Immobilization of Candida antarctica lipase B by covalent attachment on chitosan-based hydrogels using different support activation strategies

Biochemical Engineering Journal 60 (2012) 16–24

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Immobilization of Candida antarctica lipase B by covalent attachment on chitosan-based hydrogels using different support activation strategies J.A. Silva a , G.P. Macedo a , D.S. Rodrigues b , R.L.C. Giordano c , L.R.B. Gonc¸alves a,∗ a Grupo de Pesquisa e Desenvolvimento de Processos Biotecnológicos, Universidade Federal do Ceará, Departamento de Engenharia Química, Campus do Pici, Bloco 709, Fortaleza, CE, 60455-760, Brazil b Embrapa Agroenergia–Parque Estac¸ão Biológica–PqEB s/n, Brasïlia, DF, 70770-901, Brasil c Universidade Federal de São Carlos, Departamento de Engenharia Química, Rodovia Washington Luiz, km 235, São Carlos, SP, CEP 13565-905, Brazil

a r t i c l e

i n f o

Article history: Received 22 April 2011 Received in revised form 26 August 2011 Accepted 18 September 2011 Available online 1 October 2011 Keywords: Chitosan Chitosan–alginate complex Immobilized enzymes Lipase Biocatalysis

a b s t r a c t Candida antarctica lipase B immobilization by covalent attachment on chitosan and on chitosan–alginate complex previously activated by different strategies was studied. Hydroxyl and amine groups of support were activated using glycidol and glutaraldehyde. Ethylenediamine (EDA) was also used in the activation process. FT-IR analysis confirmed the reaction of these activating agents with the supports. Several activation–immobilization strategies were performed and the best derivatives showed activities of 422.44 ± 50.4 and 378.30 ± 34.70 U/g-support for chitosan and chitosan–alginate complex, respectively, slightly less in comparison to the commercial immobilized lipase Novozym 435 (529.78 ± 11.7 U/gsupport). Best results of thermal stability (incubation at 60 ◦ C) and operational stability (repeated cycles of synthesis of butyl oleate) were obtained for enzyme immobilized on chitosan–alginate, activated with 2% glutaraldehyde. This derivative was 33 times more thermally stable than the soluble enzyme, and it did not lose its initial activity after 8 cycles of a 12-h synthesis of butyl oleate. Chitosan, activated with 72% glycidol, EDA and 5% glutaraldehyde, showed less operational (loss of 16.7% of its initial activity) and thermal stabilities (only 12.5 times more thermally stable than soluble enzyme). Conversion of 100% was obtained in a 12-h reaction of butyl oleate synthesis, using the best derivatives (lipase immobilized on chitosan–Gly72%–EDA–Glu5% and on chitosan–alginate–Glu2%). © 2011 Elsevier B.V. All rights reserved.

1. Introduction Lipases (glycerol ester hydrolases) catalyze, as their natural function, the hydrolysis of triglycerides. However, lipases may be used in vitro to catalyze many different reactions, often very different from the natural ones (regarding conditions, substrates, etc.). Thus, lipases may be used in the industry not only to modify oils and fats, but also to synthesize fatty esters used as cosmetics or surfactants and to produce many different intermediates for organic synthesis, e.g. resolution of racemic mixtures [1], detergent formulations, synthesis of biosurfactants, the oleochemical, dairy and agrochemical industries, paper manufacture, nutrition, cosmetics, and pharmaceutical processing [2]. Among fatty esters of industrial interest, 1-butyl oleate is used as biodiesel, additive to decrease the viscosity of diesel in winter use, poly vinyl chloride plasticizer, water-resisting agent and in hydraulic fluids [3–6]. Usually, the synthesis of butyl oleate is conducted in the presence of concentrated sulfuric acid through a chemical route, which has many

∗ Corresponding author. Tel.: +55 85 33669611; fax: +55 85 33669610. E-mail address: [email protected] (L.R.B. Gonc¸alves). 1369-703X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2011.09.011

disadvantages, such as formation of by-products, dispose of acid wastewater, equipment erosion by the concentrated acid and others [7]. Therefore, the enzymatic syntheses become more and more interesting, since enzymes have high selectivity, specificity and activity in mild reaction conditions. Furthermore, lipases have broad availability, low cost, mild reaction conditions, no need for co-factors and substrate specificity [8]. To fully exploit the technical and economical advantages of lipases, it is recommended that they be used in an immobilized state to reduce the cost and the poor stability of the soluble form. Immobilization also facilitates the separation of products, enhances lipase properties such as thermal stability and activity, and provides more flexibility with enzyme/substrate contact by using various reactor configurations [9]. Lipase has been immobilized through several methods, namely adsorption and covalent attachment [10,11], cross-linking, adsorption followed by cross-linking, and physical entrapment using many commercial carriers [12]. In the present work, two polymers were evaluated for Candida antarctica lipase B immobilization. The first one was chitosan, ␤-(1-4)-2-amino-2-deoxy-d-glucose, which is a natural polymer obtained from chitin by N-deacetylation. Since the N-deacetylation grade can vary, chitosan products with different degrees of

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Scheme 1. Schematic illustration of the different immobilization strategies, using: activation with glutaraldehyde – Strategies I and II (A), activation with glycidol – strategy III (B), activation with glycidol, glutaraldehyde and EDA – Strategies IV and V (C), and activation with glycidol, EDA and glutaraldehyde – strategy VI (D). GTA: glutaraldehyde; GOX: glyoxyl; GDL: glycidol.

deacetylation are available [13]. Chitosan can also be used for the preparation of various hybrid polymers, by forming polyelectrolyte complex products with natural polyanions. The strong electrostatic interaction of amine groups of chitosan with the carboxyl groups of other polymers, like sodium alginate – the sodium salt of polyuronic acid containing variable proportions of 1-4 linked ␤-d-mannuronic acid and ␣-l-guluronic acid – lead to the formation of a chitosan–alginate hybrid gel. The chitosan–alginate complex is a stronger gel than pure chitosan and therefore has longer lasting activity under drastic conditions of temperature and mechanical stirring [14]. In addition to its low cost, chitosan has several advantages as a support, including its lack of toxicity and chemical reactivity, allowing for the easy fixation of enzymes [10]. A common method of protein immobilization is the covalent linkage of the enzyme to polymeric materials, like chitosan, using bi-functional cross-linking reagents, such as glutaraldehyde, which establishes intermolecular bonds between amino groups of the protein and those of the polymer. The linkage yield may be enhanced if the less reactive hydroxyl groups of the chitosan molecule are activated to react with amine groups of the enzyme. The method of immobilization involving the amino and hydroxyl groups of chitosan is named “binary immobilization” [15]. Hung et al. [15] used glutaraldehyde and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) to activate the amine and hydroxyl groups, respectively, for the covalent immobilization of lipases. In this work, the binary immobilization of CALB on chitosan and chitosan–alginate complex was explored, using glutaraldehyde, glycidol (2,3-epoxy-1-propanol) and ethylenediamine (EDA) as activating agents. Theoretically, the use of EDA in the support activation procedure increases the distance between the enzyme and the support, which may improve enzyme access and, therefore, increasing lipase immobilization yield. This diamine reactant is linked to the aldehyde groups present in the support after activation with glutaraldehyde and/or glycidol. The other amine group of EDA remains available for the linking of enzyme molecules,

