Microporous and Mesoporous Materials 163 (2012) 343–355

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Microporous and Mesoporous Materials

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Synergistic effect in the catalytic activity of lipase Rhizomucor mieheiimmobilized on zeolites for the production of biodiesel

Adriano de Vasconcellos a,1, Alex S. Paula a,1, Roberto A. Luizon Filho a, Lucas A. Farias b, Eleni Gomes a,Donato A.G. Aranda c, José G. Nery a,⇑a Departamento de Física, Instituto de Biociências, Letras e Ciências Exatas, UNESP – Universidade Estadual Paulista, Campus de São José do Rio Preto, São Paulo, 15054-000, Brazilb Laboratório Nacional de Luz Síncrotron, Campinas – São Paulo 13084-971, Brazilc Greentec/Escola de Química, Universidade Federal do Rio de Janeiro, CEP 21945-970, Rio de Janeiro, RJ, Brazil

a r t i c l e i n f o

Article history:Received 22 November 2011Received in revised form 5 June 2012Accepted 23 July 2012Available online 31 July 2012

Keywords:Lipase enzymeZeolite PEnzyme immobilizationTransesterification reactionsBiodiesel

1387-1811/$ - see front matter � 2012 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.micromeso.2012.07.043

⇑ Corresponding author. Tel.: +55 17 3221 2490; faE-mail address: nery@ibilce.unesp.br (J.G. Nery).

1 These authors contributed equally to this work.

a b s t r a c t

Gismondine (P) ion exchanged with Cu2+, Zn2+, and Ni2+ were used as solid supports for the immobiliza-tion of the lipase Rhizomucor miehei enzyme. Physicochemical characterization of the zeolite–enzymecomplexes were performed by X-ray diffraction (XRD), scanning electron microscopy (SEM), atomic forcemicroscopy (AFM) and attenuated total reflectance–Fourier transform infrared (ATR–FTIR). These bio-catalysts were used for the transesterification reaction of soybean oil to biodiesel. Divalent cation speciesand thermal treatment of the zeolitic supports had different effects on several parameters underinvestigation. In terms of the enzyme activity, the zeolite–enzyme complexes prepared with Ni-P weresuperior in comparison to the other ones and a synergistic effect for the zeolite–enzyme complex(Ni-P/200-ENZ) was observed for the transesterification reactions of soybean oil to biodiesel. The totalamount of methyl esters produced by the complex Ni-P/200-ENZ was of 56.2%, while the same concen-tration of the immobilized enzyme in its free form yielded only 39.3% of methyl esters, and Ni-P/200 in itspure form also yielded a very low amount of methyl esters (20%). The other zeolite–enzyme complexes(Zn-P/200-ENZ, Cu-P/200-ENZ, and Na-P/200-ENZ) presented a completely different behavior in compar-ison to the Ni-P/200-ENZ complexes. The total yields of methyl esters generated by these biocatalystswere very low and no synergistic effects were observed. A correlation between the cation atomic radiusand the enzymatic activity of the zeolite–enzyme complexes was observed. It was noticed that the biggerthe atomic radius of the extra-framework cation, smaller was the experimental enzymatic activitycoefficient.

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1. Introduction

Intense search for alternative fuels has stimulated and intensi-fied the efforts of the scientific community to look at the cheapand available biomass as a feedstock for the production of cleanrenewable energy. In this aspect, biodiesel plays a major role be-cause it is a biodegradable and renewable form of energy, consist-ing of alkyl ester of fatty acids [1]. Biodiesel can be made by thetransesterification of vegetable oils or animal fats with short alco-hols such as methanol and ethanol under the presence of a catalyst.The transesterification reaction consists of a sequence of three con-secutive reversible reactions, which include conversion of triglyc-erides to diglycerides, followed by the conversion of diglyceridesto monoglycerides. In each step of the overall reactions one ester

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molecule is generated and all the glycerides are converted intoglycerol [2,3].

Several excellent reviews discussing the advantages and disad-vantages of the different types of catalysts used in the biodieselproduction have been published in the last decades [4–13]. Regard-ing the use of different catalysts, according to these authors thetransesterification process for the production of biodiesel can beclassified in four different categories: (a) base-catalyzed transeste-rification [4,5], (b) acid-catalyzed transesterification [6–8], (c) en-zyme-catalyzed transesterification [9–11], and (d) supercriticalalcohol transesterification [12,13]. However, in practical termsmost of the large scale industrial production of biodiesel is per-formed through the homogeneous base-catalyzed process [4,5],using NaOH, KOH or sodium methoxide (NaOCH3) as base cata-lysts. Homogeneous base-catalyzed processes have a high reactionrate at a low temperature (60 �C), usually are more efficient andless corrosive compared to the other processes [6–8]. Althoughbase catalysts have all these advantages, their use have also seriousdrawbacks in the large scale production of biodiesel, especially

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when low quality feedstock (vegetable oils sources with high con-tents of water and free fatty acids) are used. In this case, the use ofbase catalysts will increase the probability of soap and emulsionformation, which will or can generate a large amount of wastewa-ter during the process of cleaning and separation of the glycerol(subproduct) and biodiesel (product) [8,10].

Due to these drawbacks, it is interesting from the economic andenvironmental point of view the development of new and moreefficient catalysts which could be industrially employed in the bio-diesel production using cheap, low quality and abundant biomassfeedstocks. Enzymes-catalyzed transesterification process is apromising and attractive alternative [14,15]. Enzymes exhibit sev-eral advantages compared to the homogeneous base catalysts suchas high catalytic activity for the reaction under moderate reactionsconditions, ability to process vegetable oils that contain a high de-gree of free fatty acid and water, therefore decreasing the probabil-ity of forming soap and emulsion [14–16]. Besides all theadvantages of the enzymatic process in comparison to the otherones, the main stumbling blocks for employing the enzymatictransesterification process in large industrial scale for biodieselproduction are the enzymes expensive costs, their separation andreuse after the reaction, and the deactivation of their active sitesby the glycerol [17]. One possible manner of overcoming theseobstacles is through the immobilization of the enzymes on solidcarries. This strategy is usually used to improve and increase ther-mal stability of enzymes, to gain better operation control, to facil-itate product recovery without catalysts contamination, todecrease of the enzyme inhibition by some of the reaction products[18]. Immobilization also increases enzyme selectivity towardsnon-natural substrates and improve the functional properties ofthe immobilized enzymes compared to soluble enzymes [19].

