Bioresource Technology 101 (2010) 5508–5516

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Bioresource Technology

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Evaluation of the catalytic properties of Burkholderia cepacia lipaseimmobilized on non-commercial matrices to be used in biodiesel synthesisfrom different feedstocks

Patricia C.M. Da Rós, Guilherme A.M. Silva, Adriano A. Mendes, Julio C. Santos, Heizir F. de Castro *

Engineering School of Lorena, University of São Paulo, P.O. Box 116, 12602-810 Lorena, São Paulo, Brazil

a r t i c l e i n f o a b s t r a c t

Article history:Received 12 February 2009Received in revised form 16 February 2010Accepted 16 February 2010Available online 17 March 2010

Keywords:LipaseImmobilizationBiodieselBabassu oilTallow beef

0960-8524/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.biortech.2010.02.061

* Corresponding author.E-mail address: heizir@dequi.eel.usp.br (H.F. de Ca

The objective of this work was to produce an immobilized form of lipase from Burkholderia cepacia (lipasePS) with advantageous catalytic properties and stability to be used in the ethanolysis of differentfeedstocks, mainly babassu oil and tallow beef. For this purpose lipase PS was immobilized on two dif-ferent non-commercial matrices, such as inorganic matrix (niobium oxide, Nb2O5) and a hybrid matrix(polysiloxane–polyvinyl alcohol, SiO2–PVA) by covalent binding. The properties of free and immobilizedenzymes were searched and compared. The best performance regarding all the analyzed parameters(biochemical properties, kinetic constants and thermal stability) were obtained when the lipase wasimmobilized on SiO2–PVA. The superiority of this immobilized system was also confirmed in the transe-sterification of both feedstocks, attained higher yields and productivities.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Immobilization of enzymes plays an import role within appliedbiotechnology. The main reason for immobilizing enzymes is theability to isolate the biocatalyst from the reaction product andreuse it in order to increase its productivity. For lipase (triacylglyc-erol acyl hydrolase E.C. 3.1.1.3)-catalyzed reactions, immobiliza-tion can help in providing non-aqueous conditions necessary forester and interesterification reactions (Christensen et al., 2003).

Different techniques for the immobilization of lipase have beentried and reviewed by several workers (Villeneuve et al., 2000)including deposition onto solid supports (Oliveira et al., 2000; Per-son et al., 2000), covalent binding (Soares et al., 1999; Miranda etal. 2006) and entrapment within a polymer matrix or hydrophobicsol–gel materials (Soares et al., 2004; Reetz, 2002). However, theselection of the support is a prominent factor influencing the enzy-matic reactions (Zanin and Moraes, 2004). Comparative studiesindicated that the same lipase molecule can present very differentcatalytic properties after immobilization onto different supports(Gomes et al., 2006). Furthermore, most studies have observed thatone or more properties of immobilized enzymes are different fromthose of the soluble form.

In previous works (Miranda et al., 2006; Santos et al., 2008a),low cost supports have been demonstrated to have high compati-

ll rights reserved.

stro).

bility to immobilize lipase from several sources, resulting in highdegree of recovery of the enzymatic activity and high operationalstability. Among the tested matrixes, promising results have beenreached by an inorganic matrix (niobium oxide, Nb2O5) and a hy-brid matrix (polysiloxane–polyvinyl alcohol, SiO2–PVA). In addi-tion to their low cost, these supports have several advantages foruse as a matrix, including its lack of toxicity and chemical reactiv-ity with the enzyme.

The aim of the present work was to produce an immobilizedform of lipase from Burkholderia cepacia with advantageous cata-lytic properties and stability. The lipase was immobilized on twonon-commercial matrixes by covalent binding and the influenceof several parameters on the activity of the immobilized lipasepreparation has been studied. To test the catalytic activity of thoseimmobilized derivatives, the transesterification reaction of nonedible lipids, in particularly babassu oil and tallow beef, with eth-anol being selected.