which makes it ready for immobilization [16]. To the best of our knowledge, the strategy of using ethylenediamine after chitosan activation with glutaraldehyde and/or glycidol, aiming to immobilize lipase on the beads, has not been reported in the literature yet. Last but not least, studies of thermal and operational stabilities were also made in order to evaluate the potential applications of the prepared biocatalysts.

2. Materials and methods 2.1. Materials Powdered chitosan with 85.2% degree of deacetylation was purchased from Polymar Ind. Ltda (Ceará, Brazil) and sodium alginate, from Vetec (São Paulo, Brazil). Soluble C. antarctica lipase B (Novozym 525, EC 3.1.1.3) (CALB), and Novozym 435 (immobilized CALB) were kindly donated by Novozymes Latin America Ltd. (Paraná, Brazil). Powdered chitosan, 85.2% deacetylation degree, was purchased from Polymar Ind Ltda (Ceará, Br). Glycidol 96% GC (2,3-epoxy-1-propanol), p-nitrophenyl butirate (pNPB) and pnitrophenol (pNP) were purchased from Sigma–Aldrich (St. Louis, USA). Ethylenediamine and glutaraldehyde solution Grade II 25% was purchased from Vetec (São Paulo, Brazil). All other reagents (analytical grade) were purchased from Synth (São Paulo, Brazil) and Vetec (São Paulo, Brazil).

2.2. Preparation and modification of support for enzyme immobilization In this work, 4.0 wt.% chitosan and 2.5–2.5 wt.% chitosan–alginate complex were used for CALB immobilization [17].

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Scheme 2. Schematic illustration of the reactions used to activate chitosan with different activating agents (glycidol, glutaraldehyde, EDA and a combination of them).

2.2.1. Glycidol activation Activation of chitosan and chitosan–alginate complex gels with glycidol was carried out by etherification (glyceril-support) followed by oxidation with sodium periodate to generate glyoxyl groups (GOX), according to the methodology described elsewhere [18].

diagrams of the activation–immobilization strategies studied in this work.

2.2.2. Glutaraldehyde activation [19] Chitosan and chitosan–alginate complex gels (1 g) were suspended in 9 mL of sodium bicarbonate buffer, pH 10.0, containing 2% (v/v) or 5% (v/v) of glutaraldehyde, according to each immobilization strategy (Schemes 1 and 2). The mixture was kept under agitation for 60 min at 25 ◦ C. Finally, supports were washed with distilled water to remove residue from the activating agent.

2.2.4.2. Immobilization strategy III. The support was activated with 7.2 mL of glycidol per 10.0 g of support, followed by lipase immobilization (Scheme 1B)

2.2.3. EDA cross-linking EDA cross-linking was achieved by reacting 40 mL of a 2 M ethylenediamine solution, pH 10.0, with 10 g of gel activated with glycidol and/or glutaraldehyde [20], see Scheme 2. 2.2.4. Activation–immobilization strategies Six strategies for activation–immobilization were performed using chitosan and chitosan–alginate hydrogels. Scheme 1 shows

2.2.4.1. Immobilization strategies I and II. The support was activated with glutaraldehyde, using 5% and 2% (v/v) concentration, respectively (Scheme 1A), followed by immobilization of the enzyme

2.2.4.3. Immobilization strategies IV and V. The support was activated with glutaraldehyde 5% (v/v) and 2% (v/v), respectively, followed by activation with 4.8 mL of glycidol per 10.0 g of support. Then, a linkage of one of the amino groups of EDA to aldehyde groups generated in the support was performed, followed by additional activation with glutaraldehyde 5% (v/v). Finally, lipase was immobilized in the activated support (Scheme 1C) 2.2.4.4. Immobilization strategy VI. Support was activated with 7.2 mL of glycidol per 10.0 g of support. Afterwards, one of the amino groups of the ethylenediamine molecule was bound to aldehyde groups generated in the support, followed by activation with

J.A. Silva et al. / Biochemical Engineering Journal 60 (2012) 16–24

glutaraldehyde 5% (v/v). Finally, lipase was immobilized in the activated support (Scheme 1D) Scheme 2 illustrates the reactions between chitosan and the reagents that participated in the activation processes: glycidol, glutaraldehyde and EDA. 2.3. Protein determination Protein content was determined according to the Bradford method [21] using bovine serum albumin (BSA) as a standard. 2.4. Assay of enzymatic activity The hydrolytic activity of soluble and immobilized CALB (Atd ) was determined using p-nitrophenyl butirate as substrate, in 2propanol, at pH 8.0 and 25 ◦ C, according to the methodology described in the literature [21], with some modifications. 2.5. Immobilization of CALB All of the immobilization studies were carried out in a batch reactor, under gentle agitation, at 25 ◦ C, by contacting the enzyme in 100 mM bicarbonate buffer, pH 10.0, and the previously activated support, for 6 h. In all experiments, a load of 1.8 mg of protein per gram of support was used. To determine immobilization yield, samples of supernatant were withdrawn and hydrolytic activities were measured. A blank assay was also conducted to evaluate a possible enzyme deactivation under the immobilization conditions. For this purpose, a solution of CALB was placed in a reactor under the same conditions of immobilization, but in the absence of support. 2.5.1. Immobilization yield After measuring the initial (Ati ) and the final (Atf ) enzyme activity in the supernatant and in the blank assay, the remaining enzyme activity in the supernatant (Ati − Atf ) and the immobilization yield (IY) could be calculated, using Eq. (1) [21]. IYimob (%) =

Ati − Atf × 100 Ati

(1)