Several solid materials such as ceramics, kaolinites, silica, cellu-lose, polymers, and zeolites have been used as support for enzymesimmobilization [18,19]. Zeolites are crystalline aluminosilicateswith periodic arrangements of cages and channels of moleculardimensions that find extensive industrial uses as catalysts, adsor-bents, ion exchangers [20–22]. Most of zeolites properties are re-lated to the possibility of generating and regulating their acid–base, hydrophobic–hydrophilic character and also their strongselective adsorption affinities. Due to these properties, there areseveral studies reporting the use of zeolites in their pure phasenot only as heterogeneous catalysts for transesterification of tri-glycerides to biodiesel [23–27], but also as a solid support for en-zyme immobilization [28–30]. In this case, the aim of theimmobilization is to create a zeolite–enzyme complex that canbe used as biocatalysts for the transesterification of triglyceridesto biodiesel. It is clear from these studies [28–30] that both thezeolites and the enzymes can themselves alone catalyze thetransesterification reaction. Each one of them have their own par-ticularity that determine the rate and yield of the final product.However, when both catalysts are combined through the immobi-lization process important zeolite parameters (surface area, porediameter, mechanical strength, thermal stability, chemical durabil-ity, the zeolitic support hydrophobic/hydrophilic character, andnature of the extra framework cations) and enzyme parameters(amount of immobilized enzymes, nature of the enzyme active site,and enzymatic activity) can affect the product yield of the reactioncatalyzed by the zeolite–enzyme complexes [31]. Although thereare several studies [18,19,28–31] reporting the immobilization ofenzymes on zeolites, until now to the best of our knowledge thereis no report clearly showing the synergistic effect of both catalystsin the transesterification reaction of vegetable oils to biodiesel. Inthis study, we report a systematic investigation of a system com-posed by zeolites P ion exchanged with several different cations,which were employed as solid supports for the immobilization ofthe lipase from Rhizomucor miehei. The catalytic activity of the pure

zeolites, the pure enzyme and the zeolite–enzyme complexes weretested for the transesterification reaction of soybean oil to biodie-sel under several conditions. The catalytic results were correlatedto the zeolite crystallographic structures, nature of the cation dom-inant medium, cation atomic radius, amount of immobilized en-zyme and enzymatic activity. As it was expected, both thezeolites and enzyme in their pure forms have shown catalyticactivities for the specific reaction under investigation, however insome cases it was observed a clear synergistic behavior of the zeo-lite–enzyme complexes, since the amounts of the methyl estersformed by the zeolite–enzyme complexes were higher than themethyl esters yield formed by the enzymes and zeolites used sep-arately in their pure phases.

2. Experimental

2.1. Materials

The reagents used for the transesterification reactions were re-fined soybean oil containing 0.5 wt.% of free fatty acid and 1% ofwater obtained from Cargill Oil Company (Brazil) and anhydrousmethanol (Sigma–Aldrich, 99.8%) and they were used as received.The reagents used for the esters hydrolysis reactions were 4-nitro-phenyl palmitate, Arabic gum, Triton X-100, isopropyl alcohol. Theinorganic salts and organic bases used for the preparation of thezeolitic matrixes were purchased from Sigma Aldrich and usedwithout further purification. The enzyme used was PALATASE20000L (Sigma–Aldrich), a purified 1,3-specific lipase from R. mie-hei lipase (EC 3.1.1.3), produced by submerged fermentation of agenetically modified Aspergillus oryzae.

2.2. Zeolites preparation

Zeolite P with high aluminum content was prepared following aprocedure reported in the literature [32,33]. In a typical synthesisequimolar amounts of a sodium aluminate suspension (preparedfrom Cabosil-5, Aldrich, purum, and NaOH, Fisher. A.C.S., first stir-red at room temperature for 24 h, then, kept unstirred at 100 �C for76 h and a silicate suspension (made from sodium silicate solutionSiO2 wt%., Fluka, and NaOH, Fisher, A.C.S., were mixed at 60 �C andheated to 150 �C for 5 days (Teflon lined digestion bottle). The pre-cipitate was filtered, rinsed with NaOH containing water (pH 10)and vacuum dried. Equilibration at constant humidity for 3 days(desiccator satured with KOH yielded the product in its fully hy-drated form). This sample was named Na-P zeolite, and it was con-verted to its Cu, Zn and Ni forms by ion exchange with theappropriate salt solution. In a typical ion exchange experiment 1gram of the as made Na-P was put in a Teflon lined digestion (ParrInstruments) together with 30 mL of 0.5 M Cu(NO3)2, Zn(NO3)2 orNi(NO3)2 solution and after 96 h at 100 �C the products were fil-tered, rinsed, vacuum dried, and again equilibrated under constanthumidity. The samples were named Cu-P, Zn-P and Ni-P.

2.3. Thermal treatment of the zeolitic support

Prior to the enzyme immobilization experiments, all the zeolitessamples (Na-P, Cu-P, Zn-P and Ni-P) were submitted to a thermaltreatment in order to remove the adsorbed molecules water onthe surface of the zeolites and also to observe the crystallographicstability of the zeolites under critical temperature. The sampleswere heated in air stream at 200 �C according to the following tem-perature ramps: 25–100 �C (1 h), 100–150 �C (1/2 h) and 150–200 �C (1/2 h). After reaching the desired temperature (200 �C),the samples were kept in this temperature range for 1 h. These sam-ples were named Na-P/200, Cu-P/200, Zn-P/200 and Ni-P/200,

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respectively. In parallel experiments, samples were also heated inair stream at 550 �C according to the following temperature ramps:25–100 �C (1 h), 100–150 �C (1/2 h), 150–200 �C (1/2 h) and 200–550 �C (3 h). After reaching 550 �C the samples were kept in thistemperature range for 5 h. These samples were named Na-P/550,Cu-P/550, Zn-P/550 and Ni-P/550, respectively.

2.4. Catalysts characterization

All the samples prepared in the previous sections were charac-terized by XRD, SEM, ATR–FTIR and AFM. XRD data were collectedwith a Rigaku RotaFlex RU200B (Tokyo, Japan) on a rotating anodesource with a flat-plate Bragg–Brentano geometry, operating withCu Ka radiation (wavelength = 1.5418 Å) at 50 kV and 100 mA, andequipped with a graphite monochromator. The powder diffractionpatterns were recorded in the range 2a = 5–80� with a step scan of0.02� and at a rate of 10 s/step. SEM images were recorded using aXL30 FEG instrument, and before the analysis, a thin coating ofgold was deposited onto the samples. ATR–FTIR spectra were col-lected using a Smart Orbit ATR (Thermo Scientific) diamond acces-sory and a Nicolet 6700 spectrometer (Thermo Scientific) equippedwith a DTGS detector. All samples were scanned for 64 times at thespectral resolution of 4 cm�1 between 4000 and 500 cm�1. Elemen-tal chemical analysis for Si, Al, Na, Cu, Ni and Zn of all zeolitic sup-ports were determined by Inductively Coupled Plasma AtomicEmission Spectroscopy (ICP-AES, Chemical Analysis Labs-Sao PauloUniversity Facilities-USP). Thermogravimetric analyses were per-formed on a TA Instruments Hi Res TGA 2950 thermogravimetricanalyzer equipped with an EGA furnace. Samples were analyzedin platinum pans at a heating rate of 10 �C/min over the tempera-ture range 25–1000 �C in an atmosphere of air flowing at 180 mL/min. Sample masses ranged from 1.5 to 2.5 mg. AFM experimentswere performed at the Brazilian National Laboratory of Synchro-tron Light (LNLS). The data were collected using a Dimension3000 scanning probe microscope (SPM) equipped with a Nano-Scope IIIa SMP controller (digital Instruments Inc.). Tapping modeAFM images were acquired in ambient air using TESP tapping-mode etched silicon probes.