2. Methods

2.1. Materials

Burkholderia cepacia (Lipase PS) was from Amano Enzyme Inc.(Nagoya, Japan). Tetraethoxysilane (TEOS), glutaraldehyde 25%(v.v�1) solution and epichlorohydrin were acquired from AldrichChemical Co. (Milwaukee, WI, USA). Hydrochloric acid (minimum36%), ethanol (minimum 99%), polyvinyl alcohol (MW 72,000)

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and polyethylene glycol (molecular weight 1500) were supplied byReagen (RJ, Brazil). Commercial olive oil (low acidity) was pur-chased in a local market. Babassu oil was kindly supplied by Cognis(Jacarei, SP, Brazil) having the following composition in fatty acids:(w/v): 3.5% octanoic, 4.5% decanoic, 44.7% lauric, 17.5% myristic,9.7% palmitic, 3.1% steriac, 15.2% oleic and 1.8% linoleic with aver-age molecular weight 709.90 g/mol. Beef tallow was supplied byFertibom (Catanduva, SP, Brazil) having the following compositionin fatty acids: (w/v): 3.9% myristic, 28.7% palmitic, 30.0% steriac,34.4% oleic and 3.2% linoleic with average molecular weight856.76 g/mol. Solvents were of standard laboratory grade (Synth,SP, Brazil). All the other reagents were of analytical degree.

2.2. Support synthesis

A polysiloxane–polyvinyl alcohol (SiO2–PVA) hybrid compositewas prepared by the hydrolysis and polycondensation of tetraeth-oxysilane according to the methodology previously described(Paula et al., 2008). The spheres were subsequently ground in theball mill and classified to attain particles with 0.308 mm ofdiameter (�40/+60 MESH Tyler standard sieves). Specific detailsconcerning other support morphological properties are given else-where (Santos et al., 2008b). Activation of SiO2–PVA particles wascarried out with epichlorohydrin at 2.5% (w/v) pH 7.0 for 1 h atroom temperature, followed by exhaustive washings with distilledwater. Hydrous niobium oxide was prepared according to method-ology previously described by Miranda et al. (2006) consistingof sintering niobium pentoxide with a fivefold excess byweight of potassium carbonate at 1000 �C for 6 h, followed by addi-tion of hot water and nitric acid 1.0 M. The material was dried at50 �C for 24 h. The support was submitted to a pre-treatment withnitric acid (1%) at 75 �C for 1 h to open the matrix pores and thensilanized with c-aminopropyltriethoxysilane (c-APTS) and acti-vated with glutaraldehyde (Miranda et al., 2006).

2.3. Immobilization procedure

Activated supports were soaked into hexane under stirring(100 rpm) for 1 h at 25 �C. Hexane excess was subsequently re-moved and lipase was added at a ratio of 1:5 g of enzyme per gramof support, correspondent to 12,000 activity units per gram of sup-port. PEG-1500 solution was added together with the enzyme solu-tion at a fixed amount (5 mg g�1 of support). Lipase-supportsystem was maintained in contact for 16 h at 4 �C under static con-ditions. The immobilized lipase derivatives were filtered (nylonmembrane 62HD from Scheiz Seidengazefabrik AG, Thal Schweiz,Switzerland) and thoroughly rinsed with hexane. Hydrolytic activ-ities of free and immobilized lipase were assayed by the olive oilemulsion method according to the modification proposed bySoares et al. (1999). One unit (U) of enzyme activity was definedas the amount of enzyme that liberates 1 lmol of free fatty acidper min under the assay conditions. The results were expressedin activity units per gram of solid (free enzyme preparation orimmobilized derivative).

2.4. Determination of optimal enzymatic activities and kineticparameters

Enzymatic activities (for free and immobilized lipases) as afunction of the variables temperature and pH were investigatedaccording to a 22 full factorial design with three replicates in thecenter points. Star points were added to the experimental designwhen necessary to compose second order models. The activitieswere assayed by the hydrolysis of olive oil emulsion at a fixed pro-portion oil/water 1:1 (Soares et al., 1999). The pH values wereachieved using appropriate phosphate buffer solutions (0.1 M). Re-

sults from the experimental design were analyzed using Statisticaversion 5 (StatSoft Inc., USA).

The influence of substrate concentration on the hydrolyticactivities was also analyzed in the olive oil hydrolysis assay vary-ing the proportion of oil in the emulsion from 10% to 50%. Michae-lis–Menten constant (Km) as the concentration of substrate atwhich half of the maximum reaction rate (Vmax) is reached was cal-culated with aided by computational program Enzfitter version1.05 published by Elsevier-Biosoft, 1987. In all cases, enzyme activ-ity was measured as the initial reaction rate (0–5% hydrolysis) toavoid the possible inhibition that might take place due to theappearance of reaction products.