2.5.2. Recovery activity (Atr ) The theoretical activity (Att ) of immobilized lipase on the support could be calculated using the amount of enzyme offered per gram of support (Atoff – U/g support) and the immobilization yield, see Eq. (2). After measuring the activity of the immobilized enzyme (Atd – U/g support), the recovery activity [17] was calculated using Eq. (3). Att = IY × Atoff

(2)

At Atr = d × 100 Att

(3)

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After immobilization, immobilized CALB was dried by placing it in contact with three solvent mixtures in different proportions for 2 min each. The removal of water present in 1 g of each derivative (biocatalyst) was performed in three steps using the solvent mixtures: (i) 2 mL of ethanol:H2 O (20:80, v/v); (ii) 2 mL of ethanol:hexane: oleic acid (20:40:40, v/v/v) and (iii) 2 mL of hexane:oleic acid (80:20, v/v). At each step, the extraction solution was added to a funnel with a sintered glass plate containing the derivative, and the mixture was kept for 2 min. Then, the extraction solution was separated from the derivatives by vacuum filtration. After the third step, the derivatives were stored at 4 ◦ C. Before using the dried derivatives for hydrolysis or synthesis, they were vacuum washed with 5 mL of hexane. 2.7. Operational stability of the immobilized CALB The operational stability of the derivatives was performed by submitting 0.020 g of immobilized CALB, dried as described in Section 2.6, to subsequent cycles of butyl oleate synthesis. The reaction was carried out in a batch reactor, maintained at 37 ◦ C under constant agitation. The reaction volume was 2.5 mL and the derivative mass was 0.020 g. Before each new cycle, the derivative was washed to remove products and unreacted substrates. 2.8. Butyl oleate synthesis The esterification of 1-butanol and oleic acid in n-heptane, catalyzed with the immobilized enzymes, was investigated under orbital agitation, at 37 ◦ C. The reaction was conducted in flasks containing 0.050 mol/L of substrates (oleic acid/butanol molar ratio of 1/1), in the presence of 2.5 mL of anhydrous n-heptane without added water. The reaction was initiated by adding 0.02 g of immobilized enzyme. At fixed intervals, 20 ␮L aliquots of the reaction mixture were withdrawn, mixed with 180 ␮L of hexane and then analyzed by gas chromatography–mass spectrometry (GC–MS). The experiments were carried out in triplicate. Controls without enzyme were also checked. 2.9. Gas chromatography–mass spectrometry GC–MS data were recorded with a QP 5000 Shimadzu. The gas chromatograph was fitted with a capillary column (AT-5; 30 m × 0.25 mm). Operating conditions: column oven temperature programmed at 230 ◦ C for 3 min and then at 260 ◦ C for 3 min at 6 ◦ C/min; injector/detector temperature 250◦ /280◦ , respectively; electron voltage, 70 eV. Helium was used as the carrier gas at a flow rate of 1.5 mL/min. The sample volume injected was 1 ␮L. 2.10. Fourier transform infrared spectroscopy (FT-IR) analysis

2.6. Assay of thermal stabilities of soluble and immobilized CALB Soluble or immobilized CALB was incubated in 100 mM sodium phosphate buffer, pH 8.0, at 60 ◦ C, for 21 h. Periodically, samples were withdrawn and their residual activities were assayed by the hydrolysis of p-nitrophenyl butirate, previously described. The initial activity was taken as 100%. The deactivation constant and half-life (t1/2 ) for each immobilized derivative was calculated according to the Sadana and Henley model [22], using Microcal Origin version 8.1. With the exponential nonlinear decay model and its parameters, it was possible to determine the inactivation constant (kd ) and the half-lives (t1/2 ) of the derivatives. Stabilization factors were obtained as the ratio between half-lives of the derivatives and the soluble CALB.

To investigate the activation support treatment, FT-IR analysis was conducted. The FT-IR spectra were obtained, in KBr tablets, containing 1% of the analyte (powder), in a Bomem spectrometer (model M102). 3. Results and discussion 3.1. FT-IR analysis FT-IR spectra have been widely used as a tool to identify the presence of certain functional groups or chemical bonds on a solid surface in material modifications because each specific chemical bond often shows a unique energy absorption band [23]. In

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3.2. Lipase immobilization on chitosan: study of different strategies of support activation

Fig. 1. FT-IR spectra of (a) chitosan, (b) chitosan activated with glutaraldehyde, (c) chitosan activated with glycidol.

this study, FT-IR analysis was used to examine the characteristic chemical structures of the three types of hydrogel beads: chitosan (spectrum in Fig. 1a), chitosan activated with glutaraldehyde (spectrum in Fig. 1b) and chitosan activated with glycidol (spectrum in Fig. 1c). The major peaks for chitosan are: (a) 3379.41 cm−1 (–OH and –NH stretching vibrations), 1644.97 cm−1 (–NH bending vibration in–NH2), 1439.35 cm−1 (–NH deformation vibration in–NH2 ), 1379.8 cm−1 (–CH symmetric blending vibration in –CHOH–), 1077.16 cm−1 (–CO stretching vibration in –CH–O–CH), and 894.9 cm−1 (–CN stretching vibration). The spectrum in Fig. 1a–c shows some major changes due to the reaction of glutaraldehyde and glycidol with chitosan. The peak 3379.41 cm−1 disappears, and the peaks 1644.97 cm−1 and 1439.35 cm−1 shifted to 1649.38 cm−1 and 1430.79 cm−1 respectively, and showed decreased intensity, indicating that some –OH and –NH groups were altered, due to the reaction with glutaraldehyde. The low reactivity of glycidol molecules allowed reaction only with –OH groups, which were in a higher quantity and had a lower steric hindrance in the polymer, as shown in Fig. 1, where the peaks related to the –NH bending vibration in –NH2 were unchanged. The peak-related OH groups (around 3439.58 cm−1 ) remained in the spectrum (c), probably due to the incomplete oxidation of glyceryl groups generated after the step of etherification with glycidol. There was a slight leveling out of peak 1077.16 cm−1 in both spectrums (a and b), indicating the appearance of aldehyde groups. These changes are related to the formation of chemical bonds with nitrogen and oxygen, indicating that the amine groups and hydroxyl groups were involved in the reaction with glutaraldehyde and glycidol, respectively. However, glutaraldehyde molecules react with hydroxyl groups and amino groups, while glycidol reacts only with hydroxyl groups.