2.5. Lipase immobilization on zeolites

Lipase enzyme (PALATASE 20000L, molecular size 31600 Da, pI3.8) was immobilized on the previously prepared zeolitic supportsfollowing a procedure adapted from the literature [19,31,34]. Anamount of enzyme (2 mg/mL) and zeolites (0.2 g) were mixed in5 ml of 0.3 M phosphate buffer pH 7, and stirred at 600 rpm for16 h at room temperature. The zeolite–enzyme complexes wereseparated by centrifugation at 10,000 rpm, washed twice withde-ionized water and dried at 25 �C overnight and stored at 4 �Cfor 2 days before the transesterification reaction. The concentra-tion of the immobilized enzyme was determined following themethod described by Bradford [35] using bovine serum albuminas standard. The concentration of enzyme adsorbed on the zeoliticmatrix, Pg (mg/g), was calculated using the following Eq. (1):

Pg ¼C0V0 � Cf V f

wð1Þ

where C0 is initial protein concentration (mg/mL), Cf the proteinconcentration of the filtrate (mg/mL), V0 the initial volume of lipasesolution (mL), Vf the volume of filtrate (mL) and w is the weightof zeolitic support used (g) [19]. These samples were designedNa-P/200-ENZ, Cu-P/200-ENZ, Zn-P/200-ENZ, and Ni-P/200-ENZ(zeolitic supports thermally treated at 200 �C) and Na-P/550-ENZ,Cu-P/550-ENZ, Zn-P/550-ENZ, and Ni-P/550-ENZ (zeolitic supportsthermally treated at 550 �C).

2.6. Enzyme activity assay

The enzymatic activities of the free lipase and the zeolite–enzyme complexes were determined by the method developedby Winkler and Stuckmann [36] and updated by Krieger et al.[37]. The method consists of measuring the micromoles of 4-nitrophenol released from the hydrolysis of p-4-nitrophenyl palmi-tate (p-NPP). Following this method a stock solution of the p-NPP(3 mg/mL) was prepared using HPLC grade isopropyl alcohol (solu-tion A). Another solution (solution B) comprised of (0.5%) Arabicgum, (2%) Triton X-100, diluted in phosphate buffer (pH 7,0.05 M) was prepared, and then 1 mL of solution A was added to9 mL of solution B. This mixture or final solution was named sub-strate. The activity of free enzyme was measured according to thefollowing procedure: In 900 lL of the substrate solution it wasadded 100 lL of free enzyme diluted in the phosphate buffer,and then the mixture was incubated for 1 min at 37 �C. The quan-tity of 4-nitrophenol released was determined photometrically(410 nm) and the assays were performed in triplicate, and one unitof lipase activity was define as the amount of enzyme required toproduce 1 lmol of 4-nitrophenol released from 4-NPP per minuteunder the assay conditions. The lipolytic activity of free enzyme,U, was calculated according to Eq. (2).

U ¼ Abs � V t

e � Xe � T

� �� 103

� �� D ð2Þ

where U, unit of enzyme activity is expressed as U/mL, where oneunit of activity is defined as the formation of 1 lmol p-nitrophenolreleased from the p-NPP/mL min under the assay conditions; Abs,absorbance of sample at 410 nm; Vt, total volume of reaction(mL); Xe, volume of enzyme solution (mL) or mass of the immobi-lized enzyme; e, coefficient of molar extinction (L mol�1 cm�1); T,incubation time; 103, factor correction of coefficient (e); D, sampledilution, if necessary.

The activities of zeolite–enzyme complexes were measuredaccording to the following procedure: 2–10 mg of the zeolite–en-zyme complexes were mixed with 100 lL of phosphate buffer(pH 7) and 900 lL of substrate, and then this mixture incubatedfor 1 min at 37 �C. The reaction was interrupted by adding250 lL of a solution of sodium carbonate (0.1 M) with 5% TritonX-100 to the previous mixture, and cooling it down to minus 4 �Cwith the help of an ice/ethanol bath. The solution was centrifugedat 10,000 rpm to separate the solids and the supernatants andabsorbance measurements at 410 nm of the supernatants wereimmediately performed against a blank reaction without enzyme.

2.7. Syntheses of the biodiesel

The transesterification reactions of the soybean oil to biodieselwere performed with the three different catalysts groups: freeenzyme, pure zeolites (Na-P/200, Cu-P/200, Zn-P/200, Ni-P/200)and the zeolite–enzyme complexes (Na-P/200-ENZ, Cu-P/200-ENZ, Zn-P/200-ENZ, Ni-P/200-ENZ) and (Na-P/550-ENZ, Cu-P/550-ENZ, Zn-P/550-ENZ, Ni-P/550-ENZ). Several experiments wereperformed in order to find the appropriate reactions conditionswhich would allow a comparison between the three catalystsgroups. Transesterification reactions catalyzed by the free enzymeswere performed with an oil:methanol ratio of 1:5 at 37 �C whichwas the optimum temperature for this enzyme for this specificreaction, and methanol was slowly added in order to preclude theinactivation of the enzyme. After 72 h of reaction the products (bio-diesel, glycerol) were separated by centrifugation at 10,000 rpm.Transesterification reactions catalyzed by pure zeolites (Na-P/200,Cu-P/200, Zn-P/200, Ni-P/200) were performed with an oil:metha-nol ratio of 1:10 at 37 �C for 72 h and after the reaction the products

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(biodiesel, glycerol) were separated by centrifugation at10,000 rpm. Transesterification reactions catalyzed by zeolite–en-zyme complexes (Na-P/200-ENZ, Na-P-550-ENZ, Cu-P/200-ENZ,Cu-P/550-ENZ, Zn-P/200-ENZ, Zn-P/550-ENZ, Ni-P/200-ENZ, andNi-P/550-ENZ) were performed with an oil:methanol ratio of 1:5at 37 �C/72 h. The progress of the transesterification reactions werefollowed by thin layer chromatography (TLC) based on a proceduredescribed by Yang et al. [38]. At predetermined time intervals, asmall volume (100 lL) of reaction mixture was collected, andmixed with 500 lL hexane for 2 min. After separation by centrifu-gation, 3 lL of the upper layer was applied to a silica gel plate. Asolution of hexane/ethyl acetate/acetic acid (90:10:1) was used asdeveloping solvent and a solution of methanol/sulfuric acid (1:1)was used as color reagent. After spraying the color reagent over sil-ica gel plate, followed by the development solvent, the silica gelplate was heated at high temperature and then analyzed.

Fig. 1. XRD patterns of the zeolite gismondine in its sodium form (Na-P) and ionexchanged with copper, zinc and nickel cations.

2.8. Gas chromatography

The methyl esters yields of the transesterification reactionswere assayed by gas chromatography in a Thermos Ultra Chro-matograph provided with flame ionization detector employingsilica capillary column 30 m length and 0.25 mm inner diameter,packed with poly (ethylene glycol) (0.25 lm film thickness). Solu-tion of methyl ester (0.1 lL) in hexane containing approximately1% esters was injected under the following conditions: the carriergas was helium at a flow rate of 2 mL/min. The injector and detec-tor temperatures were 250 �C. Oven temperature started at 50 �Cfor 1 min, increased up to 250 �C at a rate of 5 �C/min and heldfor 10 min.

