Process Biochemistry 41 (2006) 114–119 www.elsevier.com/locate/procbio
The novel hexadecyltrimethylammonium bromide (CTAB) based organogel as reactor for ester synthesis by entrapped Candida rugosa lipase Francesco Lopez a,b,*, Francesco Venditti a,b, Giuseppe Cinelli a, Andrea Ceglie a,b b
a Department of Food Technology, University of Molise, Via De Sanctis, I-86100 Campobasso, Italy Consorzio Interuniversitario per lo sviluppo dei Sistemi a Grande Interfase (CSGI), c/o Department of Food Technology, University of Molise, I-86100 Campobasso, Italy
Received 31 March 2005; received in revised form 17 May 2005; accepted 18 May 2005
Abstract The novel microemulsion based organogel (MBG) prepared with cationic surfactant hexadecyltrimethylammonium bromide (CTAB) was used as support for the immobilization of lipase from Candida rugosa. In this study, we found that lipase entrapped in this system is able to catalyze the esterification reaction of pentanol with caprylic acid in hexane. The maximal pentyl caprilate production of about 94% was reached in about 215 h. Recycling the immobilized enzyme was shown to be feasible, demonstrating that lipase retains its activity for several cycles. Remarkably, the enzymatic activity of lipase immobilized in the cationic MBG remained almost stable at 30 8C for at least 2 months. Since highly reproducible results and quantitative production of pentyl caprylate was obtained at low costs, our data suggest that the novel CTAB microemulsion based gel represents a reliable tool for biotechnological applications and a commercially attractive system. The devised matrix may be generally applicable to other biologically interesting reaction systems, i.e. to bioconversion processes in organic solvents. # 2005 Elsevier Ltd. All rights reserved. Keywords: CTAB; Microemulsion based organogel; Lipase; Pentyl caprylate; Pentanol; Enzyme immobilization
1. Introduction The introduction of gelatin-based organogels in the 1980s [1,2] was of outstanding importance for the improvement of most the biotechnological processes performed in apolar media. Microemulsion based gels (MBGs) are usually prepared by mixing an aqueous gelatin solution and a w/o microemulsion [3–6]. Gelatin is obtained by thermal degradation and hydrolysis of collagen and has been largely used as gelling agent in water for pharmaceutical, food, cosmetic and other industrial applications [7]. * Corresponding author. Tel.: +39 0874404632; fax: +39 0874404652. E-mail address:
[email protected] (F. Lopez). 1359-5113/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2005.05.009
Microemulsions are defined as transparent, isotropic, thermodynamically stable solutions of oil and water stabilized by surfactant molecules [8]. The use of microemulsion systems in many types of biotransformation processes has been documented extensively. Starting from studies describing reactions effected with enzymes solubilized in microemulsion systems [9], to reactions performed in non aqueous media [10], a plethora of literature is available in the field of biotransformations [5,11]. Recently, immobilization of enzymes on suitable supports has represented the main goal of biotechnological applications; in particular, the new technology is dealing with supports that allow high activity and high stability of products coexisting with low costs.
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The main advantage offered by organogels comes from the possibility of utilizing systems in a solid (MBGs) rather than a liquid form (microemulsions). In particular, their potential in biotechnological applications stems from the fact that the guest molecules solubilized in MBGs can be recycled, with apparent economical benefits. Furthermore, such kind of supports are easy to handle and suitable for storage and manipulation processes. Until recently, almost all the reported organogels containing gelatin were prepared using reverse micelles made of the anionic surfactant bis-2-ethylhexylsodiumsuccinate (AOT) [1,3,5]. In light of the increasing interest in the interactions between polymer and surfactants in general, and interactions between oppositely charged molecules and polymer in particular [12], the possibility of using a positive charged surfactant appears of critical importance. It is in fact well known that the nature of the polar head group of the surfactant can exert a great impact on reaction rates and equilibrium position of desiderate chemical reactions. Recently, a new organogel based on cationic surfactant has been developed in our laboratory [13]. Remarkably, this kind of support represents a great improvement in the development of new tools to study the protein–surfactant interactions and, more interestingly, from a biotechnological point of view, it could allow the encapsulation of negatively charged hosts, like nucleic acid [14] and protein. In previous studies, we have characterized the microemulsion CTAB/water/hexane/pentanol system [15] and the hydrolyzing activity of lipase from Candida rugosa solubilized in this quaternary microemulsion system [16]. In the present work, we analyzed the behavior of lipase from C. rugosa when immobilized in the MBG CTAB/ water/hexane/pentanol system stabilized by gelatin, with a particular interest in the ‘‘synthetic ability’’ of the enzyme, i.e. the esterification process of caprylic acid with pentanol in hexane. The choice of lipase as enzyme stems from two reasons: the first is that lipases acting at the lipid–water interfaces are well suited for water-insoluble substrates and the second, more interesting for biotechnological applications, is that lipases are among the most widely used enzymes for esterification of a large variety of substrates [17–19]. In order to optimize the esterification process and overcome the problem of low diffusion rate of substrates described in the MBG [20], we used lipase immobilized in a slightly modified form of the matrix, consisting of a ‘‘pulverized’’ form of dried MBG CTAB/water/hexane/ pentanol system stabilized by gelatin. Last, but not least, we have also tested the possibility of recycling the immobilized enzyme, demonstrating that lipase retains its activity for several cycles. Such a possibility is crucial, since it would allow for regeneration of enzymes, one of the most challenging problems to be solved in industrial processes.