Six strategies for the activation–immobilization of lipase on chitosan were studied, with the objective of obtaining a derivative with high activity and stability (operational and thermal stability at 60 ◦ C). Table 1 shows the results of derivative activity (Atd ) and immobilization yield. Blank experiments were conducted in parallel with immobilization to observe how the enzyme was deactivated during the 6 h of assay. For this purpose, a solution of CALB was placed in a reactor under the same conditions of immobilization, but in the absence of support. An average 18.0% loss of enzyme activity was observed. Immobilization yield (IY = 15.8%), and derivative and recovery activity were very low for strategy III, when chitosan was activated with 72% (v/w) of glycidol, as can be seen in Table 1. Three factors may have contributed to this fact. First, glyoxyl groups (aldehyde) resulting from the activation are less reactive than aldehyde groups of glutaraldehyde. Second, the high concentration of glyoxyl groups (aldehyde) in the support enables its interconnection (aldol reaction), since they are close to each other, which causes the deactivation of some of these groups [24]. Last but not least, the glutaraldehyde cross-linking in chitosan particles produces surface hydrophobicity [26], which would favor lipase immobilization since it present an exceptionally high affinity for more hydrophobic supports [1]. In strategies I and II, where, respectively, 5% (v/v) and 2% (v/v) of glutaraldehyde were used as an activating agent, good results of derivative activity (up from 270 U/g) and immobilization yield (over 50%) were obtained. This behavior may be explained by the fact that glutaraldehyde is more reactive than glycidol. By using glutaraldehyde, the linkage can be done using the terminal amine groups of the protein, while, with glyoxil groups, the ␧-amino groups of the lysine residues have to be available to allow immobilization [10]. Furthermore, the high concentration of amine groups in chitosan (85.2% degree of deacetylation) explains the high degree of activation obtained with glutaraldehyde, since it can react directly with the amino groups of chitosan. Rodrigues et al. [10] obtained much higher values of aldehyde group concentration when chitosan was activated with glutaraldehyde (1692 ␮mol/g dried support) rather than glycidol (687 ␮mol/g dried support). When 5% of glutaraldehyde was used, a higher immobilization yield was achieved – 74.78%, compared to 59.52% when 2% of glutaraldehyde was used. This is because the number of active groups on the surface of the support was higher, since the concentration of glutaraldehyde is one of the factors that controls the degree of cross-linking in chitosan particles [26]. However, the derivative activity was lower (277.77 U/g) compared to that for 2% of glutaraldehyde (394.49 U/g). It was expected that a higher immobilization yield resulted in larger enzyme loads in the support, since a larger amount of enzyme molecules disappeared from the

Table 1 Immobilization parameters of CALB–chitosan derivatives: recovery activity (Atr ), immobilization yield (IY), enzyme activities of the supernatant (soluble enzyme) before (Ati ) and after (Atf ) immobilization, and derivative activity (Atd ). Immobilization conditions: 10 mL of enzymatic solution, in bicarbonate buffer 100 mM, pH 10.0, contacted with 1 g of support at 25 ◦ C, for 6 h. Enzyme load: 1.8 mg protein/g support. Immobilization strategy I II III IV V VI Novozym 435

Ati (U/mL) 117.56 ± 117.50 ± 144.43 ± 133.50 ± 125.37 ± 133.14 ± –

3.5 3.5 4.3 4.0 3.8 4.0

Atf (U/mL) 29.65 ± 47.59 ± 121.50 ± 57.10 ± 6.85 ± 65.17 ± –

0.9 1.4 3.6 1.7 0.2 1.9

Atd (U/g) 277.77 394.49 6.17 286.85 413.72 422.44 529.78

± ± ± ± ± ± ±

33.9 39.2 0.8 23.8 40.8 50.4 11.7

Atr (%)

IY (%)

31.6 56.4 2.7 37.5 39.9 62.2 –

74.8 59.5 15.8 57.3 94.5 51.0 –

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Table 2 Immobilization parameters of CALB–chitosan–alginate derivatives: recovery activity (Atr ), immobilization yield (IY), enzyme activities in the supernatant (soluble enzyme) before (Ati ) and after (Atf ) immobilization, and derivative activity (Atd ). Immobilization conditions: 10 mL of enzymatic solution, in bicarbonate buffer 100 mM, pH 10.0, was contacted with 1 g support at 25 ◦ C, for 6 h. Enzyme load: 1.8 mg protein/g support. Immobilization strategy

Ati (U/mL)

I II III IV V VI

111.72 121.22 144.52 125.30 117.07 173.21

± ± ± ± ± ±

3.3 3.6 4.3 3.8 3.5 5.2

Atf (U/mL) 53.10 23.14 92.84 39.10 18.59 121.70

supernatant. The lower derivative activity may be caused by the distortion of the enzyme structure or by the reduction of pore diameters after cross-linking, due to the high reactivity of glutaraldehyde. This activation agent can also react with other groups of the enzyme [26] and the distortions in enzyme tertiary structure may be caused by the formation of a larger number of links between active groups of the enzyme and the support [17,27]. It is also reported [25] that increasing the degree of cross-linking may enhance the hydrophobicity in microspheres of chitosan, which may affect the enzyme catalytic activity, due to structural distortions in the protein, depending on the interaction strength between enzyme and support used [28]. It should also be emphasized that a system containing high loads of enzyme may be subject to diffusion limitations and steric hindrance, which may also explain the lower derivative activities observed when 5% of glutaraldehyde was used. Other authors [25] report that different physical characteristics of chitosan microspheres are achieved when they are prepared with different degrees of cross-linking with glutaraldehyde. For instance, the diffusion coefficient (D) of a drug (centchroman) decreased from 8.08 × 10−12 cm2 s−1 to 0.24 × 10−12 cm2 s−1 when increasing the concentration of glutaraldehyde from 2% to 6%. In this case, the rate of hydrolysis reaction, used to determine activity, is not only governed by kinetics; it is also influenced by the barrier that the substrate must pass through to reach the active site of the enzyme. The enzyme molecule itself acts as a barrier for diffusion [29]. Steric hindrance is characterized by the difficulty of access of the substrate molecule within the active site, since the enzyme can be immobilized with the wrong orientation, obstructing the active site [30]. Strategies IV, V and VI followed almost the same activation procedure, with one difference: the initial activation step of strategies IV and V was performed with 5% (v/v) and 2% (v/v) glutaraldehyde, respectively. High enzyme loads (above 280 U/g) were obtained when these two immobilization strategies were used. By using strategy V, a high immobilization yield (94.54%) and enzymatic activity of the derivative (413.72 U/g-support) was achieved. These two immobilization strategies also promoted hydrophobization of the chitosan surface, since a large hydrocarbonic chain was generated at the end of the activation process. As lipases have a great affinity with non-polar substances, the higher interaction between them and the support caused an increase in immobilization, as can be seen in Table 1 (strategy V). Hydrophobization step can be also observed for all strategies in which glutaraldehyde was used as activating agent (I, II, IV, V and VI). However, hydrophobization procedure is higher for Strategies I and II than Strategies IV, V and VI, due to the 2 amine molecules inserted after amination step with EDA, which increases the swelling degree of the support. Another aspect should be considered when glutaraldehyde is used to activate aminated supports: below each glutaraldehyde molecule, there are amino groups that can confer some ionic exchanger properties to the support. Therefore, a physical adsorption may occur before the covalent