3. Results and discussion

3.1. XRD characterization of the different zeolitic supports

One of the most interesting aspect of zeolite gismondine is its ex-tremely flexible framework which is very sensitive to the severalfactors such as the nature of the extra-framework cations, the degreeof dehydration and chemical composition of its unit cell. The highestpossible symmetry of a gismondine type framework is a tetrahedralone with a space group I41/amd, and a theorical root system of sub-symmetries can be generated by successive removal of symmetryelements. A significant number of these subsymmetries has beenobserved in real minerals or zeolite like materials [32,39]. Analysisof the XRD patterns of the as made Na-P ion exchanged with Cu2+,Zn2+, and Ni2+ (Fig. 1) will show that the symmetries of Cu-P, Ni-Pand Zn-P samples have changed in comparison with the originalone of Na-P. Originally the crystallographic structure of Na-P wasrefined in the space group C2/c (a = 14.239 Å, b = 9.9836 Å, c =10.007 Å, b = 134.217�) as previously reported by Albert et al. [33].The complete X-ray characterization of the Cu-P, Zn-P and Ni-P sam-ples is still an ongoing work, but the preliminary indexation of theXRD pattern of Cu-P using TREOR program has resulted in a mono-clinic unit cell with approximated dimensions of a = 10.003 Å,b = 10.618 Å, c = 9.667 Å, and b = 93.35�. This unit cell dimensionsare very similar to the unit cell values of Mn-P, Ca-P, Sr-P, and Ba-Ppreviously reported in the literature [39]. The unit cell parametersfor Ni-P kept the same values of the Na-P (a = 14.239 Å, b =9.9836 Å, c = 10.007 Å, b = 134.217�) indicating that the incorpora-tion of nickel into the channels and cavities of Na-P was too smallto induce a significant modification of the structure as it was ob-served for Cu-P. Indexation of a reliable unit cell for Zn-P samplewas not possible due to the overlap of diffraction peaks. Elementalchemical analyses (ICP-AES) and TGA experiments (H2O) haveconfirmed the approximated chemical composition for each one of

following samples: Na-P(Na8Si8Al8O32�xH2O), Ni-P(Na6Ni0.6Si8-Al8O32�xH2O), Zn-P(Zn4Al8Si8O32�xH2O) and (Cu4Al8SiO8O32�xH2O).The chemical composition found for Ni-P and Zn-P are consistentwith the chemical composition expected for zeolites with GIS topol-ogy [40]. It is interesting to note that the SiO2/Al2O3 ratio (seeTable 1) of Na-P (�1.14), Ni-P (�1.05) and Zn-P (�1.10) have notchanged due to the ion exchange treatment, however the SiO2/Al2O3 ratio (�1.38) for the Cu-P is higher than the other ones. Be-cause of the high SiO2/Al2O3 ratio of Cu-P, it is expected a contractionof the unit cell compared to the other zeolites, since the Si-O bonddistance is shorter than the Al–O bond distances. One possible expla-nation for the increase in the SiO2/Al2O3 ratio could be related to thelow pH of the Cu-P ion exchange medium (pH 4.3), compared to theNi-P (pH 6.7), and Zn-P (pH 6.1) media observed during the ion ex-change experiments. Therefore, the higher pH observed for the Cu-P system was responsible for the effective Al framework extraction.The unusual high SiO2/Al2O3 ratio observed for the Cu-P has drawnour attention to the complexity of zeolitic systems enriched withtransition metals, such as Cu2+, and the possibility of partial reduc-tion of the cupric ions to cuprous ions during the thermal treatments[41–43]. This behavior has been reported in the literature for Yzeolites where the presence of Cu2+ and Cu+ has been probed usingroom temperature carbon dioxide, carbon monoxide, pyridine and2,6-dimethyl-pyridine physisorption and infrared spectroscopictechniques [44,45]. Although in this study physisorption and spec-troscopic experiments have not been performed in order to detectthe possible formation and existence of cuprous ions in the Cu-Psamples, the XRD and chemical analysis data give a clear evidencethat from the crystallographic point of view the Cu-P sample haskept its GIS framework. Thermal treatments performed at 200 �Con all samples aiming to eliminate the water molecules adsorbedon the zeolite surfaces have caused no significant effect on the crys-tallographic structures of Na-P, Cu-P, Zn-P and Ni-P as shown by XRDpatterns of these samples (Figs. 2–5). Neither the chemical composi-tions of these samples were affected after the thermal treatments at200 �C (see Table 1). However the same did not occur when the sam-ples were subjected to the thermal treatment at 550 �C. The originalNa-P structure at 550 �C (Fig. 2) was transformed into a dense irre-versible NaAlSiO4 phase identified as Nepheline (JCPDS-35-0424).This phase was classified as irreversible, because in contact withwater molecules, the structure did not return to the original Na-P.

This information is very important because during the enzymeimmobilization procedure, the zeolitic supports formed at 550 �C

Table 1Chemical compositions of the zeolitic supports by ICP-AES.

Sample SiO2 (%) Al2O3 (%) Na2O (%) ZnO (%) CuO (%) NiO (%) H2O (%)

Na-P/RT 35.90 31.37 17.43 – – – 16.0Cu-P/RT 24.02 17.40 0.08 – 31.69 – 27.0Zn-P/RT 26.93 24.49 1.10 27.70 – – 17.70Ni-P/RT 32.84 31.38 16.52 – – 2.39 16.90Na-P/200 �C 35.90 31.37 17.43 – – – –Cu-P/200 �C 24.02 17.40 0.08 – 31.69 – –Zn-P/200 �C 26.93 24.49 1.10 27.70 – – –Ni-P/200 �C 32.84 31.38 16.52 – – 2.39 –Na-P/550 �C 42.17 36.66 20.51 – – – –Cu-P/550 �C 26.36 19.93 0.03 – 37.02 – –Zn-P/550 �C 35.99 27.68 0.89 35.98 – – –Ni-P/550 �C 41.90 39.87 15.84 – – 2.33 –

Fig. 2. XRD patterns of the Na-P zeolite at different temperature range.

Fig. 3. XRD patterns of the Cu-P zeolite at different temperature range.

Fig. 4. XRD patterns of the Zn-P zeolite at different temperature range.

Fig. 5. XRD patterns of the Ni-P zeolite at different temperature range.

A. de Vasconcellos et al. / Microporous and Mesoporous Materials 163 (2012) 343–355 347

were put in contact with an aqueous solution, therefore in order tocorrelate parameters such as the amount of immobilized enzyme,enzymatic activity and zeolitic support structure, it was importantto be sure of the real structure of the solid support.

Cu-P and Zn-P samples treated at 550 �C (Fig. 3) were also con-verted into a completely amorphous and irreversible phases. In the

case of Cu-P, some of the reflections of the new phase could beattributed to cuprite (JCPDS-5-667). Some of the reflections ofthe new irreversible phase formed at 550 �C (Fig. 4) for Zn-P wereattributed to wurtzite (JCPDS-36-145). An interesting and unex-pected behavior has occurred with the Ni-P thermally treated at

348 A. de Vasconcellos et al. / Microporous and Mesoporous Materials 163 (2012) 343–355

550 �C, since there was no significant transformation to any irre-versible new phase; the material kept its original structure, andonly a slight loss of crystallinity was perceptible (Fig. 5). The factthat Ni-P was able to keep most of its crystallographic structureat such a high temperature compared to the other ion exchangedzeolites are still under investigation, but this unexpected and inter-esting result was able to provide us with a good model to study andcorrelate the amount of immobilized enzyme and enzymatic activ-ity to the structure of the inorganic solid support.