2.5. Thermal stability

Soluble (1.0 mL) and immobilized derivatives (0.1 g) were incu-bated in the presence of 1 mL buffer phosphate pH 6.5 (100 mM) at60 �C for different time intervals. Samples were removed and as-sayed for residual activity as previously described, taking an un-heated control to be 100% active. The rate of denaturation (kd)and half-life time (t1/2) were calculated by Eqs. (1) and (2),respectively

Ain ¼ Ain0 expð�kd � tÞ ð1Þt1=2 ¼ ln 0:5=� kd: ð2Þ

2.6. Surface area determinations

The surface area measurements were performed by adsorption,using nitrogen as the adsorbate. The samples were previously de-gassed to below 50 mm Hg at room temperature and the analyseswere performed at 77 K, using liquid nitrogen. The equilibriuminterval was 5 s. The surface area was calculated using the B.E.T.method. Pore volume and area distributions based on the BJH cal-culation (Ramos et al., 1998) were evaluated by the B.E.T apparatussoftware (NOVA 1200-Quantachrome).

2.7. Fourier transform infrared spectroscopy (FTIR)

Samples were submitted to FTIR analysis using the equipmentSpectrum One, Perkin Elmer (Waltham, MA, USA). Spectra were ob-tained in the 400–4000 cm�1 wave number range.

2.8. Scanning electron microscopy (SEM)

Conformational changes, such as surface cavities in the support,set by the activating agents insertion were observed by scanningelectron microscopy (SEM). Micrographs of support surfaces wereobtained by using a LEO1450VP scanning electron microscope(Schott Zeiss from Brazil, SP, Brazil).

2.9. Biodiesel synthesis and reaction monitoring

The reactions were performed in closed reactors with a capacityof 25 mL containing 12 g of substrate consisting of either babassuoil or tallow beef and anhydrous ethanol, without the addition ofsolvents, at fixed molar ratio oil to alcohol (1:12). The mixtureswere incubated with the immobilized derivatives at proportionsof 20% (w/w) in relation to the total weight of reactants involvedin the reaction media. The experiments were carried out at 50 �C.Reactions were performed for a maximum period of 48 h underconstant magnetic agitation of 150 rpm. For the time course stud-ies, an aliquot of reaction medium was taken at various time inter-vals and analyzed by gas chromatography following previousestablished conditions (Urioste et al., 2008). All samples were

5510 P.C.M. Da Rós et al. / Bioresource Technology 101 (2010) 5508–5516

measured in triplicate. The transesterification yield was defined asthe ratio between the produced and theoretical esters concentra-tions � 100%. Theoretical ester concentrations were calculated bytaking into consideration the raw material fatty acid compositionand its initial weight mass in the reaction medium (Moreiraet al., 2007; Urioste et al., 2008).

3. Results and discussion

3.1. Support morphological characteristics and immobilization yield

The morphology, distribution and size of the particles for pureand activated supports were studied by micrographics obtainedby scanning electron microscopy (SEM) as shown in Fig. 1(a–d).The method of B.E.T was also used as tool for evaluation the spe-cific surface area, mean pore diameter and pore volume averageof these samples.

SEM allowed verifying morphological differences between pureand activated SiO2–PVA supports (Fig. 1a,b). For pure support, atypical flat surface of amorphous material was observed (Fig. 1a)having the following properties as determined by BET method:average pore diameter (22.91 Å); surface area BET (461 m2/g) andporous volume (0.275 cm3/g). After the activation, small fissureswere verified, which may help the fixation of the enzyme on thesupport (Fig. 1b).

Fig. 1c shows that the pure niobium oxide is formed by particleshaving irregular size and surface showing different arrangementswith a specific surface area of 42.15 m2/g and pore volume of0.029 cm3/g. After the support silanization with c-APTS and activa-tion with glutaraldehyde (Fig. 1d) the particles became more com-pacted, not showing any more empty spaces, a situation that was

Fig. 1. Scanning electron micrographs (SEM): (a) SiO2–PVA pure, (b) SiO2–PVA activateglutaraldehyde.

also confirmed by the specific surface area and pore volume values.The activation with glutaraldehyde reduced the surface area to33.57 m2/g and pore volume to 0.027 cm3/g, probably due to thecross-linking reaction in the support surface.

In the immobilization procedure, a fixed lipase loading (200 mgof lipase per gram of support, correspondent to 12,000 units) wasused, generating immobilized derivatives on SiO2–PVA and Nb2O5

with similar hydrolytic activities, respectively, 3632 and 3228 U/g, correspondent to immobilization yields of 29.73% and 26.42%.This may suggest that under the tested conditions, both supportshad similar compatibility with the enzyme, although trough differ-ent interaction mechanisms.

