Study of lipase immobilization on zeolitic support and

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ORIGINAL ARTICLE

Study of lipase immobilization on zeolitic support and transesterification reaction in a solvent free-system Anastasia MacArio , Girolamo Giordano, Leonardo Setti , Attilio Parise, Juan M. Campelo, José M. Marinas & Diego Luna ... show less Pages 328-335 | Published online: 11 Jul 2009

Department of Organic Chemistry, University of  Download citation  http://dx.doi.org/10.1080/10242420701444256 Cordoba, Cordoba, Spain

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Abstract In order to understand the role of the acid–base, electrostatic and covalent interactions between enzyme and support, the catalytic behavior of the Rhizomucor miehei lipase (RML) immobilized on zeolite materials has been studied. The highest lipase activities were obtained when this enzyme, immobilized by adsorption, interacts through acid–base binding forces with In this article

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the support surface, resulting in activation of the enzyme catalytic center. http://www.tandfonline.com/doi/full/10.1080/10242420701444256?scroll=top&needAccess=true

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the support surface, resulting in activation of the enzyme catalytic center.


Due to the interest in biodiesel production by mild enzymatic transesteri cation, this heterogeneous biocatalyst has been used in transesteri cation of fatty acids contained in olive oil. The results show a high oleic acid conversion for several reaction cycles with a higher total biodiesel productivity compared to that using the free enzyme. Keywords:Lipase, zeolites, adsorption, covalent binding, biodiesel, transesteri cation

Introduction Conventional biodiesel technology involves the use of inorganic homogeneous basic catalysts (sodium hydroxide, potassium hydroxide, and sodium or potassium methoxide) and methanol. The main disadvantage of a homogeneous basic catalyst is the presence of saponi cation reactions during the transesteri cation process, which reduces biodiesel production e ciency. With a high free fatty acid content in the oil, soap formation can be avoided by using acid catalysis, hence the biodiesel yield increases. Nevertheless, acid-catalyzed transesteri cation is much slower than the base-catalyzed reaction, and needs more extreme temperature and pressure conditions (Freedman et al.  1984; Schwab et al.  1987). In contrast, enzymatic transesteri cation avoids soap formation, involves neutral pH, and is less energy consuming. Using an immobilized enzyme, it is possible to combine the advantages of enzymatic transesteri cation with those of heterogeneous catalysis. There are several methods for the enzyme immobilization: adsorption, covalent binding, cross-linking and containment behind a barrier (e.g. micro-encapsulation, entrapment and con nement). The zeolites and related materials show potentially interesting properties which are easy to modulate, such as hydrophobic/hydrophilic behavior, acid/base character, mechanical and chemical resistance, crystal morphology and size, external and total surface area and pore diameter. Their easy water dispersion/recuperation represents an additional advantage. Therefore, the compositional and structural variety of molecular http://www.tandfonline.com/doi/full/10.1080/10242420701444256?scroll=top&needAccess=true

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advantage. Therefore, the compositional and structural variety of molecular


sieves o ers a powerful tool for tuning the carrier adsorption properties. Recent studies have demonstrated that lipases adsorbed on hydrophobic supports exhibit a clear hyper-activation compared to the soluble enzyme (Fernandez-Lafuente et al.  1998; Palomo et al.  2002) due to interfacial activation. This interfacial activation is caused by a conformational change resulting from adsorption. From X-ray crystallographic studies (Brady et al.  1990; Derewenda et al.  1992), the crystal and molecular structure of a triacylglycerol lipase has been elucidated. This suggests that the most suitable immobilization method for this enzyme has to involve the “lid” region in order to activate its catalytic center. Immobilization by adsorption enables contact between the enzyme and support without using intermediate molecules. Consequently, if the support surface has physicochemical properties that allow enzyme lid opening, the adsorbed lipase could have an “open” conformation. In covalent attachment, the “bridgemolecule” used to functionalize the support, could interact with any amino acid constituent of the lipase without allowing “interfacial activation”. However, covalent attachment leads to more stable enzyme immobilization than that obtained by adsorption. There have been several studies of the lipase immobilization on di erent kinds of support, including zeolites (HugeJensen et al.  1988; Gonçalves et al.  1996; Oliveira et al.  1997; de Fuente et al.  2001; Ikeda et al.  2001; Charusheela et al.  2002; Grazu et at.  2005), but no data are available concerning the Rhizomucor miehei lipase (RML) immobilization on zeolite. The aim of this work was to study the e ects of acid–base, electrostatic and covalent interactions on the conformation of immobilized RML. Silicalite-1 obtained by di erent synthesis routes and delaminated zeolite ITQ-2 have been prepared as lipase-supports. In order to evaluate the catalytic behavior of these heterogeneous biocatalysts, ester hydrolysis and transesteri cation reactions have been chosen as catalytic tests. Finally, the results have been compared with those obtained using the free enzyme and the lipase http://www.tandfonline.com/doi/full/10.1080/10242420701444256?scroll=top&needAccess=true

