoint covalent linked deriva-tive (Gx-CCL), as well as with the cross linked derivatives. Inthose derivatives the enzyme surface was modified by chemicalamination (as is described in Section 2.2.2) in order to increaseprotein binding sites to the polymers, thus increasing the rigid-ity of its structure. The increase in stability of O-CCL-NH2-DxSO4and O-CCL-NH2-DxCHO compared to its counterpart O-CCL couldbe clearly attributed to the protection of the enzyme structureby the polymer. However, unexpectedly O-CCL-NH2-DxSO4 pre-sented a higher thermal stability than O-CCL-NH2-DxCHO despitethe stronger covalent bond of the aldehyde dextran polymer to thelipase of this derivative. One possible reason for the stabilization isthe high hydrophilyc microenvironment provided by the polymerthat could also increase enzyme stability, a phenomenon previously

observed [36].

At 60 ◦C (Fig. 1d) all derivatives were strongly affected by ther-mal inactivation, showing in all cases within 24 h of incubation lessthan 20% residual activity.

114 C. Pizarro et al. / Journal of Molecular Catalysis B: Enzymatic 78 (2012) 111– 118

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ig. 1. Thermal stability of CCL and its derivatives. The incubation was carried out

f duplicates. (�) CCL, ( ) O-CCL, (©) O-CCL-NH2-DxSO4, ( ) O-CCL-NH2-DxCHO

.3. Stability of CCL and its derivatives in solvents and co-solvents

In biphasic reaction systems, pure oils are viscous and thusifficult to work with, so using mixtures of oil/organic solvents/co-olvents could simplify the reaction design. Common immiscibleolvents used for that purpose are cyclohexane and hexane and

o-solvents partitioned into the aqueous phase are mainly polyolse.g., polyethylene glycol).

Fig. 2 shows the residual activity of CCL and its deriva-ives at different times of incubation in the evaluated solvents.

able 2inetic parameters of inactivation of CCL and its derivatives.

Biocatalyst Inactivation mechanism Parameter

CCL Series type in two stages k1 (h−1)

k2 (h−1)

a

T1/2 (h)

O-CCL Series type in two stages k1 (h−1)

k2 (h−1)

a

T1/2 (h)

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T1/2 (h)

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b

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a: it does not adjust to inactivation mechanism, nm: not measured, RA: residual activitnactivation of the second stage, T1/2: half-life, a: residual activity fraction of the enzyme

onformational state.

M sodium phosphate buffer, pH 7, non-reactive conditions. Results are the meanNBr-CCL, ( ) Gx-CCL.

Cyclohexane and hexane had an inactivation effect on the sol-uble enzyme, showing the lack of protection provided by theimmobilization. In addition, it is observed that O-CCL and CCL-O-NH2-DxSO4 were significantly activated when they were incubatedin those solvents. This is in agreement with the stabilizing effectof the solvent on the hydrophobic interactions of the enzyme

derivative supporting the conformational changes which increasethe interfacial activation of lipolytic enzymes. Even though, O-CCL-NH2-DxCHO derivative is also produced by hydrophobicinteractions to the agarose support, no activation was observed.

Temperature

30 ◦C 40 ◦C 50 ◦C 60 ◦C

0.064 1.094 1.757 nm0.003 0.016 0.029 nm0.540 0.449 0.125 nm52.1 2.0 0.5 nmna 0.516 0.567 0.414na 0.001 0.005 0.092na 0.872 0.758 0.250>90% RA a 150 h 559.0 90.2 2.5na 0.001 0.002 0.363>90% RA a 150 h 693.1 346.6 1.9na 0.005 0.027 0.726na 0.480 0.470 0.050>90% RA a 150 h 651.6 106.4 1.0na 0.894 2.046 0.357na 0.001 0.009 0.085na 0.907 0.709 0.200>90% RA a 150 h 596.7 39.3 2.6na 0.004 0.006 0.215na 0.490 0.480 0.086>90% RA a 150 h 983.0 543.0 3.7

y, k1: constant for thermal inactivation of the first stage, k2: constant for thermalintermediate conformational state, b: residual activity fraction of the enzyme final

C. Pizarro et al. / Journal of Molecular Catalysis B: Enzymatic 78 (2012) 111– 118 115

Fig. 2. Stability of CCL and its derivatives in solvents. The incubation was carriedopN

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Fig. 3. Stability of CCL and its derivatives in co-solvents. The incubation was carriedout in 25 mM sodium phosphate buffer, pH 7, non-reactive conditions, dielectric

ut in non-reactive conditions, ratio W/O 1:1, W: sodium phosphate buffer 25 mM,H 7, O: solvent. Results are the mean of duplicates. (�) CCL, ( ) O-CCL, (©) O-CCL-H2-DxSO4, ( ) O-CCL-NH2-DxCHO, (�) CNBr-CCL, ( ) Gx-CCL.

he latter may be explained by the larger structure rigidity pro-uced by the covalent binding of the aldehyde dextran polymero the enzyme surface, making it less susceptible to the solventnterfacial activation effect.