3.2. Effects of the thermal treatments of the zeolitic supports on theimmobilization and catalytic activities of the enzymes

Table 2 shows the effect of thermal treatments on the immobi-lization and catalytic activities of the R. miehei lipase immobilizedon Na-P, Cu-P, Zn-P, and Ni-P thermally treated at 200 and 550 �C.It can be noticed that the percentage of immobilized enzymes onthe Na-P/200 and Na-P/550 supports were practically the same(12% and 11.8%, respectively). Analysis of the XRD diffraction pat-terns for both samples (Fig. 2) reveals that both structures are quitedifferent. For the Cu-P system, the highest amount of immobilizedenzyme was observed for the Cu-P/550 (20%) compared to the 16%of the Cu-P/200 support. XRD data for both samples (Fig. 3) haveshown that at 550 �C, Cu-P was transformed into a completelyamorphous and irreversible structure, while Cu-P/200 kept theGIS framework. The same analysis can be extended to the Zn-Psamples. Zn-P/550 (Fig. 4) has a completely different structure incomparison Zn-P/200, however it was able to immobilize around19% of enzyme compared to the 12.5% of the Zn-P/200. Ni-P treatedat 200 and 550 �C practically immobilized the same amount of en-zymes (11.2% and 10.1%, respectively), but differently from theother zeolitic supports the crystallographic structure of Ni-P at200 and 550 �C are practically the same (Fig. 5).

Based on the present data, a straightforward correlation be-tween crystallographic structure and the amount of immobilizedenzyme can be made: all zeolitic supports which were able to re-tain the gismondine topology have immobilized similar amountenzyme (Na-P/200 � 12%, Cu-P/200 � 16%, Zn-P/200 � 12.5%, Ni-P/200 � 11%, Ni-P/550 � 10.1%). Table 2 shows also a clear correla-tion between the zeolitic support structures and the enzymaticactivity of the lipases. According to Table 2, the highest valuesfor enzymatic activity were observed also for the zeolite–enzymecomplexes have kept their original gismondine framework, as ob-served in the case of Na-P, Cu-P, Zn-P and Ni-P samples thermallytreated at 200 �C. The enzymatic activity measured for these fourdifferent complexes were the following: 4.8 (Na-P/200-ENZ), 12.6(Cu-P/200-ENZ), 9.0 (Zn-P/200-ENZ) and 26.6 (Ni-P/200-ENZ) U/mg-enzyme.

These values were sharply reduced for all the zeolites thermallytreated at 550 �C: 2.8 U/mg-enzyme (Na-P/550-ENZ); 2.6 U/mg-en-zyme (Cu-P/550-ENZ); 6.6 U/mg-enzyme (Zn-P/550-ENZ) and to13.7 U/mg-enzyme (Ni-P/550-ENZ). Based on these data attemptswere made in order to correlate the experimental results obtained

Table 2Percentage of immobilized enzyme on the different zeolitic supports and it respective enz

Zeolite Support thermal treatment (�C)

Na-P 200550

Cu-P 200550

Zn-P 200550

Ni-P 200550

for the enzymatic activity in function of the atomic radius of thedominant extra-framework cation present in the zeolitic supports.Fig. 6 shows that the bigger the atomic radius, smaller is the exper-imental enzymatic activity coefficient. Therefore, based on theseexperimental data (see Table 2), it is possible to state that in thecase of zeolitic supports with GIS framework, the amount of immo-bilized enzymes depends on the zeolitic support crystallographicstructure, and the enzymatic activity is closely related to the atom-ic radius of the dominant extra-framework cation of the zeoliticsupport. Part of the results observed in this study are in accordancewith other results reported in the literature. Yagiz et al. [19] havealso observed similar correlation between the amount of immobi-lized enzymes and the zeolitic support structures after performingseveral enzyme immobilization experiments on zeolitic supportswith different types of framework such as zeolites A (LTA)), Xand Y (FAU), mordenite (MOR), and ZSM-5 (MFI). On the otherhand, systematic studies elaborated by Serralha et al. [30] on thesame zeolites (A, X, Y and ZSM-5) did not show a straightforwardcorrelation between the structure of zeolitic support and theamount of immobilized enzymes, although they were able to cor-relate the enzymatic activities with the charge density of the ex-tra-framework cation. According to their conclusions, extra-framework cations with high charge density will tightly bound tothe water molecules, and as consequence the water and enzymaticactivities will be decreased [30]. Our results have lead us to a dif-ferent conclusion. In our system (zeolite P) the charge density ofthe extra-framework cations were increased in the following orderNa < Cu < Zn < Ni, however the highest value for enzymatic activitywere observed for Ni-P instead of Na-P, which is an indication thatthe enzymatic activity is more likely to be correlated with theatomic radius of the dominant extra-framework cation than withits charge density.

3.3. SEM and AFM characterization of the zeolitic supports

SEM and AFM images were taken to investigate the morphologyand topography of the as synthesized zeolite Na-P/200, Ni-P/200,and Ni-P/200-ENZ. SEM data show that the basic morphology ofthe zeolite Na-P/200 (Fig. 7a) was not modified neither by theion exchange with the nickel (Fig. 7b) nor by the immobilizationof the enzyme on the zeolite surface (Fig. 7c) and the typical mor-phology of crystalline low-silica Na-P was kept [46]. However, AFMimages (Fig. 8) have shown small imperfections on the zeolitic sup-ports surfaces due to the enzyme immobilization, and these imper-fections were reflected in the different degree of roughness eachone of the samples: Na-P (6.19 nm), Ni-P/200 (8.56 nm) and Ni-P/200-ENZ (13.94 nm).

3.4. Effect of the cation on enzymatic activity

It is well know that enzymes in the presence of certain cationscan have their enzymatic activity reduced, and also it is well estab-lished that heterogeneous catalysts such as zeolites can leach out

ymatic activity.

Immobilization (%) Enzymatic activity (U/mg enzyme)

12.0 ± 0.3 4.811.8 ± 0.3 2.8

16.0 ± 0.3 12.620.0 ± 0.4 2.6

12.5 ± 0.6 9.019.1 ± 0.4 6.6

11.2 ± 0.6 26.610.1 ± 0.6 13.7

Fig. 6. Enzymatic activity in function of the cation atomic radius.

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alkaline cations into the solution during the reaction. Even thoughchemical analyses (ICP-AES) for all the zeolitic supports treated at200 �C have shown that there was no leaching during the transe-sterification reactions, the influence of the Cu, Ni and Zn on theenzymatic activity of R. miehei was systematically tested.

Several studies in the literature have reported that each specificlipase enzyme will respond to the presence of Cu, Ni and Zn cationsin different manners. In some cases it will stimulate or inhibit theactivity of the enzyme. Riaz et al. [47] have studied the effects ofvarious metal ions in the lipase activity of Bacillus sp. FH5 and itwas observed that the presence of Cu2+, Zn2+, Ni2+ in a concentra-tion of 1 mM has caused a reduction of 20–30% of the enzyme’sactivity. Borkar et al. [48] have studied the hydrolysis of trioleinwith Pseudomonas aeruginosa SRT9, and their results have shownthat the presence of Cu2+ or Zn2+ decreased the relative activityof this enzyme around 37.3% in comparison to the original valuein a system free of these cations. Sharma and co-workers [49] havestudied the hydrolosis reaction of p-nitrophenyl butyrate (pNBP)using lipases Bacillius sp. RSJ-1 as catalyst, and in this specific casethe lipase enzymatic activity was slightly reduced (12%) in thepresence of Ni2+ (1 mM concentration), and strongly reduced inthe presence of 1 mM of Cu2+ and Zn2+ (56% and 47% of activityreduction, respectively).