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compared with those obtained using the free enzyme and the lipase


covalently attached to the functionalized sepiolite/AlPO4 (80–20 wt%) material.

Experimental Materials The enzyme used was PALATASE 20000L (Novo), a puri ed 1,3-speci c lipase from RML (EC 3.1.1.3), produced by submerged fermentation of a genetically modi ed Aspergillus oryzae. Other reagents used were commercially available from Sigma Chemical Co. The chemicals used for the preparation of the supports were: precipitated silica gel, tetrapropylammonium bromide (TPABr), sodium hydroxide or sodium uoride and distilled water. The reagents used for the esters hydrolysis reaction were myristic acid (98%), methyl myristate (99%), copper acetate, acetone and toluene. The reagents used for the transesteri cation reaction were olive oil from Calabria, Italy (76% wt/wt in oleic acid), anhydrous methanol (99.9%), and n-hexane (95%).

Synthesis of the supports Silicalite-1 type zeolites with di erent physico-chemical surface properties were prepared in alkaline and uorine systems as described in the literature (Hayhurst et al.  1988; Patarin et al.  1989). Professor Avelino Corma, from Instituto de Tecnología Química (Universidad Politécnica de Valencia, Spain), kindly supplied the delaminated zeolite ITQ-2. The sepiolite/AlPO4 material was prepared according to the published procedure (Bautista et al.  1998).

Enzyme immobilization The RML enzyme and the calcined support (weight ratio: free enzyme/support equal to 2.5) were mixed in 50 mL of 0.2 M phosphate http://www.tandfonline.com/doi/full/10.1080/10242420701444256?scroll=top&needAccess=true

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enzyme/support equal to 2.5) were mixed in 50 mL of 0.2 M phosphate


bu er pH 7, and stirred at 250 rpm for 24 h at 0°C. The support with immobilized lipase was separated by ltration, washed twice with deionized water and dried at 25°C overnight. The total protein concentration of the initial solution and the supernatant was calculated using UV absorption at 280 nm (Peterson  1987). The calibration curve, determined on a PerkinElmer UV spectrophotometer, was obtained using bovine serum albumin (BSA) as a standard. A linear regression equation was applied to the data: Concentration = 2.2433*Absorbance, with a correlation coe cient of 0.999. The amount of protein adsorbed on the support (WIP, (mg)) was determined from the following mass balance: WIp=C 0V0−C fVf, where C 0 is the initial protein concentration (mg mL−1); V0 is the volume of the initial solution (mL); C f and Vf are the protein concentration (mg mL−1) and the volume (mL) of the supernatant, respectively. The covalent attachment of lipase on functionalized sepiolite/AlPO4 was carried out following the published procedure (Bautista et al.  1998).