± ± ± ± ± ±

1.6 0.7 2.8 1.2 0.6 3.6

Atd (U/g) 268.96 378.30 27.59 179.21 270.94 167.67

± ± ± ± ± ±

3.2 34.7 23.9 9.7 1.3 5.7

Atr (%)

IY (%)

45.9 38.6 5.3 20.8 27.5 32.5

52.5 80.9 35.8 68.8 84.1 29.7

reaction since the supports could be considered as heterofunctional matrices [31]. High immobilization yield and enzymatic activity of the derivative was expected for strategy IV, but it was not observed. The initial concentration of glutaraldehyde (5%) influenced the process of lipase immobilization. It has been mentioned before that the diffusion coefficient of a drug (centchroman) was almost 34 times smaller when concentration of glutaraldehyde was increased from 2% to 6% [25]. Probably, the high degree of cross-links between aldehyde groups of glutaraldehyde reduced the porosity of the material, thus hindering the access of EDA molecules and, consequently, the continuity of the support activation process [10]. Strategy VI, in which 72% (v/w) of glycidol was used, followed by linking EDA to the glyoxyl groups generated and then linking the glutaraldehyde (5% v/v) to the amino groups of chitosan, proved to be the best activation–immobilization strategy, taking into account the derivative activity, which was 422.44 U/g-support. First, EDA molecules bind to glyoxyl (aldehyde) groups in the support. Finally, glutaraldehyde reacts with the other end of EDA, which makes the support very reactive for enzyme immobilization. This modification of the support with ethylenediamine was used to obtain a bifunctional support, with moieties that were able to physically adsorb the proteins and groups that enable its covalent immobilization [32]. Furthermore, some authors [28] describe that high reactivity of reactive groups of supports was attained by increasing space arm. The best performance of supports prepared by using strategy IV, compared to strategies IV and V, can be also related to the increasing in the space arm (strategies IV and V) due to the glutaraldehyde molecule used in step one, which gave the highest reactivity of the aldehyde groups favoring the reaction of vicinal aldehyde groups (cross-linking by reaction of vicinal aldehyde groups), and reducing the density of aldehyde groups on those supports. 3.3. Lipase immobilization on chitosan–alginate The same activation–immobilization strategies studied for chitosan were repeated for chitosan–alginate complex. Although the chitosan–alginate complex prepared here presented a lower quantity of amino groups, the aim of using this support to immobilize enzymes in this study was to evaluate thermal and operational stability, as well as enzymatic activity per weight of support. As the polyelectrolyte gel formed between chitosan and alginate is more resistant than the gel formed only with chitosan [14], it was expected that this support would have greater mechanical strength and, consequently, greater mechanical stability. Table 2 shows the results of CALB immobilization on chitosan–alginate complex, for different activation– immobilization strategies. It can be observed that, in most immobilization assays, the behavior of this support was different from the one prepared only with chitosan, which was expected. After complexation of chitosan with alginate, there was a modification in the physical–chemical characteristics of the material. Different effects on enzyme activity are expected when using

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Table 3 Derivatives activity before (Ati ) and after (Atf ) the drying process. Derivative

Immobilization strategy

Ati (U/g)

Atf (U/g)

Chitosan–Gly72%–EDA–Glu5% Chitosan–Glu2% Chitosan–Glu2%–Gly48%–EDA–Glu5% Chitosan–alginate–Glu2%

VI II V II

422.44 394.49 413.72 378.30

211.23 205.23 210.36 190.86

different supports, due to different enzyme orientations of the molecule on the support [33]. The enzyme reacts with the support in different areas (areas with more hydrophobic residues or more negative charges, for instance), with different effects on enzyme activity. It can be observed that the derivatives obtained when using strategies I, II and V presented similar activities. The derivative obtained using strategy III, involving activation with 72% (v/w) glycidol followed by lipase immobilization, showed low activity, probably due to the same reasons presented for the derivative prepared with chitosan. The best results for enzyme activity in this support were obtained using strategies I and II, in which glutaraldehyde was used as an activating agent (5% and 2%, respectively), and strategy V, where the support was activated with glutaraldehyde 2% (v/v), followed by activation with glycidol 72% (v/w), linking of the EDA to the glyoxyl group that was generated and finally, activation with 5% (v/v) glutaraldehyde. The highest activity value was obtained using strategy II (378.30 U/g-support).

3.4. Thermal stability of derivatives The derivatives produced from strategies II, V and VI, using chitosan as support, and the derivative produced from strategy II, using chitosan–alginate complex, were subjected to thermal stability at 60 ◦ C tests for 21 h. Before performing these studies, there was a step involving drying the derivatives to remove water. Table 3 presents the derivatives selected and enzymatic activities before (Ati ) and after (Atf ) the process of drying described in the experimental procedures. Other authors [34], when investigating the immobilization of CALB in silica with controlled porosity, have also observed loss of activity after drying the derivatives with acetone, even when the process was carried out for short periods of time. To prevent loss of activity, the authors suggest the addition of albumin, polyethylene or other polymers during this stage, in order to protect the enzyme. Whetje et al. [35] reported that the loss of activity is also related to the amount of water withdrawn from the biocatalyst. Results of thermal stability at 60 ◦ C were compared with the stability of the soluble enzyme and of a commercial derivative (Novozym 435), at the same temperature. For each thermal stability assay, the half-life (t1/2 ) and the stabilization factor were determined. Analyzing Fig. 2, it is clear that the thermal stability of the derivatives is much higher than the stability of the soluble enzyme. When the enzyme is in its soluble form, it presents some flexibility, which means that its active site undergoes conformational changes that are often irreversible, causing inactivity. When immobilized, a more rigid form is acquired because of linkages to the support. This stiffness decreases the enzyme’s flexibility, maintaining the form of the active site, which is responsible for its activity [30]. Comparing the profiles of derivative activity prepared in this work (chitosan–Gly72%–EDA–Glu5% and chitosan–alginate– Glu2%) with a commercial derivative (Novozym 435), it can be observed that the decrease in activities of the three derivatives is similar, within the experimental error (Fig. 2).