In this study several enzymatic assays were performed in orderto check the effects of the Cu2+, Ni2+ and Zn2+ cations on the en-zyme activity. The activity of the free enzyme was measured forthe three different cations using a solution of copper, zinc and nick-

Fig. 7. SEM of the zeolitic support: (a) Na-P/

el in three different concentrations (0.1, 10 and 20 mM). Fig. 9shows the results of this control study. According to the data theenzymatic activity has decreased with the increase of the copperconcentration (Fig. 9a). The enzymatic activity obtained at concen-trations (1, 10 and 20 mM) were (93.8%, 75.3% and 36.9%, respec-tively) compared to initial activity (100%). These data are a clearindication that the Cu2+ cations can be a strong inhibitor for theR. miehei lipase at high concentration (20 mM), however at a lowerconcentration (1 mM) the effects of Cu2+ cations are not so relevantin terms of enzymatic activity reduction (6.2%). Enzymatic activitydata for Zn2+ cations at three different concentrations (1, 10 and20 mM), have shown that there was a small decrease in enzymeactivity since the final value were 87.8%, 85.5%, and 87.4% com-pared to the initial measured activity in the absence of this cationin solution (100%) (Fig. 9b). This small decreasing of the enzymaticactivity indicates that the Zn2+ cations are not a strong inhibitor ofthe enzyme under investigation. The enzyme activity in the pres-ence of Ni2+ cations were also measured at three different concen-trations (1, 10 and 20 mM), and the decrease of the enzyme activitywas also very low (95.0%, 93.8%, and 88.0%, respectively). It isworth to notice that even at a considerable high concentration ofNi2+ (20 mM), the total activity loss was only 12% of initial activity(Fig. 9c). These data suggest that the enzyme is slightly inhibitedby the presence of cation nickel in solution. Therefore, the finalconclusion of these enzymatic assays lead us to conclude that evenin case of Ni2+ and Zn2+ leaching during the immobilization pro-cess, the presence of these cations would not significantly reducethe efficiency of the enzymatic catalyst, because even at a highconcentration (20 mM), the presence of these cations in solutionhave caused very little interference in the enzymatic activityexperimentally determined.

3.5. Effect of the immobilization on the optimal temperature andthermal stability of the enzymes

Enzymes have an optimum temperature in which they exhibittheir optimum catalytic activity. In this study, enzymatic activitiestests were carried out at temperatures ranging from 32 to 53 �C forthe free enzyme R. miehei and its optimal temperature was foundat 44 �C (Fig. 10a). Increasing the temperature above the optimumtemperature (44 �C) will cause a sharp decrease of enzyme activitywhich can be attributed to the deactivation and denaturation of theenzyme. At 46 �C there was a decrease of 10% of the enzyme’sactivity and at 53 �C a swift denaturation of the enzyme has oc-curred and the enzyme’s activity was reduced in 80%. These resultsare consistent with previous results reported in the literature forthe R. miehei’s optimum temperature, which was found to bein the temperature range of 30–50 �C at a pH of 8.0 [50]. The

200, (b) Ni-P/200 and (c) Ni-P/200-ENZ.

Fig. 8. AFM and 3D surface images of the zeolitic supports (a and d) Na-P/200; (b and e) Ni-P/200 and (c and f) Ni-P/200/ENZ.

Fig. 9. Enzymatic inhibition of the free enzyme in the presence of different cations:(a) copper (N), (b) zinc (d) and (c) nickel (j).

Fig. 10. Effect of temperature on the enzymatic activity. (a) Rhizomucor mieheilipase free (d) and immobilized on different zeolitic supports: (b) Na-P (j), (c) Cu-P(N), (d) Zn-P (s) and (e) Ni-P (D).

350 A. de Vasconcellos et al. / Microporous and Mesoporous Materials 163 (2012) 343–355

catalytic activities of the enzyme were also measured in theirimmobilized forms in order to learn if the enzyme immobilizationon zeolitic supports would affect the original enzyme’s optimumtemperature.

Thermophilicity assays for the zeolite–enzyme complexes(Na-P/200-ENZ, Cu-P/200-ENZ, Zn-P/200-ENZ, Ni-P/200-ENZ) weremeasured in 37–52 �C temperature range and the results weresimilar to the ones obtained for the free enzyme. Therefore,the R. miehei immobilization on the different zeolitic supportshad no effect in the enzyme’s thermophilicity (see Fig. 10b–e),and these results are in accordance with other previous resultsreported in the literature [29,51,52].

Gonçalves et al. [29] have studied the immobilization of cutin-ase on two different solid supports (NaY zeolite and the polyamideAccurel PA6) have also observed that the maximal optimal temper-ature were the same for both solid supports (30 �C). Dizge and co-workers [53] have investigated the immobilization of lipase fromThermomyces lanuginosus onto styrene-divinyl bezene-polyglutar-aldehyde (STY-DVB-PGA) copolymer and have also observed thatthe enzyme immobilization on this polymeric support did not af-fect thermophilicity of T. lanuginosus enzyme. On the other hand,Morana et al. [51] have reported that the immobilization of BetaXylosidase on Alginate has caused a temperature a small shift from

A. de Vasconcellos et al. / Microporous and Mesoporous Materials 163 (2012) 343–355 351

85 �C (optimal temperature for free enzyme) to 90 �C for the poly-mer–enzyme complex.

In order to know how if the thermal stability of the R. miehei li-pase would be affected or improved by its immobilization on thedifferent zeolitic supports, several experiments were performedfor both the free enzyme and the zeolite–enzyme complexes at44 �C which is the optimal temperature experimentally found forthe R. miehei lipase in this investigation. The experiments wereperformed at six different time intervals (0, 60, 120, 180, 240 and300 min) for free enzyme and at four different time intervals (0,60, 80, 180 and 300 min) for the following zeolite–enzymes com-plexes: Na-P/200-ENZ, Cu-P/200-ENZ, Zn-P/200-ENZ, and Ni-P/200-ENZ.

Fig. 11a shows the thermal stability profile for the free enzyme.It can be noticed that after 60 min the activity has decreaseapproximately 40% and then it has remained constant until theend of the experiment, 300 min later. This sharp decrease of theenzyme’s activity can be attributed to the disruption of the non-covalent interactions of the enzyme, which are responsible for itsstability in the native conformation [53].

Fig. 11b–e shows the thermal deactivation rate experimentallymeasured at 44 �C for Na-P/200-ENZ, Cu-P/200-ENZ, Zn-P/200-ENZand Ni-P/200-ENZ zeolite–enzyme complexes. All the zeolite–en-zyme complexes have showed the same behavior by retainingapproximately 100% of their initial activities until the end of theexperiments. It means that the immobilization has increased thethermal stability of the enzyme compared to its thermal stabilityin the free form. These results are in agreements with several otherstudies reported in the literature [29,54,55].