For the epoxy support (SiO2–PVA) which is able to react withdifferent nucleophilic groups on the protein surface such as amino,hydroxyl or thiol with minimal chemical modification of theprotein (Mateo et al., 2000), the lipase was first hydrophobicallyadsorbed on the support and then a covalent immobilization be-tween nucleophile groups of the lipase and the epoxy groups ofthe support was favored (Mateo et al., 2002). For the silanizedNb2O5 activated with glutaraldehyde, the amino group of enzymereacts with glutaraldehyde to form Schiff bases (C@N bound).The lower pore volume and specific surface area in relation tothe epoxy-SiO2–PVA, suggested that Nb2O5 is a less attractive ma-trix for lipase immobilization. Therefore, additional tests were car-ried to allow a more appropriate choice of the support forimmobilization of this particular lipase.

3.2. Biochemical properties and kinetic constants

A 22 full factorial design was performed to attain the optimalconditions of pH and temperature for hydrolytic activities of free

d with epichlorohydrin, (c) Nb2O5 pure and (d) Nb2O5 silanized and activated with

Table 1Experimental design and results according to the central composite ‘‘22 + star”factorial design carried out to evaluate the influence of the variables pH andtemperature in the hydrolytic activities for free and immobilized lipases.

Runs Variables(coded valuesin parenthesis)

Hydrolytic activity (U/g)

pH (X1) T (X2)(�C)

FreeLipase

Immobilizedon SiO2–PVA

Immobilizedon Nb2O5

1 6.5 (�1) 40 (�1) 33,956 1468 20002 8.5 (+1) 40 (�1) 32,812 2650 14453 6.5 (�1) 60 (+1) 44,564 1381 20334 8.5 (+1) 60 (+1) 48,204 2717 14575 7.5 (0) 50 (0) 54,080 1902 15826 7.5 (0) 50 (0) 52,156 1962 16397 7.5 (0) 50 (0) 54,756 1911 16318 6.1 (�1.41) 50 (0) 44,2009 8.9 (+1.41) 50 (0) 50,02410 7.5 (0) 36 (�1.41) 24,28411 7.5 (0) 64 (+1.41) 32,552

Table 3Model equations fitted to the experimental data points, considering the lipase activity(A, U/mg) as a function of pH (X1) and temperature (X2) coded values.

Models R2

Free lipase Afree enzyme = 53,655 + 1343. X1 -2734.X1

2 + 4721.X2 -12137.X22

0.9545

Immobilized onSiO2–PVA

Alipase-POS-PVA = 1199 + 630.X1 0.9774

Immobilized onNb2O5

Alipase-Nb2O5 = 1684–283.X1 0.9255

Table 4Analysis of variance (ANOVA) for the model that represents the hydrolytic activity ofthe free lipase PS as a function of pH (X1) and temperature (X2).

Source Sum of Squares(x 10�3)

Degree ofFreedom

MeanSquare(x 10�3)

F p

X1 14,375 1 14,375 7.8993 0.1067X1

2 41,883 1 41,883 23.0152 0.0408*

X2 177,793 1 177,793 97.6999 0.0101*

X22 825,221 1 825,221 453.47 0.0022*

Lack of fit 44,997 4 11,249 6.1815 0.1441Pure Error 3640 2 1820Cor total 10,69,960 10

* Significant at 95% confidence level; X1 and X2 represent the variables pH andtemperature, respectively.

Table 5Analysis of variance (ANOVA) for the model that represents the hydrolytic activity ofthe lipase PS immobilized on SiO2–PVA as a function of pH (X1) and temperature (X2).

Source Sum ofSquares

Degree ofFreedom

MeanSquare

F p

X1 15,85,081 1 15,85,081 1513.926 0.0006*

Lack offit

34,556 3 11,519 11.002 0.0845

PureError

2094 2 1047

Cortotal

16,21,731 6

* Significant at 95% confidence level; X1 and X2 represent the variables pH andtemperature, respectively.

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and immobilized lipases. The design is presented in Table 1, to-gether with the experimental results. For the free enzyme, starpoints were added to the design to compose a second order model,since for this system a linear model was not satisfactory (R2 of 0.35with significant lack of fit at 95% confidence level, data not shown).

As displayed in Table 1, the influence of the studied variables onthe hydrolytic activities of the lipase was different for free andimmobilized forms. For free lipase, hydrolytic activities variedfrom 24,284 to 54,756 U/g and the highest activities were obtainedwhen central level of temperature and central or alkaline pH valueswere used (assays 5, 6, 7, 9). Values for hydrolytic activity of thelipase PS immobilized on SiO2–PVA varied from 1381 to 2717 U/gand the highest activities were obtained at alkaline pH levels (as-says 2 and 4). For lipase PS immobilized on Nb2O5 values for hydro-lytic activity varied from 1445 to 2033 U/g and the highest activitywas obtained at the lowest level for the pH (6.5).