Fig. 2. Thermal deactivation profile for soluble and immobilized CALB at 60 ◦ C and pH 8.0. Soluble enzyme Ati = 1945.69 U/mL (), Novozym 435 Ati = 529.78 U/g (), CALB immobilized by strategy VI (chitosan–Gly72%–EDA–Glu5%) Ati = 211.23 U/g (), CALB immobilized by strategy II (chitosan–Glu2%) Ati = 205.23 U/g ( ), CALB immobilized by strategy V (chitosan–Glu2%–Gly48%–EDA–Glu5%) Ati = 210.36 U/g () and CALB immobilized by strategy II (chitosan–alginate–Glu2%) Ati = 190.86 U/g (䊉). The lines represent the trend of the Sadana and Henley model [16].

Table 4 shows the results of thermal deactivation at 60 ◦ C of soluble CALB, a commercial biocatalyst (Novozym 435), and the biocatalysts prepared in this work (chitosan–Gly72%–EDA–Glu5%, chitosan–Glu2%–Gly48%–EDA–Glu5% and chitosan–Glu2%, chitosan–alginate–Glu2%). The derivative that showed a higher stabilization factor (SF) was chitosan–alginate–Glu2%, being approximately 33 times more stable than the soluble enzyme, and twice more stable than commercial immobilized CALB–Novozym 435 (SF = 17.1). This is probably due to the fact that the chitosan–alginate complex allows a greater number of linkages between the enzyme and the support, increasing the rigidity. The chitosan–alginate complex is formed in an organized way, which gives the support a better geometric congruence with the enzyme. The poor “geometric congruence” between the enzyme and the support is suggested as a major impediment in the stabilization of protein structures [36]. The derivative chitosan–Gly72%–EDA–Glu5% was 12.5 times more stable than soluble enzyme. The derivatives chitosan–Glu2% and chitosan–Glu2%–Gly48%–EDA–Glu5% showed low stabilization factors. These results led to the selection of derivatives chitosan–Gly72%–EDA–Glu5% and chitosan–alginate–Glu2% for further studies of operational stability in organic medium on butyl oleate synthesis. The stability of these derivatives at high temperatures represents how the enzyme undergoes changes in its active site, through loss of activity [30]. The enzyme may have undergone changes in Table 4 Thermal deactivation at 60 ◦ C and pH 8.0, of soluble CALB, Novozym 435, CALB derivatives: chitosan–Gly72%–EDA–Glu5%; chitosan–Glu2%; chitosan–Glu2% –Gly48%–EDA–Glu5% and chitosan–alginate–Glu2%. Biocatalyst

kd (h−1 )

Soluble CALB Novozym 435 Chitosan–Gly72%–EDA–Glu5% Chitosan–Glu2% Chitosan–Glu2%–Gly48%–EDA–Glu5% Chitosan–alginate–Glu2%

7.02 0.92 1.02 1.08 1.06 0.34

± ± ± ± ± ±

1.2 0.3 0.4 0.1 0.1 0.1

t1/2 (h)

Stabilization factor (SF)

0.10 1.71 1.25 0.64 0.70 3.30

1.00 17.1 12.5 6.4 7.0 33.0

J.A. Silva et al. / Biochemical Engineering Journal 60 (2012) 16–24

Fig. 3. Operational stability in the synthesis of butyl oleate to Novozym 435 Ati = 529.78 U/g (), chitosan–Gly72%–EDA–Glu5% Ati = 211.23 U/g (䊉) and chitosan–alginate–Glu2% Ati = 190.86 U/g (). Each cycle had a duration of 12 h with orbital stirring, and was maintained at a temperature of 37 ◦ C. Reagents: oleic acid and n-butanol at 0.050 mol/L−1 in n-heptane. The lines represent the trend of experimental data.

its structure due to the temperature’s direct influence and even become loosened from the support, as it may have been distorted by a change in the structure of the support. As the chitosan–alginate gel has higher mechanical stability, this probably contributed to the derivative chitosan–alginate–Glu2% showing greater thermal stability. 3.5. Operational stability of derivatives Fig. 3 shows the study of the operational stability of derivatives chitosan–Gly72%–EDA–Glu5%, chitosan–alginate–Glu2% and Novozym 435, using subsequent synthesis of butyl oleate as a model reaction. Enzyme reuse was evaluated because it is of great importance in its use for batch and continuous processes. After 8 cycles, each lasting 12 h, it was noted that chitosan–alginate–Glu2% derivative and the Novozym showed less loss of activity after the cycles of synthesis (chitosan–alginate–Glu2% derivative maintained its initial activity, while Novozym had a loss of only 2.8% of its initial activity), demonstrating that these derivatives had a better operational stability when compared to the chitosan–Gly72%–EDA–Glu5% derivative. However, chitosan–Gly72%–EDA–Glu5% also presented good operational stability, because it lost only 16.7% of its initial activity. This results is in agreement with other reports from the literature [7], the authors observed a slight decrease of enzyme activity (<15%) after 18 cycles and comment that the repeated use of immobilized lipase has been improved significantly compared with the soluble Rhizopus arrhizus lipase. 3.6. Synthesis of butyl oleate Butyl oleate was synthesized with success using the two derivatives produced in this work, as well as the commercial derivative (Data not shown), since a 100% conversion of oleic acid after 12 h of reaction. This result is in agreement with other reports from the literature [7,37], depending of the source of lipase used, high conversion (>80%) was achieved after 4 h of reaction. It can be seen in Fig. 4 that after 12 h of reaction, the yield was 100% for both derivatives, although the reaction using the

23

Fig. 4. Kinetics of synthesis of butyl oleate using the derivatives chitosan–Gly72%–EDA–Glu5% Ati = 358.13 U/g (䊉) and chitosan–alginate–Glu2% Ati = 362.59 U/g (), at 37 ◦ C and for 24 h in orbital stirring. Reagents: oleic acid and buthanol at 0.050 mol/L−1 in n-heptano. Reaction volume = 2.5 mL. Derivative mass = 0.020 g.