Catalytic test For the hydrolysis of methyl myristate to myristic acid, pre-determined amounts of the immobilized (adsorbed and covalently attached) lipasesupport, was added to 5 mL of 0.2 M phosphate bu er pH 7 containing 0.25 mL of 0.7 M methyl myristate in acetone. The mixture was stirred (250 rpm) at 37°C in a thermostatic bath and the amount of myristic acid produced was determined colorimetrically (Lowry et al.  1976). For the transesteri cation reaction, the parameters used, including temperature, water and methanol content, were those suggested in previous studies as optimal conditions (Ma et al.  1999; Soumanou et al.  2003; Salis et al.  2005). In particular, the molar ratio of olive oil:methanol of 1:5 was used in order to have a high reaction rate and to minimize di usion limitations without inducing enzyme inhibition. At every sampling point, 10 mL of n-hexane was added to the reaction to extract the esters, and the http://www.tandfonline.com/doi/full/10.1080/10242420701444256?scroll=top&needAccess=true

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mL of n-hexane was added to the reaction to extract the esters, and the


mixture was separated by centrifugation. The recovered catalyst was washed with n-hexane, dried at room temperature and stored at 0°C until subsequent use. The products were analyzed by gas chromatography using an Agilent 6890 GC with a Supelcowax 10 column. For quantitative analysis, methyl pentadecanoate was used as standard ester.

Regeneration of the support The support containing the used biocatalyst could be easily regenerated by a simple thermal treatment at 380°C for 5 h. After this regeneration process, thermal analysis on the regenerated support was carried out on a Netzsch STA 409 instrument, in order to verify the complete absence of organic compounds.

Results and discussion Characteristics of the supports Silicalite-1 synthesis was performed in alkaline or uorine media, in order to obtain supports with di erent physico-chemical surface properties. Zeolites synthesized in alkaline systems usually have a high number of silanol groups (≡SiOH) named defect groups (Bordiga et al.  2001), which possess moderate Brönsted acidity (Louis et al.  2004). In contrast, Silicalite-1 synthesized in uorine media are relatively defect-free (Camblor et al.  1999), and the uorine ions remain in small cages of the MFI structure even after the calcination process (Fyfe et al.  2001).

29Si-NMR

analysis

carried out on samples S1 and F-S2 con rmed the presence of silanol groups solely on the S1 support surface. In order to characterize these silanol groups, FT-IR analysis was carried out and showed the presence of terminal silanols, exclusively. The delaminated zeolites (as well as the ITQ-2) were produced from lamellar 6/18

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The delaminated zeolites (as well as the ITQ-2) were produced from lamellar precursor zeolites giving a completely hydroxylated and well-ordered external surface (Corma et al.  1999). The FT-IR spectrum of the ITQ-2 sample used in this work clearly showed the presence of exclusively terminal silanol groups on the surface (Onida et al.  2003). The three kinds of support used for lipase adsorption were characterized by the presence of terminal silanols for Silicalite-1 materials synthesized in the alkaline system and the delaminated zeolite ITQ-2, and by the complete absence of ≡SiOH groups on the surface of Silicalite-1 synthesized in uorine media. For the latter material, the uoride ions on the support surface (con rmed by atomic absorption: 1.63% wt of F−) produced a strong negative charge. The silanol groups present on the S1 and ITQ-2 surface had weak Brönsted acidity, stemming from the NH3-TPD analysis carried out on both samples (NH3 desorption temperature >330°C), in agreement with the literature (Corma et al.  1999; Onida et al.  2003; Louis et al.  2004). Finally, the sepiolite/AlPO4 materials were functionalized by –OH surface nucleophilic substitution (Bautista et al.  1996).

Lipase immobilization Enzyme adsorption on the di erent supports is summarized in Table I. The highest enzyme amount was retained on the F-S2 type support (69%), which employs electrostatic binding forces between the enzyme and support. In particular, the residual negative charge of the uoride media on the external support surface can interact with the positive charge of the enzyme amino groups (Scheme 1a). In contrast, the defect groups present on the support surface of the S1 and ITQ-2 materials result in retention of much less enzyme (39 and 27%, respectively). We presume that for these materials, the acid silanol groups can interact with the amino- or carboxyl groups of the enzyme, according to Scheme 1b. Scheme 1. Probable enzyme support interactions: (a) electrostatic binding for F-S2 support; (b) acid-base binding for S1 and ITQ-2 supports. http://www.tandfonline.com/doi/full/10.1080/10242420701444256?scroll=top&needAccess=true

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for F-S2 support; (b) acid-base binding for S1 and ITQ-2 supports.