Results from Table 1 were used to estimate the main variableeffects and their interactions (Table 2). For free lipase, the statisti-cal analysis of the results showed significant quadratic effects forboth studied variables, besides a significant and positive linear ef-fect for the variable pH, at 95% of confidence level. For the immo-bilized derivatives, on the other hand, only the linear effect of pHwas significant at this confidence level.

From these results, statistical models could be composed withthe coefficients correspondent to the significant effects. The coeffi-cients related to non significant effects were excluded from themodel, except when it was necessary to maintain hierarchy (Myersand Montgomery, 1995). The resulting equations are shown in Ta-ble 3.

The analysis of variance of the models (Tables 4–6) indicatedthat the lack of fit was not significant at 95% of confidence level,and the R2 values showed in Table 3 indicated that the modelscan explain more than 90% of the experimental variability. Thus,

Table 2Estimated effects, standard errors and p-values for the hydrolytic activity of free and imm

Variables Free lipase Immobilized

Effects Standard Error p Effects

Mean 53,655 ±779 0.0000* 1999X1 (Linear) 2685 ±955 0.1067 1259X1 Quadratic) �5469 ±1140 0.0408*

X2 (Linear) 9443 ±955 0.0100* �10X2 (Quadratic) �24,275 ±1140 0.0022*

X1. X2 2392 ±1349 0.2182 77

* Significant at 95% confidence level; X1 and X2 represent the variables pH and tempera

the fitted equations displayed were considered adequate fordescribing the activity as a function of the studied variables andwere used to plot the response surfaces as showed in Fig. 2(a–c).

By considering the adjusted models, optimized conditionsdetermined for each evaluated system were: free enzyme, 50 �Cand pH 7.5; immobilized derivative on SiO2–PVA, 40 to 60 �C andpH 8.5; immobilized derivative on Nb2O5, 40–60 �C and pH 6.5.To confirm these models, assays were carried out under the estab-lished conditions and the differences between the experimentaland theoretical values were lower than 8%.

obilized lipase on SiO2–PVA and Nb2O5, obtained in the experimental design.

lipase on SiO2–PVA Immobilized lipase on Nb2O5

Standard Error P Effects Standard Error p

±12 0.0000* 1684 ±12 0.0000*

±32 0.0007* �566 ±31 0.0030*

±32 0.7865 23 ±31 0.5418

±32 0.1403 �11 ±31 0.7661

ture, respectively.

Table 6Analysis of variance (ANOVA) for the model that represents the hydrolytic activity ofthe lipase PS immobilized on Nb2O5 as a function of pH (X1) and temperature (X2).

Source Sum ofSquares

Degree ofFreedom

MeanSquare

F p

X1 319,790 1 319,790 335.79 0.0030*

Lack offit

23,850 3 7950 8.35 0.1089

PureError

1905 2 952

Cor total 345,545 6

* Significant at 95% confidence level; X1 and X2 represent the variables pH andtemperature, respectively.

5512 P.C.M. Da Rós et al. / Bioresource Technology 101 (2010) 5508–5516

The results for free enzyme were in agreement with the ex-pected behavior attaining specific optima conditions for pH andtemperature. These conditions are presented as a ‘‘top” in the cor-respondent response surface (Fig. 2.a), and the temperature at thispoint (50 �C) is the same that the optimum previously reported byother authors (Pencreac’h et al., 1997) for lipase from Burkholderiacepacia, formerly known as Pseudomonas cepacia.

In relation to the immobilized derivatives, the immobilizationprocedure resulted in thermal stable systems in the range of 40–60 �C without significant changes in their activity (Fig. 2a and b).The increase in thermal stability is expected for immobilized sys-tems. However, no significance effect of temperature was observedfor B. cepacia lipase immobilized on both tested supports. This maysuggest that diffusion limitation occurred in hydrolysis reactioncarried with these immobilized derivatives, in despite of the parti-cle size was small enough to avoid this.

With relation to the optimum pH, the values for the immobi-lized systems were shitted in the opposite way. The lipase immo-bilized on SiO2–PVA had the optimum pH value shifted to morealkaline conditions, whereas the immobilization on Nb2O5 shiftedto more acid pH optimum value.