chitosan–Gly72%–EDA–Glu5% derivative had been slightly faster in the initial hours (Fig. 4). The analysis of butyl oleate was made using gas chromatography coupled with mass spectrometry. We observed the disappearance of the chromatographic band corresponding to oleic acid (MM = 282 g/mol, tR = 5.95 min) and the appearance of the band on the butyl oleate (MM = 338 g/mol, tR = 8.25 min) during the reaction time, confirming the formation of the product. Reinforcing these results, the fragmentation pattern of butyl oleate shown here was the same found at the National Institute of Standards and Technology (NIST) [38]. The mass/charge ratio and intensities (%) of mass spectrum peaks obtained were, respectively: 55.10 (100), 56.10 (54.12) 57.15 (73.77), 67.15 (32.60), 69.10 (67.44), 83.15 (53.39), 84.10 (34.68), 97.15 (40.96), 111.15 (22.81), 137.15 (6.49), 152.20 (5.16), 180.25 (6.14), 222.20 (6.02), 264.25 (11.85), 265 (12.36), 338.30 (1.79). 4. Conclusions FT-IR analysis showed that activation with glutaraldehyde and glycidol, used in some activation strategies, did, in fact, happen, proving that the reactions occurred. Analyzing the results of the different activation–immobilization strategies studied in this work, both chitosan and chitosan–alginate complex are good supports for immobilizing C. antarctica lipase B. The best derivative activity value was 422.44 ± 50.4 U/g, with chitosan as support, prepared according to strategy VI. The best result, 378.30 ± 34.7 U/g, was obtained for chitosan–alginate complex, prepared according to strategy II. Seeing that the enzymatic load was low (1.8 mg of protein per weight of support), these results are satisfactory when compared with the activity of a commercial derivative, Novozym 435 (529.72 ± 11.7 U/g). The derivatives that showed the best results for thermal stability at 60 ◦ C were chitosan–Gly72%–EDA–Glu5% and chitosan–alginate–Glu2%, with stabilization factors of 12.5 and 33.0, respectively. The stabilization factor of derivative chitosan–alginate–Glu2% was higher than that of Novozym 435 (17.1). Therefore, this derivative has great potential for commercial use. After 8 cycles of butyl oleate synthesis (12 h each), chitosan–alginate–Glu2% was the most stable derivative

24

J.A. Silva et al. / Biochemical Engineering Journal 60 (2012) 16–24

prepared in this work, retaining all of its original activity. Similar results were obtained with Novozym 435, which retained 97.2% of its activity after the same time span. chitosan–Gly72%–EDA–Glu5% did not show as good an operational stability as the other two derivatives, retaining around 83.3% of its initial activity after 8 cycles. After 12 h of reaction using the best derivatives selected in this work, a 100% conversion of oleic acid was possible. This was an excellent result, whereas the same reaction using the commercial derivative obtained the same percentage of conversion. Although the commercial derivative showed greater enzymatic activity (529.72 ± 11.7 U/g) than the derivatives produced in this work (chitosan–Gly72%–EDA–Glu5%: 358.13 ± 5.95 U/g and chitosan–alginate–Glu2%: 362.59 ± 4.46 U/g), similar conversion results were obtained, indicating their viability in the synthesis of esters. Acknowledgments The authors would like to thank the Brazilian research-funding agencies FINEP, CNPq and CAPES. References ˜ G. Fernández-Lorente, C. Mateo, R. Fernández-Lafuente, [1] J.M. Palomo, G. Munoz, J.M. Guisán, Interfacial adsorption of lipases on very hydrophobic support (octadecyl–Sepabeads): immobilization, hyperactivation and stabilization of the open form of lipases, J. Mol. Catal. B: Enzym. 19–20 (2002) 279–286. [2] R. Sharma, Y. Chisti, U.C. Banerjee, Production, purification, characterization, and applications of lipases, Biotechnol. Adv. 19 (2001) 627–662. [3] Y.-Y. Linko, M. Lamsa, X. Wu, E. Uosukainen, J.S.P. Linko, Biodegradable products by lipase biocatalysis, J. Biotechnol. 66 (1998) 41–50. [4] C.E. Orrego, J.S. Valencia, C. Zapata, Candida rugosa lipase supported on high crystallinity chitosan as biocatalyst for the synthesis of 1-butyl oleate, Catal. Lett. 129 (2009) 312–322. [5] I.B.-B. Romdhane, Z.B. Romdhane, A. Gargouri, H. Belghith, Esterification activity and stability of Talaromyces thermophilus lipase immobilized onto chitosan, J. Mol. Catal. B: Enzym. 68 (2011) 230–239. [6] E. Séverac, O. Galy, F. Turond, C.A. Pantel, J-S. Condoret, P. Monsan, A. Marty, Selection of CalB immobilization method to be used in continuous oil transesterification: analysis of the economical impact, Enzyme Microb. Technol. 48 (2011) 61–70. [7] S.-G. Wang, W.-D. Zhang, Z. Li, Z.-Q. Ren, H.-X. Liu, Lipase immobilized on the hydrophobic polytetrafluoroethene membrane with nonwoven fabric and its application in intensifying synthesis of butyl oleate, Appl. Biochem. Biotechnol. 162 (2010) 2015–2026. [8] R. Dalla-Vecchia, M.G. Nascimento, V. Soldi, Aplicac¸ões sintéticas de lipases imobilizadas em polímeros, Quim. Nova 27 (2004) 623–630. [9] F.M. Gomes, E.B. Pereira, H.F. Castro, Immobilization of lipase on chitin and its use in nonconventional biocatalysis, Biomacromolecules 5 (2004) 17–23. [10] D.S. Rodrigues, A.A. Mendes, W.S. Adriano, L.R.B. Gonc¸alves, R.L.C. Giordano, Multipoint covalent immobilization of microbial lipase on chitosan and agarose activated by different methods, J. Mol. Catal. B: Enzym. 51 (2008) 100–109. [11] A.I.S. Brígida, A.D.T. Pinheiro, A.L.O. Ferreira, G.A.S. Pinto, L.R.B. Gonc¸alves, Immobilization of Candida antarctica lipase B by covalent attachment to green coconut fiber, Appl. Biochem. Biotechnol. 136 (2007) 67–80. [12] V.M. Balcão, A.L. Paiva, F.X. Malcata, Bioreactors with immobilized lipases: state of the art, Enzyme Microb. Technol. 18 (1996) 392–416. [13] S.S. Betigeri, S.H. Neau, Immobilization of lipase using hydrophilic polymers in the form of hydrogel beads, Biomaterials 23 (2002) 3627–3636. [14] F.L. Mi, H.W. Sung, S.S. Shyu, Drug release from chitosan–alginate complex beads reinforced by a naturally ocurring cross-linking agent, Carbohydr. Polym. 48 (2002) 61–72. [15] T.C. Hung, R. Giridhar, S.H. Chiou, W.T. Wu, Binary immobilization of Candida antarctica lipase on chitosan, J. Mol. Catal. B: Enzym. 26 (2003) 69–78.