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2. Materials and methods 2.1. Chemicals Hexadecyltrimethylammonium bromide, 1-pentanol, hexane, caprylic acid, gelatin type A from porcine skin (bloom 300), Lipase VII from C. rugosa were purchased from Sigma. CTAB was three times re-crystallized from anhydrous ethanol, and stored over dried silica gel under vacuum. Water was twice distilled in an all-quartz device. Lipase VII (1100 U/mg) was used without any further purification. Hexane and pentanol were used as received. 2.2. Preparation of CTAB MBGs containing lipase The preparation of the MBGs and the parameters used to define the microemulsions here used were the same as described in Lopez et al. [13]. To define the microemulsion composition, we use as parameters the molar ratios, i.e. water/CTAB (W0), pentanol/ CTAB (P0) and the overall concentration of CTAB. For our purpose, samples were prepared as follows: gelatin was weighted in a glass vial for a final overall concentration of 7% (w/v) and the appropriate amount of potassium phosphate buffer solution (50 mM, pH 6.0) was added. The microemulsion CTAB/buffer solution/hexane/pentanol (1.5 ml) at W0 = 5, P0 = 4.8, [CTAB] = 0.1 M obtained as already described [13] was then added to the mixture. The solution was stirred at 60 8C for 20 min and continuously stirred during the cooling process till the temperature reached 30 8C. Lipase (0.33 mg) from a 6 mg/ml stock solution in potassium phosphate 50 mM, pH 6.0 was then added (at 30 8C) to 1.5 ml of the microemulsion and gelatin solution during the cooling phase. The final W0 of the sample was 65. The lipase containing gel was then transferred in a 25 ml vial, and reduced into small pieces. Finally, the solvent was removed by evaporation resulting in a dry gelatin film entrapping the enzyme. Such a film was finely scratched with a spatula. Hereafter, we will refer to this powder as ‘‘pulverized’’ organogel. 2.3. Enzymatic reaction The enzymatic reaction was started at 30 8C by adding caprylic acid and pentanol in hexane (15 ml) to the lipase containing gel. The concentrations of caprylic acid and pentanol were 5 mM. At regular time intervals, samples were withdrawn from the reaction medium and analyzed by gas chromatography. After the first run of the enzymatic reaction (more than 200 h), the solvent was removed with N2 and the pulverized MBG was washed with fresh hexane, kept in solvent at 30 8C for 8 h and centrifuged. The procedure was repeated at least three times to remove traces of ester and free enzyme. After removing the hexane, the organogel was placed into new vials to be re-used.
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The enzymatic reaction catalyzed by free lipase was started by adding the enzymatic solution to the vial containing pentanol and caprylic acid in hexane used in the same proportion as described above. Gas chromatography (GC) was performed on a VG Trio 2000 instrument equipped with a SPB-5 fused silica capillary column (30 m, 0.2 mm film thickness, Supelco), connected to an on-column injector through retention gap and used with the following temperature program: 90– 280 8C, 10 8C/min. Nitrogen served as carrier gas and the injector and detector temperature was 280 8C.