Changes on the optimum pH values to more alkaline values isan expected behavior for immobilized lipases, in the view of theestablished serine-based nucleophilic attack aided by a histidineworking as a base (Villeneuve et al., 2000). Such behavior is ex-pected because at least partial opening of the lid upon immobiliza-tion is likely to expose the his at the active site more directly tosolution hydrogen ions, and so only less acidic conditions yieldthe unprotonated form of the imidazolium ring required for a basicbehavior (Balcão et al., 1996; Perez et al., 2007). The oppositeperformance exhibited by the immobilized derivative on Nb2O5

can be attributed to the positive character of the matrix, whichcan create a more basic microenvironment around the lipase mol-ecules. In this case, the optimum pH for the protein molecule canbe reached near to the support surface only when more acid con-ditions are available in the bulk medium.

Fig. 2. Surface responses for the hydrolytic activity of the Burkholderia cepacialipase in the free form (a) immobilized on SiO2–PVA, (b) immobilized on Nb2O5, (c)as a function of the variables pH and temperature according to the fittedmathematical models.

3.3. Kinetic parameters

Under the chosen conditions, the kinetic parameters weredetermined for each lipase preparation. Data for hydrolytic activi-ties under different substrate concentrations are shown in Fig. 3,which indicated that the Michaelis–Menten equation can be usedto adjust data to calculate km and Vmax values.

Generally, km values increased upon immobilization. This maybe attributed to either structural changes in the enzyme uponimmobilization or changes in the accessibility of the active siteto the substrate. However, in this study, the km value of the immo-bilized lipase decreased upon immobilization on SiO2–PVA parti-cles (from 4018 mM for the free enzyme to 1882 mM for the

0 372 744 1116 1488 1860

0

1x104

2x104

3x104

4x104

5x104

6x104

Free PS lipase

Rat

e (fr

ee P

S, U

/g)

Fatty acids (mM)

0

500

1000

1500

2000

2500

3000

PS Immobilized on SiO2 -PVA PS Imobilized on Nb2 O5

Rat

e (im

mob

ilized

PS,

U/g

)

Fig. 3. Hydrolytic activities of the lipase in free form (right axis) and immobilizedon SiO2–PVA and Nb2O5 (left axis) as a function of the substrate concentration(expressed in total fatty acids content in olive oil/water emulsions).

0 20 40 60 80 100 120

0

20

40

60

80

100

Free Lipase

Lipase immobilized on SiO2-PVA Lipase immobilized on Nb2 O5

Res

idua

l act

ivity

(%

)Time (min)

Fig. 4. Thermal deactivation for free lipase (s), SiO2–PVA immobilized (d) lipaseand Nb2O5 immobilized (?) lipase (Experiments were carried out in 0.1 Mphosphate buffer pH 6.5 at 60 �C).

Table 7Half-life time (t1/2) and the rate of denaturation (kd) for free and immobilized lipases.

Lipase form Rate of denaturation (kd,h�1)

Half-life time (t1/2,h)

Free lipase 1.39 0.36

P.C.M. Da Rós et al. / Bioresource Technology 101 (2010) 5508–5516 5513

immobilized derivative). However, the immobilized derivative onNb2O5 allowed lower substrate access in the porous support, indi-cating that the affinity towards the substrate was reduced. Theinternal surface with smaller porous diameter verified in Nb2O5

promoted diffusional limitation of the substrate. In addition, thehigh reactivity of the activating reactant in relation to the epoxygroups also might have led to excessive cross-linking in the matrixand the formation of small pores. As a consequence, an increase onthe km value for the immobilized derivative on Nb2O5 occurred(km = 5267 mM).

Immobilized on SiO2–PVA

0.11 6.24

Immobilized on Nb2O5 0.25 2.77

Table 8Biochemical and kinetics properties of Burkholderia cepacia lipase (free and immo-bilized derivatives on SiO2–PVA and Nb2O5).

Parameters Free ImmobilizedSiO2–PVA

ImmobilizedNb2O5

Optimum pH 7.5 8.5 6.5Optimum temperature

(�C)50 40–60 40–60

Half-life time (t1/2) at60 �C, (h)

0.36 6.24 2.77

Kinetic parametersKm (mM) 4018 1883 5267Vmax (U/g) 56,441 3873 6323

3.4. Thermal stability

Experiments were performed to determine the thermal deacti-vation constants (kd) and the half-life time for free and immobi-lized derivatives under thermal effect at 60 �C and results aredisplayed in Fig. 4. Patterns of heat stability indicated that theimmobilization process tends to stabilize the enzyme. In this case,half-life time (t1/2) of the enzyme is shown to be inversely propor-tional to the rate of denaturation (kd). The half-life time (t1/2) is thetime which takes for the activity to reduce to a half of the originalactivity. These results are shown in Table 7.