[16] R. Fernandez-Lafuente, C.M. Rosell, V. Rodriguez, C. Santana, G. Soler, A. Batista, J.M. Guisán, Preparation of activated supports containing low pK amino groups. A new tool for protein immobilization via the carboxyl coupling method, Enzyme Microb. Technol. 15 (1993) 546–550. [17] A.A. Mendes, H.F. Castro, D.S. Rodrigues, W.S. Adriano, P.W. Tardioli, E.J. Mammarella, R.C. Giordano, R.L.C. Giordano, Multipoint covalent immobilization of lipase on chitosan hybrid hydrogels: influence of the polyelectrolyte complex type and chemical modification on the catalytic properties of the biocatalysts, J. Ind. Microbiol. Biotechnol. 38 (2011) 1055–1066. [18] J.M. Guisán, Aldehyde–agarose gels as activated supports for immobilizationstabilization of enzymes, Enzyme Microb. Technol. 10 (1988) 375–382. [19] W.S. Adriano, E.H. Costa-Filho, J.A. Silva, R.L.C. Giordano, L.R.B. Gonc¸alves, Stabilization of penicillin G acylade by immobilization on glutaraldehyde-activated chitosan, Braz. J. Chem. Eng. 22 (2005) 529–538. [20] H.C.T. Cardias, C.C. Grininger, H.C. Trevisan, J.M. Guisan, R.L.C. Giordano, Influence of activation on the multipoint immobilization of penicillin G acylase on macroporous silica, Braz. J. Chem. Eng. 16 (1999) 141–148. [21] M. Kordel, B. Hofmann, D. Schomburg, R.D. Schimid, Extracellular lipase of Pseudomonas sp strain ATCC 21808—purification, characterization, crystallization, and preliminary-X-ray diffraction data, J. Bacteriol. 173 (1991) 4836–4841. [22] A. Sadana, J.P. Henley, Analysis of enzyme deactivations by a series-type mechanism: influence of modification on the activity and stability of enzymes, Ann. N. Y. Acad. Sci. 501 (1987) 73–79. [23] N. Li, R. Bai, A novel amine-shielded surface cross-linking of chitosan hydrogel beads for enhanced metal adsorption performance, Ind. Eng. Chem. Res. 44 (2005) 6692–6700. [24] N.L. Allinger, M.P. Cava, D.C. Jongh, C.R. Johnson, N.A. Lebel, C.L. Stevens, Química Orgânica, second ed., Editora LTC, Rio de Janeiro, 1976. [25] K.C. Gupta, F.H. Jabrail, Effects of degree of deacetylation and cross-linking on physical characteristics, swelling and release behavior of chitosan microspheres, Carbohydr. Polym. 66 (2006) 43–54. [26] A. Manrich, C.M.A. Galvão, C.D.F. Jesus, R.C. Giordano, R.L.C. Giordano, Immobilization of trypsin on chitosan gels: use of different activation protocols and comparison with other supports, Int. J. Biol. Macromol. 43 (2008) 54–61. [27] V. Bulmus, K. Kesenci, E. Piskin, Poly(EGDMA/AAm) copolymer beads: a novel carrier for enzyme immobilization, React. Funct. Polym. 38 (1998) 1–9. [28] B.D. Ribeiro, A.M. Castro, M.A.Z. Coelho, D.M.G. Freire, Production and use of lipases in bioenergy: a review from the feedstocks to biodiesel production, Enzyme Res. (2011), doi:10.4061/2011/615803 (article ID 615803, 16 pp.). [29] L.R.B. Gonc¸alves, R.L.C. Giordano, R.C. Giordano, Effects of diffusion on the kinetics of maltose hydrolysis using glucoamylase immobilized on macroporous silica, Braz. J. Chem. Eng. 14 (04) (1997) 341–346. [30] M.D. Trevan, Immobilized Enzymes, first ed., John Wiley & Sons Ltd., England, 1980. [31] L. Betancor, F. Lopez-Gallego, A. Hidalgo, N. Alonso-Morales, G. DellamoraOrtiz, C. Mateo, R. Fernandez-Lafuente, J.M. Guisan, Different mechanisms of protein immobilization on glutaraldehyde activated supports: effect of support activation and immobilization conditions, Enzyme Microb. Technol. 39 (2006) 877–882. [32] C. Mateo, G. Fernandez-Lorente, O. Abian, R. Fernandez-Lafuente, J.M. Guisan, Multifunctional epoxy supports: a new tool to improve the covalent immobilization of proteins. The promotion of physical adsorptions of proteins on the supports before their covalent linkage, Biomacromolecules 1 (2000) 739–745. [33] C. Mateo, R. Torres, G. Fernandez-Lorente, C. Ortiz, M. Fuentes, A. Hidalgo, F. Lopez-Galego, O. Abian, J.M. Palomo, L. Betancor, B.C.C. Pessela, J.M. Guisan, R. Fernandez-Lafuente, Epoxy-amino groups: a new tool for improve immobilization of proteins by epoxy method, Biomacromolecules 4 (2003) 772–777. [34] R.M. Blanco, P. Terreros, M.F. Pérez, C. Otero, G. González, Functionalization of mesoporous silica for lipase immobilization characterization of the support and the catalysts, J. Mol. Catal. B: Enzym. 30 (2004) 83–93. [35] E. Whetje, P. Adlercreutz, B. Mattiasson, Improved activity retention of enzymes deposited on solid supports, Biotechnol. Bioeng. 41 (1993) 171–178. [36] R. Fernandez-Lafuente, D.A. Cowan, A.N.P. Wood, Hyperstabilization of a thermophilic esterase by multipoint covalent attachment, Enzyme Microb. Technol. 17 (1995) 366–372. [37] M. Iso, B. Chen, M. Eguchi, T. Kudo, S. Shrestha, Production of biodiesel fuel from triglycerides and alcohol using immobilized lipase, J. Mol. Catal. B: Enzym. 5 (2001) 3–58. [38] http://webbook.nist.gov/chemistry (accessed on 15.02.2010).