3.6. Soybean oil transesterification

The transesterification reactions of soybean oil to biodiesel cat-alyzed by free enzyme, pure zeolites and the zeolite–enzyme com-plexes were followed by TLC (thin layer chromatography) and fullyanalyzed by GC (gas chromatography). Analyses by TLC of the reac-tion products catalyzed by the free enzyme have shown that themajority of soybean oil was converted to biodiesel using anamount of free enzyme equivalent to 64.45 U (Fig. 12a, inset II).The unit of enzymatic activity U was defined in section 2.6 (Eq.2). Reactions with 9.02 U of free enzyme (amount of the immobi-

Fig. 11. Enzymatic stability at 44 �C. (a) Rhizomucor miehei lipase free (d) andimmobilized on different zeolitic supports: (b) Na-P (j), (c) Cu-P (N), (d) Zn-P (s)and (e) Ni-P (D).

lized enzyme onto the Zn-P zeolite) have resulted in a small con-version of soybean oil to biodiesel (Fig. 12b, inset III). Reactionscatalyzed with 7.22 U of free enzyme (amount of the immobilizedenzyme onto the Ni-P zeolite) have converted only a small amountof soybean oil to biodiesel (Fig. 12b, inset IV). According to the GCanalyses the reactions catalyzed with 64.45 U of free enzyme haveresulted in 95% methyl esters conversion after 72 h, while the reac-tions catalyzed with 9.02 U of free enzyme have resulted in 42.5%of methyl esters conversion under the same conditions, and thereactions catalyzed with 7.22 U of free enzyme have resulted in39.3% conversion of methyl esters (Fig. 13).

TLC analyses of the reactions catalyzed by Na-P/200, Na-P/200-ENZ and Na-P/550-ENZ have indicated that no significant conver-sion of soybean oil to biodiesel (Fig. 12c, insets V–VII) have occurred.The same results were observed for pure Cu-P/200, Cu-P/200-ENZ,and Cu-P/550-ENZ catalysts (Fig. 12d, insets VIII–X). Shortly, Na-Pand Cu-P and their zeolites-complexes derivatives were not effi-cient catalysts for the transesterification reaction of vegetable oilsto biodiesel. The products of these reactions catalyzed by these zeo-lite–enzyme complexes were not analyzed by GC, due to the verylow yield of methyl esters as revealed by the TLC data.

Qualitative TLC analyses of the reactions catalyzed with Zn-P/200 have indicated that only small fractions of the vegetable oilwere converted to biodiesel (Fig. 12e, inset XIII). However, Zn-P/200-ENZ and Zn-P/550-ENZ complexes were able to convert a largeamount of the vegetable oil to biodiesel (Fig. 12e, inset XI–XII).Quantification of the methyl esters (GC analyses) formed by thesethree catalysts are shown in Fig. 14. Pure Zn-P/200 yielded a lowamount of methyl esters (20.3%), while the enzyme in its free form(9.02 U) has yielded of 42.5% methyl esters. Zn-P/200-ENZ and Zn-P/550-ENZ complexes have yielded 39.7% and 29.6% of methyl es-ters, respectively.

TLC analyses (Fig. 12f – insets XIV–XVI) of the reactions cata-lyzed by Ni-P/200-ENZ, Ni-P/550-ENZ and Ni-P/200, have indicatedthat Ni-P/200 was able to catalyze a low amount of soybean oil tobiodiesel (Fig. 12f – inset XVI), which were equivalent to a 20% ofmethyl esters yield according to the GC analyses. Ni-P/200-ENZcomplex was able to convert a large amount of soybean oil to bio-diesel (Fig. 12f – inset XIV) which were equivalent to 56.2% ofmethyl esters conversion, according to GC analysis. However, reac-tions catalyzed by Ni-P/550-ENZ complex have yielded an averageconversion of 27.6% of methyl esters according to CG analysis(Fig. 14). These results clearly indicate a very interesting synergyfor this particular system, because the amount of methyl esterformed by the Ni-P/200-ENZ complex (56.2%) is higher than themethyl esters amount formed by the pure enzyme (39%), pure zeo-lite Ni-P/200 (20%) and the Ni-P/500-ENZ (27.6%).

3.7. ATR–FTIR analyses of the immobilized enzyme structure infunction of the different zeolitic supports

In order to find one possible and plausible explanation for thesynergistic effect observed in this study for the Ni-P/200-ENZ com-plex, but not for the other zeolitic supports attempts were made tocorrelate enzyme structural features and the zeolitic supports afterthe immobilization of the enzyme.

The crystallographic structures of several lipases were eluci-dated by several authors [56–59], and in general all the lipaseshave an ab hydrolase fold structure with a catalytic triad similarto the one found in serine proteases. The active site is generallyburied under a lid or flap, containing an amphiphilic a-helix, whichmakes the active site inaccessible to the substrate in the so-calledclosed conformation. Opening the lid exposes a large hydrophobicsurface to the substract, whereas the previously exposed hydro-philic domain becomes buried inside the protein. In the case of R.miehei lipase the catalytic site is formed by a triad of three residual

Fig. 12. Qualitative analysis by TLC of the methyl esters formed using the different catalysts. (a) pure soybean oil (I). Soluble catalyst: free enzyme (64.45 U) (II), (b) freeenzyme (9.02 U) (III) and free enzyme (7.22 U) (IV). Solid catalysts: (c) Na-P/200-ENZ (V), Na-P/550-ENZ (VI) and Na-P/200 (VII), (d) Cu-P/200-ENZ (VIII), Cu-P/550-ENZ (IX)and Cu-P/200 (X); (e) Zn-P/200-ENZ (XI), Zn-P/550-ENZ (XII) and Zn-P/200 (XIII); and (f) Ni-P/200-ENZ (XIV), Ni-P/550-ENZ (XV) and Ni-P/200 (XVI).

Fig. 13. Percentage of methyl esters produced with different amounts of freeenzymes.

Fig. 14. Percentage of methyl esters produced with different catalysts.

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amino acids (His257, Asp203 and Ser144) and it is concealed undera short amphipatic helix. This amphipatic helix acts as ‘‘lid’’, open-ing the active site when the enzyme is adsorbed at the oil–waterinterface [60].

The main possible interactions involved in the adsorption ofproteins on solid surfaces are hydrophobic and electrostatic inter-actions and hydrogen bonds [61]. In order to understand the inter-actions of the immobilized enzymes onto the zeolitic supports,ATR–FTIR study of the enzyme–zeolites complexes were per-

A. de Vasconcellos et al. / Microporous and Mesoporous Materials 163 (2012) 343–355 353

formed, since this technique has been extensively used to examinethe structure of proteins in solution, and also the structural fea-tures of adsorbed proteins on solids [62–64].

Fig. 15 shows the ATR–FTIR spectra of the free enzyme in solu-tion and enzyme–zeolite complexes (solid supports). In general theinterpretation of infrared spectroscopy data of zeolites or alumino-silicates can be divide in two distinct groups; the skeletal IR spec-tra (1600–500 cm�1) and the surface hydroxyl group (4000–1500 cm�1) [65]. In order to study a possible correlation betweenthe amount of immobilized enzymes and enzymatic activity infunction of the zeolitic support structure, a careful analysis of thefrequencies that occur in this region (1600–500 cm�1) wereperformed. Analyses of the ATR- FTIR spectra of the Na-P, Cu-P,Zn-P, and Ni-P zeolites in the skeletal region (1600–500 cm�1)have revealed that Na-P and Ni-P basically have kept the samestructural features. This observation is in agreement with theXRD data (see Figs. 2b and 5b). All the observed frequencies(1641, 940, 739, 657, 598, 454 cm�1) in this region are in agree-ment with the previous results reported by Albert et al. [33].However a different spectra is observed for Cu-P and Zn-P in theskeletal range (1600–500 cm�1). This is a clear indication thatthe ion exchange treatments strongly have affected the originalframework of the original Na-P and these observations are alsosupported by the XRD data (see Figs. 3b and 4b).