Immobilized derivatives revealed higher half-life time, for thelipase immobilized on SiO2–PVA in the order of 6.24 h. The half-lifefor immobilized derivative on Nb2O5 was 2.77 h, while the free en-zyme was totally denatured after 30 min incubation at 60 �C.

Epoxy SiO2–PVA derivative was more thermal stable thanNb2O5 derivative, probably due to the poor geometric congruencybetween Nb2O5 and enzyme which did not allow the formationof new bonds between the lipase and active groups in the support.As a consequence, no significant improvement in the thermal sta-bility after the immobilization was observed. The use of supportwith good geometric congruence, such as epoxy-SiO2–PVA, pro-vided the formation of strong covalent bonding of enzyme on thesupport, increasing the thermal stability of the derivative (Fernán-dez-Lafuente et al., 1995).

To better illustrate the differences in the catalytic properties ob-served between the lipase from Burkholderia cepacia immobilizedonto different materials, the biochemical and kinetic properties

of free and immobilized lipases on epoxy-SiO2–PVA and Nb2O5

activated with glutaraldehyde are summarized in Table 8.

3.5. FTIR spectra for the free lipase and immobilized derivatives

The efficiency of the methodology in relation to the lipase incor-poration on both supports was also assessed by infrared spectros-copy. This technique is used to detect the presence of covalentbonds in molecules, based on the principle that each type of cova-lent bond has a characteristic absorption wavelength represent as

4000 3500 3000 2500 2000 1500 1000 5000.0

0.5

1.0

1.5

2.0

2.5

Immobilized PS on SiO2-PVA

Support (SiO2-PVA)

Free PS lipase

Abs

orba

nce

Wave number (cm-1)

4000 3500 3000 2500 2000 1500 1000 500

0.0

0.5

1.0

1.5

2.0

2.5

Support (Nb2O5)

Immobilized PS on Nb2O5

Free PS lipase

Abs

orba

nce

Wavenumber (cm )-1

Fig. 5. FTIR spectra for the activated supports, free lipase and immobilizedderivatives.

A

0 6 12 18 24 30 36 42 480

10

20

30

40

50

60

70

PS immobilized on SiO2-PVA

PS immobilized on Nb

2 O5

Babassu oil

Ethy

l est

ers

(% w

t)Time (h)

B

0 6 12 18 24 30 36 42 480

10

20

30

40

50

60

70 Tallow beef

PS immobilized on Nb2 O5

PS immobilized on SiO2 -PVA

Ethy

l est

ers

(% w

t)

Time (h)

Fig. 6. Profile for alkyl esters formation in the transesterification reaction of thebabassu oil (a) and beef tallow (b) with ethanol using PS immobilized on SiO2–PVAand Nb2O5 as biocatalysts (molar ratio oil to ethanol 1:12 at 50 �C).

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an upward peak on a charted spectrum. Fig. 5(a,b) shows a set ofspectra for free lipase (PS), activated supports and immobilizedderivatives.

Free enzyme displayed a typical spectrum of proteins (Fig. 5a,b)with bands associated to their characteristic amide group (CONH).In this case, in the range from 1600 to 1700 cm�1, there is a amide Iband due to the double bond CO stretching, the C–N stretching andNH bending (Stuart et al., 1996).

The spectrum for SiO2–PVA showed bands at 1110–1000 cm�1

corresponding to Si–O–R asymmetric stretching (Fig. 5a). Othercharacteristic bands were observed at 950 cm�1 (Si–O–Si axialdeformation), 810 cm�1 (Si–O–Si axial deformation) and600 cm�1 (Si–O–Si angular deformation). For the immobilizedderivative (Fig. 5a), a slight additional bands in the spectrum at1600 cm�1 was observed, indicating that the covalent bond be-tween the enzyme and support occurred (NH bending).

The spectrum for Nb2O5 showed a peak at 1110–1000 cm�1 cor-responding to Si–O–R asymmetric stretching (Fig. 5b) and1600 cm�1 corresponding to C–N stretching, as a result of the sali-nization step. After immobilization, a slight increase on peak at1600 cm�1 was also verified, showing however lower intensity inrelation to that obtained by SiO2–PVA derivative.

The spectrum for Nb2O5 showed that a peak corresponding tothe hydroxyls (3200–3600 cm�1) originally present in the puresupport were broader for Nb2O5 than for SiO2–PVA. Note also that,different chemical agents were used to activate the supports and

this behavior can be related to the chemical modification under-took in the support surface caused by each of the activation agentstested. The fact of the peak of the hydroxyls was smaller in theSiO2–PVA can be related to the use of epichlorohydrin as activatingagent.