Contrary to the phenomenon of activation of the CCL deriva-ives mentioned before, with the Gx-CCL derivative incubated inyclohexane a slight decline in its residual activity was observed.his effect can be attributed to the stiffness of the enzyme structureroduced by the multipoint covalent attachment in detriment of itsctive configuration acquisition.

The activation behavior of the derivatives in hexane (Fig. 2b)s quite similar to that described in cyclohexane. However, higherercentages of activation for all derivatives were obtained, evenor those derivatives less susceptible to conformational changesor interfacial activation (Gx-CCL and CCL-O-NH2-DxCHO). The lat-er may be explained by the ability of cyclohexane for strippinghe essential monolayer of water surrounding enzyme moleculeshich are required for its catalytic activity [37]. Although both

olvents are considered moderate polarity solvents (log P valuesrom 2.0 to 4.0), there is a small difference in log P values (3.2or cyclohexane and 3.5 for hexane), which agrees with the facthat the higher polarity a solvent has, the higher will be the waterayer disruption produced by it. Accordingly, an explanation forhe different CCL derivative behavior relies in that, apparently theipase immobilization also helps avoiding water layer stripping,hus maintaining its structure with catalytic activity.

In other aspect, previous studies with CRL immobilized in kaolinarticles (a non hydrophobic type of hydrated aluminum silicatelay) [38] showed that the derivative was not activated by incu-

ation in hexane, and moreover presented a continuous decay of

ts activity. The results reported by these researchers are oppositeo that obtained by us demonstrating that the type of immobiliza-ion also affects the interaction between the solvent and the lipase.

constant of the solution, εm = 46. Results are the mean of duplicates. (�) CCL, ( )O-CCL, (©) O-CCL-NH2-DxSO4, ( ) O-CCL-NH2-DxCHO, (�) CNBr-CCL, ( ) Gx-CCL.

Moreover, the inactivation of the free enzyme by hexane is slightlylower than that occurring in cyclohexane incubation.

Results obtained using different co-solvents to study its effecton the activity of CCL and its derivatives are shown in Fig. 3. Theincubation in 50% (v/v) propanol, produced a strong inactivationof the enzyme both free and immobilized (Fig. 3a). By contrast, in70% (v/v) ethylene glycol (Fig. 3b), the hydrophobic derivatives O-CCL and O-CCL-NH2-DxSO4 were activated. The same phenomenonwas observed by incubation in 40% (v/v) PEG (Fig. 3c) showing thatthe presence of this co-solvent also facilitated the conformationalchange of lipase hyper activation.

Since incubation media with co-solvents were prepared in orderto have the same dielectric constant in every case, the resultingeffect of activation of O-CCL and CCL-O-NH2-DxSO4 in PEG can-not be ascribed to the environment polarity. This response couldbe attributed to a specific interaction of the co-solvent molecule

with the hydrophobic elements of the enzyme and its active site(or surroundings). The difference in the activation profile betweenethylene glycol and PEG further suggests that the molecule

116 C. Pizarro et al. / Journal of Molecular Catalysis B: Enzymatic 78 (2012) 111– 118

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ig. 4. Hydrolysis of fish oil with CCL derivatives. Reaction conditions: 37 ◦C, pH 7, 3olution fish oil/hexane. (�) Saturated fatty acids, (�) monounsaturated fatty acids

nteracts with PEG polymer derivatives in a different way than withhe ethylene glycol monomer molecule. Also with PEG, the activityf other derivatives remained around 100% for more than 48 h ofncubation, whereas a gradual inactivation between 30 and 60% wasbserved with ethylene glycol at same incubation period. The sol-ble enzyme was not activated with ethylene glycol or PEG and theecay of its activity was consistent with the thermal inactivationhat the lipase suffered at the temperature of incubation. The sta-ility of CCL (in soluble form) in PEG has been previously reported39], showing that the activation only affected certain enzymesoforms.

.4. Comparison of derivatives of CCL for the enzymaticydrolysis of fish oil

From results obtained in studies of thermal stability andolvent/co-solvents, reaction conditions for the hydrolysis of fishil triacylglycerols were defined as described in Section 2.2.5.

fish oil, W/O ratio 1:1, W: 40% v/v PEG in 200 mM sodium phosphate buffer, O: 1:4olyunsaturated fatty acids.

Fig. 4a–c shows the hydrolysis of the different families of fattyacids from fish oil using CCL derivatives formed by hydrophobicadsorption. It is seen that at the end of the reaction, the highestpercentages of hydrolysis of both saturated and monounsaturatedfatty acids, were obtained with O-CCL and CCL-O-NH2-DxSO4.

An interesting effect to note is that the reaction dynamics isdifferent for each family of fatty acids. The dynamics of hydrolysis ofsaturated and monounsaturated fatty acids resembles a saturationcurve, whereas for the polyunsaturated fatty acids the degree ofhydrolysis increases proportionally over time, showing a restrictioncatalytic effect.