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Table I. Enzyme adsorption results. CSV

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Covalent immobilization resulted in 99.99% of the enzyme amount being retained on the functionalized sepiolite/AlPO4, equal to the 433 mg of enzyme per gram of the support. In this case, the amino groups of the enzyme are chemically bonded with the activated support surface by a –CH = N– bond (Bautista et al.  1996).

Screening of the support for lipase immobilization: esters hydrolysis The amino acids present in the hinge region of the lipase lid have an important role, both in enzyme activation and in stabilization of the immobilized open enzyme form. Arginine (R86), a basic amino acid, is the key amino acid in the hinge region (Herrgard et al.  2000). Consequently, the involvement of R86 of the RML could play a very important role in the immobilization of this enzyme. Thus, the catalytic behavior of the immobilized enzyme could be signi cantly a ected by the di erent types of binding forces involved.

The hydrolysis of esters using the heterogeneous biocatalysts prepared by adsorption, showed that the highest methyl myristate conversion was http://www.tandfonline.com/doi/full/10.1080/10242420701444256?scroll=top&needAccess=true

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adsorption, showed that the highest methyl myristate conversion was


obtained for lipase immobilized on supports with silanol groups on their surface, such as Silicalite-1 prepared in the alkaline system (S1) and delaminated zeolite (ITQ-2) (Figure 1). Very low conversion was achieved for the enzyme adsorbed on the support prepared in F-media (F-S2). The maximum alkyl ester conversion obtained with the supported biocatalyst was 94% with the lipase/S1 catalyst, and 86% with the lipase/ITQ-2 catalyst, after 70 h of reaction time. The di erence between the lipase/S1 and lipase/ITQ-2 catalysts is attributable to di erent enzyme loadings (see Table I). Thus, although the highest enzyme loading was by covalent attachment on sepiolite/AlPO4 (433 mg of enzyme per gram of support), this only gave low methyl myristate conversion (52%). Figure 1. Methyl myristate conversion. Reaction conditions: 70 h at 37°C, catalyst amount = 0.4 g (enzyme content for each catalysts: 53 mg for lipase/ITQ-2, 60 mg for lipase/S1, 116 mg for lipase/F-S2, 173 mg for lipase/sepiolite/AlPO4).

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These results suggest that di erent interactions between lipase and support strongly in uence the nal enzyme conformation. Even though the S1 and ITQ-2 supports did not adsorb the highest amount of protein, the conversion yield values obtained were the highest. The very low activity of the lipase/F-S2 catalyst (290 mg of enzyme per grams of the support) may be http://www.tandfonline.com/doi/full/10.1080/10242420701444256?scroll=top&needAccess=true

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the lipase/F-S2 catalyst (290 mg of enzyme per grams of the support) may be


due to the fact that the high electrostatic interactions between enzyme and the support surface do not involve the amino acids of the enzyme lid. The same may be true of covalent attachment. In both cases, the enzyme may be bound in its closed form. In contrast, the acid silanol groups present on the surface of the support obtained in the alkaline system or in the delaminated zeolite can selectively interact with the basic arginine constituent of the hinge region of the lid. These interactions allow the opening of the lid and, hence, activation of the catalytic center. The results reported in Figure 2 clearly show two di erent catalytic behaviors. Lipase/S1 and lipase/ITQ-2 catalysts show decreasing extents of conversion during the reaction cycles. In contrast, the lipase/sepiolite-AlPO4 catalyst shows the same conversion value, even after four reaction cycles. This suggests that the lipase is desorbed from the S1 and ITQ-2 supports during operation. Consequently, the conversion decreases from 94 to 60% for lipase adsorbed on S1, and from 86 to 50% for lipase adsorbed on ITQ-2. However, the lipase/sepiolite-AlPO4 catalyst does not show any enzyme leaching due to the covalent binding of enzyme and support, and gives the same conversion even after four cycles. The lower conversion showed by lipase/sepiolite-AlPO4 (close to 50%) con rm that the covalent binding forces do not allow activation of the enzyme catalytic center. Finally, in Figure 3 the total productivity obtained for each tested catalyst is compared with that of the free lipase (expressed as “µg of myristic acid per mg of enzyme per hours of reaction”). Note that only lipase supported on S1 and ITQ-2 show a higher productivity than the free enzyme. Figure 2. Reusability of catalysts in the ester hydrolysis reaction. Reaction conditions for each cycle: 70 h at 37°C, initial catalyst amount 0.4 g (enzyme content for each catalysts: 53 mg for lipase/ITQ-2, 60 mg for lipase/S1, 116 mg for lipase/F-S2, 173 mg for lipase/sepiolite/AlPO4).