Regarding the resulting immobilized derivatives, it can be notedthat besides the peak at 1650 cm�1 (amino deformation), peakscorresponding to carboxyl deformation (1500 and 2800 cm�1) dis-appeared, which demonstrated that the characteristic functionalgroups of the lipase were covalently bond on the groups insertedin the supports.

It was also noted that the immobilized derivative on SiO2–PVAdisplayed a peak inside of the same existent band for the free lipase(Fig. 5a), while the immobilized derivative on Nb2O5 exhibited a

Table 9Transesterification activity of the immobilized derivatives using different feedstocks.

Lipase PS Feedstock TransesterificationYield (%)

Productivity (mgbiodiesel/g.h)

Immobilized onSiO2–PVA

Babassuoil

100 12.90

Beeftallow

89.70 12.50

Immobilized onNb2O5

Babassuoil

74.13 9.60

Beeftallow

40.20 5.60

P.C.M. Da Rós et al. / Bioresource Technology 101 (2010) 5508–5516 5515

peak displacement to the right side (Fig. 5b). This observed effect inthe spectrum for the immobilized derivative on Nb2O5 was consis-tency with its higher Km value when compared with the immobi-lized derivative on SiO2–PVA.

3.6. Biodiesel synthesis

Biodiesel synthesis was carried out as described in the materialand methods (Section 2.9). The reaction was monitored by quanti-fication of the ethyl esters as shown in Fig. 6(a and b), respectively,for babassu oil and tallow beef. The observation of these figuresindicated that, independent of the feedstock, both immobilizedderivatives were able to form the main fatty acids esters in theseraw materials. However, both reaction rates and yields were influ-enced by the support and activating agents used for immobilizingthe lipase PS.

For babassu oil, ethyl esters concentrations varied between46.3% using lipase immobilized on Nb2O5 to 61.9% for lipase immo-bilized on SiO2–PVA, corresponding to transesterification yieldsranging from 74.13% to 100% (Table 9). For biodiesel from beef tal-low, the lipase immobilized on Nb2O5 attained a transesterificationyield of 40.21%, corresponding to a productivity of 5.60 mg/g h,while the lipase PS immobilized on SiO2–PVA gave a productivityof 12.50 mg/g h, reaching a conversion of 89.70%, at the same reac-tion time. For both raw materials, the highest performance wasachieved by lipase PS immobilized on SiO2–PVA, attaining satisfac-tory yield up to 48 h. The lower performance of the lipase PSimmobilized on Nb2O5 is consistent with the assessed biochemical,kinetic and morphological properties.

4. Conclusions

Support material and immobilization strategy were found to bethe most important two factors to attain active and stable immobi-lized derivatives. Thus, exploiting good immobilization carriers hasbeen an attractive approach for enzyme engineering. This workverified the influence of two non-commercial supports to immobi-lize lipase from Burkholderia cepacia (Lipase PS), evaluating the ki-netic and biochemical parameters of the resulting immobilized andtheir subsequent application in the ethanolysis of the babassu oiland beef tallow. Silanized Nb2O5 activated with glutaraldehydeshowed lower pore volume and specific surface area in relationto the epoxy-SiO2–PVA.

Factorial designs were used to study the biocatalytic propertiesof the lipase PS free upon immobilization on SiO2–PVA and Nb2O5

allowed evaluation of the influence of pH and temperature and tomaximize the hydrolytic activity of the enzyme. Through thosetechniques mathematical models were composed which could ex-plain the variation in the activity of the Burkholderia cepacia lipaseas a function of the studied variables. The established conditionsfor maximization of the hydrolytic activity were: free enzyme-pH7.5 and 50 �C; enzyme immobilized on SiO2–PVA-pH 8.5 with a

wide temperature range (40–60 �C) and enzyme immobilized inNb2O5 pH 6.5, also with a wide temperature range (40–60 �C).The affinity of the lipase PS for the substrate (emulsion of oliveoil) was higher when immobilized on SiO2–PVA (km = 1892 mM)than immobilized on Nb2O5 (km = 5267 mM). Immobilized lipaseon SiO2–PVA was approximately 2 times more stable than theimmobilized derivative on Nb2O5 and 17 times more stable thanthe free lipase, showing that immobilization procedure in this sup-port stabilize thermically the enzyme. In the biodiesel synthesisfrom both feedstocks, results showed that the best performancewas attained with the lipase PS immobilized on SiO2–PVA, givinga transesterification yield higher than 89% in 48 h.

Acknowledgements

The authors are grateful for the financial support provided byFAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo)and CNPq (Conselho Nacional de Desenvolvimento Científico e Tec-nológico), Brazil.

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