Therefore, it is evident that the immobilization by hydropho-bic interactions does not modify the pattern of hydrolysis thathas been previously reported with CCL in soluble form [40]. Alsoa lower rate of hydrolysis with CRL in microemulsions of fish oil

triglycerides containing DHA has been observed [41]. Consistentlyin this study (Table 3), DHA was the fatty acid with a lower rate ofhydrolysis compared to other fatty acids from fish oil, when usingO-CCL to CCL-O-NH2-DxSO4 derivatives. This can be explained by

C. Pizarro et al. / Journal of Molecular Catalysis B: Enzymatic 78 (2012) 111– 118 117

Table 3Specific rate of hydrolysis of fatty acids in fish oil with different derivatives of CCL.

Fatty acid Hydrolysis rate (% hydrolysis h−1 g derivative−1)

O-CCL O-CCL-NH2-DxSO4 O-CCL-NH2-DxCHO CNBr-CCL Gx-CCL

SaturatedMyristic C14:0 18.7 12.9 4.1 1.6 1.2Palmitic C16:0 21.6 14.7 1.5 2.2 1.9Stearic C18:0 18.6 10.3 4.0 2.0 1.7

MonounsaturatedPalmitoleic C16:1n7 33.7 19.1 1.2 4.9 2.6Vaccenic C18:1n7 19.4 16.3 3.6 2.7 1.6Oleic C18:1n9 21.0 12.9 4.3 2.4 1.7Eicosaenoic C20:1n9 8.7 6.5 1.9 0.9 0.8Erucic C22:1n9 5.5 4.0 1.5 0.8 0.8Nervonic C24:1n9 7.7 4.4 1.4 1.3 2.1

PolyunsaturatedLinoleic C18:2n6 26.9 17.0 3.9 3.7 2.0Linolenic C18:3n6 6.2 6.6 2.0 1.2 1.9

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he steric hindrance in polyunsaturated fatty acids produced byultiple folds in cis-type unsaturations along its hydrocarbon chain

nd also, due to the sn-2 position of triacylglycerol which com-only occurs, making it difficult for the lipase to reach the ester

ond.In another aspect, the sulfate dextran polymer layer in O-CCL-

H2-DxSO4 derivative apparently does not cause any side effectsver the reaction rate. In contrast, the covalent binding of the alde-yde dextran polymer in the O-CCL-NH2-DxCHO derivative confersreater rigidity to the enzyme surface in detriment of its catalyticotential.

CCL derivatives formed by covalent attachment (CNBr-CCL andx-CCL) clearly did not favor fish oil hydrolysis (Fig. 4d and e) whichas highly decreased compared to that achieved with deriva-

ives immobilized by hydrophobic adsorption. No difference in theynamics of hydrolysis for each family of fatty acids was detected,howing that the effect of non-hydrolysis specificity of DHA tendso disappear.

. Conclusions

According to the results shown in this study, the kind of immo-ilization of CCL modulates both thermal stability and activation

n solvent/co-solvents media, and also its catalytic activity againsthe substrate assessed: fish oil for the production of Omega-3 fattycids.

It was established that the best derivatives for the hydrolysisf fish oil using solvated reaction media are those hydrophobicerivatives and also those with functional cross linked polymershat do not have excessively rigid lipase structure, such as O-CCLnd O-CCL-NH2-DxSO4. With these derivatives the highest levelsf hydrolysis were obtained.

Immobilizations that significantly improve the thermal stabil-ty do not necessarily improve the activity of hydrolysis. Then theerivative formed by covalent multipoint, Gx-CCL, which possesses

greater stiffness in the protein structure, is precisely the derivativeith the highest values of half-life times at all studied tempera-

ures. However, this rigidity effect makes it less susceptible to theccurrence of interfacial activation conformational changes, as washown by its activity in the presence of solvents/co-solvents and its

owest capacity for fish oil fatty acids hydrolysis.

The strategy of covering O-CCL derivatives with functionalolymers effectively increases its stability, particularly with O-CCL-H2-DxSO4 and O-CCL-NH2-DxCHO. However, fish oil fatty acids

[

[[

1.6 0.8 0.81.3 0.5 0.70.9 0.5 0.7

hydrolysis is greatly diminished with O-CCL-NH2-DxCHO deriva-tive.

Fatty acids hydrolysis rates with O-CCL and CCL-O-NH2-DxSO4derivatives show that long chain polyunsaturated fatty acids(mainly DHA) are more resistant to hydrolysis. The latter may con-stitute an excellent strategy for omega-3 fatty acids concentrationinto glycerides or its further use in the synthesis of structured tri-acylglycerols rich in Omega-3.

Acknowledgments

This work has been sponsored by PBCT-CONICYT (“Thesis inIndustries TPI-12”). We gratefully recognize PUCV and MECESUPfor the fellowship for Dr. Pizarro.

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