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Figure 3. Myristic acid productivity after four reaction cycles, each one for 70 h at 37°C. For free lipase each reaction cycle was of 24 h with 100 mg enzyme.

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Olive oil transesterification The main fatty acids present in the vegetable oil used in this work are oleic acid (76% wt/wt), palmitic acid (16.1% wt/wt) and linoleic acid (4.4% wt/wt). The methyl ester measured to monitor the transesteri cation reaction was methyl oleate. In order to compare the catalytic performances of our catalysts with those of the free lipase, catalytic tests using free enzyme and lipase/S1 catalyst were carried out. http://www.tandfonline.com/doi/full/10.1080/10242420701444256?scroll=top&needAccess=true

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lipase/S1 catalyst were carried out.


The results show the same oleic acid conversion from both catalysts after 3 h of reaction (Figure 4). This is an indirect con rmation that the physical adsorption procedure on zeolite preserves the activity of the immobilized lipase enzyme. In Figure 5, the results obtained after 3 h of reaction with lipase adsorbed by acid–base interaction on S1 and ITQ-2, and with the lipase covalently attached on the sepiolite/AlPO4, are compared. A lower oleic acid conversion was observed in the reaction catalyzed by the enzyme covalently attached on sepiolite/AlPO4. Figure 4. Methyl oleate content and oleic acid conversion as a function of time in the transesteri cation reaction catalyzed by lipase/S1 and free lipase. Reaction conditions: 40°C, 0.6 g lipase/S1 or 100 mg free lipase.

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Figure 5. Transesteri cation reactions for the di erent catalysts. Reaction conditions: 3 h at 40°C, methanol:oil ratio equal to 5:1. Catalyst amount: 0.6 g for lipase/S1 and lipase/ITQ-2 catalysts, 0.4 g for lipase/sepiolite/AlPO4 and 100 mg for free lipase.


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However, the stability of the lipase/sepiolite/AlPO4 biocatalyst is higher than that of the lipase/S1 and lipase/ITQ-2 biocatalysts. For the latter, an inevitable enzyme leaching occurs during reuse (Figure 6). Figure 6. Methyl oleate content as a function of reaction cycle. Reaction conditions for each cycle: 3 h at 40°C, methanol:oil ratio equal to 5:1. Initial catalyst amount: 0.6 g for lipase/S1 and lipase/ITQ-2 catalysts, 0.4 g for lipase/sepiolite/AlPO4.

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In order to evaluate catalytic performance, another important parameter is the productivity, expressed as “mg of methyl oleate per mg of enzyme per hour of reaction”. Figure 7 reports the biodiesel productivity for the http://www.tandfonline.com/doi/full/10.1080/10242420701444256?scroll=top&needAccess=true

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hour of reaction”. Figure 7 reports the biodiesel productivity for the


immobilized catalysts compared with that of the free lipase. It can be observed that the catalysts prepared by adsorption show the highest productivity, more than twice that obtained with the free enzyme. Only the lipase/sepiolite-AlPO4 catalyst had a lower productivity than that of the free lipase. This con rms the earlier hypothesis that the covalent binding forces reduce the catalytic activity of the immobilized lipase. Figure 7. Biodiesel productivity after three reaction cycles, each one of 3 h at 40°C. For free lipase the quantity used for each cycle was 100 mg.