These observations again reinforce the conclusion that astraightforward correlation between the amount of immobilizedenzymes and the zeolitic support structure can be made, sincethe amount of immobilized enzymes are almost the same for allzeolitic support treated at 200 �C (see Table 2). Therefore a possibleexplanation for the synergistic effect observed for Ni-P/200-ENZcomplex compared to the other supports is probably related tothe ability of the enzyme to maintain the functionality of its cata-lytic site after the immobilization, specially the stabilization of itssecondary structure.

ATR–FTIR can provide useful insights into the secondary struc-ture changes of the immobilized enzymes. FT-IR spectra of proteinshave some ‘‘finger print’’ in the region 1500–1800 cm�1. This spec-tra region covers three majors group of infrared absorption bands.One is the amide I band (C@O stretch weakly coupled with C–N

Fig. 15. ATR-FTIR spectra of the free and immobilized Rh

stretch and the N–H bending) located in the 1610–1700 cm�1 re-gion. Amide II band (C–N stretch strongly coupled with the N–Hbending) is found in the region 1530–1550 cm�1. Amide III (N–Hin plane bending coupled to C–N stretching; C–H and N–H defor-mation) occurs between 1350 and 1200 cm�1 [66–68].

The ATR–FTIR spectrum of the pure R. miehei in solution isshown in Fig. 15. It shows the maximum of the amide I band occursat 1641 cm�1. This is typical of protein with high b-sheet content,although bands in the region 1630 to 1638 cm�1 region could alsobe overlapped by the b-turns structures [69,70].

Analyses of the ATR–FTIR for the all the enzyme–zeolite com-plexes show that the adsorbed R. miehei has kept most of its b-sheet structure only for Ni-P/200/ENZ complex. The fact that thereis no difference in the positions, bandwidths, intensity and area ra-tio of the amide I, strongly suggest that when the lipase is adsorbedonto the Ni-P zeolite surface, it did not change its main structuralcomponent: the b-sheet structure. However, the same did not oc-cur for Zn-P and Cu-P zeolites. In this case there are changes inthe position, bandwidths and intensity of the adsorption band re-lated to the b-sheet structure (see inset in Fig. 15).

As stated before, the interactions involving the adsorption ofproteins on solid surfaces are only possible through hydrophobic,hydrogen bonds and electrostatic interactions. Herrgard et al.[71] have identified a functionally important electrostatic networkwhich includes the amino acids residues S144, D203, H257, Y260,H143, Y28, R80, and D91 (amino acid residue numbering is fromRmL, according to Ref. [65]). This electrostatic network consistsof residues belonging to the catalytic triad (S144, D203, H257), ofresidues located in proximity to the active site (Y260), and of res-idues which stabilize the geometry of the active site (Y28, H143),and of residues located in the lid (D91) or close to the first hinge(R80). According to their studies [71] the lid (D91) and the firsthinge (D80) are associated with the interfacial activation of lipases,which occurs when an a-helical lid opens up by rotating aroundtwo hinge regions, while the stabilization of the enzyme in its openconformation is related to the site–site electrostatic interactionenergies between the residues R86 and D61, D113 and E117.

Based on the electrostatic model proposed by Herrgard et al.[71], Macario et al. [31] have performed a set of experiments on

izomucor miehei lipase on different zeolitic supports.

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zeolite/enzyme immobilization, and they have proposed two pos-sible interaction mechanisms between the enzyme and the zeoliticsupport. The first one was based on the electrostatic interactionforces between any aminoacid constituent of the enzyme and thezeolitic support prepared in fluoride (F�) media; the second isbased on an acid–base binding force between the weak acid silanolgroups of the zeolitic support (prepared in OH� media) and thespecific basic aminoacid Arginine (R86). Their experimental resultscould be better explained using the acid–base binding force be-tween the weak silanol groups of the zeolitic support and the basicamino-acid Arginine [R86], which is a constituent of the hide regionof the enzyme lid [31].

We tried to explain the correlation between the immobilizationof the enzymes onto the zeolitic supports and their respectiveenzymatic activities (Table 2) using both models proposed by Mac-ario et al. [31]. Cu-P/200 has immobilized a relatively high amountof enzyme (16%) in comparison to the other zeolitic supports, butits final enzymatic activity was very low (12.6 U/mg-enzyme).Zn-P/200-ENZ and Na-P/200-ENZ complexes had also similarbehaviors (see Table 2). According to these data, it is reasonableto assume that during the immobilization process, the weak acidsilanol groups (Si–OH) located in the external surface of the zeo-lites were able to react with any residual basic amino acid, butnot specifically with basic residue Arginine (R86) as proposed byMacario et al. [31]. Therefore the enzyme is not immobilized inits open and active form. On the other hand, there is a nice corre-lation between the data presented in Table 2 and the acid-bindingmodel for the Ni-P/200 support. Although, the amount of immobi-lized enzyme is relatively low (11.2%) compared to the value re-ported for CuP/200 (16%) and similar to the values reported forNa-P/200 and Zn-P/ENZ (12% and 12.5%, respectively), the valueof enzymatic activity (26.6 U/mg) is far superior to the values ob-served for the other zeolitic supports. It means that during theimmobilization process, the weak acid silanol groups (Si–OH) lo-cated on the zeolite surface [72,73] were able to react with the ba-sic residue basic amino acid of the arginine (R86) and the enzymeimmobilized in its open and active form. The ATR–FTIR data show-ing the preservation of the b-sheet structure of the enzyme immo-bilized onto Ni-P and the high yield of methyl esters obtained withthis heterogeneous biocatalyst support this conclusion.

4. Conclusions

Gismondine zeolite (Na-P) ion exchanged with Ni, Cu, and Znwere used as solid supports for immobilization of the enzyme R.miehei. The zeolite–enzyme complexes were used as catalysts forthe transesterification reaction of soybean oil to biodiesel. Zeo-lite–enzyme complexes prepared with Ni-P were efficient catalystsfor this specific reaction and a synergistic effect was observed forthis zeolite–enzyme complex. This synergistic effect is probablydue to the fact that the immobilized enzyme was able to rearrangeits catalytic center in its active form onto the Ni-P zeolitic support.To the best of our knowledge this is the first study that shows a sig-nificant synergistic effect involving a specific zeolite support (Ni-P)and the R. miehei enzyme.

Acknowledgments

This work has been supported by The State of São PauloResearch Foundation (FAPESP) under Program The YoungInvestigator Award 05/54703-6 (J.G.N.) and Award 08/56973-9.We also thank CAPES and the National Council for Scientific andTechnological Development (CNPq) for financial support underthe Awards 07/478104-3 and 558880-2010-0.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.micromeso.2012.07.043.

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