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Finally, comparison with literature data shows that the immobilized lipase from Rh. miehei studied in this work exhibits comparable or higher catalytic performance than other lipases (free, immobilized, commercial or by others microorganisms) (Hse et al.  2002; Soumanou et al.  2003; Noureddini et al.  2005; Salis et al.  2005). However, it should be noted that some of the reaction parameters and fatty acids sources used in the literature comparisons di er from those used in this work. For example, Salis et al. ( 2005) obtained a triolein conversion of about 90% after 3 h reaction using immobilized Pseudomonas cepacia lipase at 40°C (molar ratio butanol:triolein of 6:1). With the commercial lipozyme (RML immobilized on an anion exchange resin), Salis et al. showed a conversion of triolein of http://www.tandfonline.com/doi/full/10.1080/10242420701444256?scroll=top&needAccess=true

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an anion exchange resin), Salis et al. showed a conversion of triolein of


about 90% only after 10 h reaction. Soumanou et al. ( 2003) report sun ower oil conversion in a solvent-free and in an n-hexane system using commercial immobilized lipase from RM at a molar ratio methanol:sun ower oil of 3:1. The immobilized RML (lipozyme) gave, at 40°C, a sun ower conversion of about 60% after 10 h of reaction in the nhexane system, and a conversion of about 45% after 24 h in the solvent-free system.

Regeneration of the support The support containing spent biocatalyst could be easily regenerated by a simple thermal treatment, as the enzyme is immobilized by adsorption. DSC analysis of the sample after protein adsorption (sample S1) showed a peak at 367.8°C, which could be attributed to the presence of protein. No peaks were observed in the DSC curve of the regenerated support (4 h at 380°C). This con rms the complete absence of organic compounds. In order to verify that the regenerated support (named R-S1) was able to adsorb new enzyme, it was used in the lipase immobilization procedure and, subsequently, in the trans-esteri cation reaction. The amount of adsorbed protein was about the same as that obtained in the initial immobilization (148 mg g−1, compared to 151 mg g−1 initially obtained). After 3 h of reaction, the methyl oleate content, using the lipase/R-S1 catalyst, was 80% and total conversion of the oleic acid had been achieved which was virtually the same as that obtained using the biocatalyst immobilized on the support for the rst time. These results con rm that because, during the regeneration process, condensation of the hydroxyl groups (Si-OH, the main enzyme attachment sites) does not occur, the same support can be used more than once to obtain an active and supported biocatalyst.

Conclusions http://www.tandfonline.com/doi/full/10.1080/10242420701444256?scroll=top&needAccess=true

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Lipase adsorption on zeolitic supports is a good procedure to obtain an active heterogeneous enzymatic catalyst. The results reported clearly show that the zeolitic materials, having a large number of Si-OH groups, are able to adsorb the lipase enzyme in its open conformation. The lipase–zeolite linkage obtained for the lipase/S1 and lipase/ITQ-2 catalysts, is due to weak acid interactions between the zeolite surface and the enzyme lid. Free lipase shows a similar catalytic behavior to, but a productivity less than half of the lipase/S1 and lipase/ITQ-2 catalysts. Electrostatic and covalent binding forces give strong lipase–zeolite interaction, allowing to high enzyme loadings, but in its closed form. Consequently, the lipase attached by these interactions has a lower catalytic performance. Nevertheless, the covalent immobilization procedure was the best method to obtain a very stable catalyst. Enzyme adsorbed on zeolitic materials was able to catalyze a number of reaction cycles, but progressively leached from the support. Further studies will investigate this problem.

Acknowledgements The authors would like to thank Professor Avelino Corma (from Instítuto de Tecnología Química (UPV-CSIC) Universidad Politécnica de Valencia, Spain) for supplying the ITQ-2 material.

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