3. Results and discussion 3.1. Overlook of the phase diagram In this work, we investigate the ability of gelatin organogels based on cationic surfactant to immobilize enzymes and optimize the yield of products for biotransformation reactions. The principal aim of the work was to investigate on the potential of using the cationic MBGs in enzyme catalyzed reaction performed in organic solvents. In Fig. 1, the partial phase diagram in the variables W0 and gelatin concentration and the composition of samples used for the enzymatic reaction are shown. As clearly
indicated in the reported graph, W0 and gelatin percentage are parameters of critical importance in order to obtain a transparent gel. At low gelatin loading, the samples are low viscous (circles). Samples at W0 < 78 are optically isotropic so they can be considered as ordinary liquids, while at higher W0 values, they are birefrangent suggesting the presence of long-range anisotropic structures. For high enough gelatin loading, samples are solid-like and do not flow under gravity (triangles) [13]. The choice of the suitable composition is the result of a reasonable compromise between the W0 and the gelatin content. The content of gelatin should be high enough to obtain a stiff gel that can be easily handled and cut. As far as the W0 is concerned, in our experiments, we utilized W0 = 65 because this value is in between the minimum water loading required for the formation of single phase MBG (W0 = 40) and the boundary with birefrangent systems (W0 > 80). Moreover, it was demonstrated in anionic MBG systems that an excess of water favors the ester hydrolysis [21]. In the inset, in correspondence of W0 = 65 and gelatin percentage (7%), parameters utilized for our purposes, the image of typical vials containing the cationic MBG before evaporation is reported in order to show either the consistence and the transparency of the matrix. On the right, a schematic representation of the interconnected network of gelatin and water rods stabilized by a monolayer of surfactant, in coexistence with a population w/o
Fig. 1. Schematic representation of the partial phase diagram for the CTAB MBGs. Circles, triangles and diamonds represent liquid, gel and two-phase systems, respectively [13,16]. Samples at W0 < 78 are optically isotropic (grey panel on the left). In the upper inset, the optimal range of the gel and the aspect of the sample after preparation and before the solvent evaporation is reported. In the inset showed below the probably schematic model by analogy with the AOT MBG proposed model.
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microemulsion, by analogy with the AOT MBG proposed by Atkinson is represented [4]. 3.2. Enzymatic activity Lipase from C. rugosa immobilized in our novel cationic organogel was used to catalyze the esterification reaction in hexane. Caprylic acid and pentanol were used as substrates, as shown in Scheme 1. Lipases are known to act at oil–water interface [22]. The interfacial activation is associated with a conformational change in the lipase molecule. This hypothesis is in accordance with X-ray crystallographic studies of lipases from different species showing a large displacement of the loop covering the active site in response to the interfacial activation [23]. Thus, it is likely that conformational rearrangement of surface loops in these enzymes in response to the oil–water interface is an essential part of their functionality. Supposing that our system is made of this rigid interconnected network of gelatin and water rods stabilized by a monolayer of surfactant, in coexistence with a population of w/o microemulsion [4], we can assume that lipase can easily be activated by the huge interfacial areas present in this network. Rees et al. already demonstrated in AOT based MBGs that pelleted matrices retain the surfactant, gelatin, water and enzymes molecules without loosing physical stability [24]. Although a number of studies has clearly indicated that pelleted and granulated organogel are a powerful tool for the yield optimization of enzymatic reactions, there are experimental evidences from Jenta et al. [20] demonstrating that in such kind of matrix, the diffusion of substrate and products is the rate limiting step. In particular, the pelleted AOT based MBGs were described by these authors as regularly sized sections obtained from cooled organogel (at 25 8C) cut in regular pieces. In the same paper, granulated organogel were described as granules ca. 0.5 mm in diameter obtained from the MBGs by using a mechanical grinding [20]. In our novel cationic based organogel, in order to obtain a low substrate diffusion distances and thus increase the reaction rate, we decided to take advantage of a slightly different procedure. For this purpose, the gel obtained by mixing 1.5 ml of the CTAB microemulsion and gelatin solution with lipase from C. rugosa (see Section 2 for details) was cut in small pieces and the vial containing the matrix was kept opened to allow the solvents evaporation. The dried gelatin film entrapping the enzyme was then scratched with a spatula. This procedure resulted in a fine powder consisting of granules with an average in diameter of ca. 1 mm.
Fig. 2. Relative concentrations of substrates and products in reaction mixture containing lipase immobilized in CTAB organogel as function of time at 30 8C. Closed symbol = pentyl caprylate; open symbol = caprylic acid. Conditions: 0.33 mg dissolved lipase in 1.5 ml of gel, concentrations of caprylic acid and pentanol were 5 mM. The composition of the CTAB MBG gel was: CTAB/buffer solution/hexane/pentanol microemulsion at W0 = 65, P0 = 4.8, [CTAB] = 0.1 M and gelatin 7%. Data point are the average for at least three experiments error bars indicate the standard deviations. Lines reported in figure are mere guides for eyes.
Fig. 2 shows the esterification process in the so obtained CTAB microemulsion-based gel of caprylic acid in hexane with pentanol at 30 8C as a function of time. The concentrations of both substrates and products in hexane analyzed by gas chromatography at different time intervals are reported. As clearly indicated in the graph, the half time of the reaction is around 40 h and both ester conversion and reagents consumption appear to go almost to completion in 200 h. Data points reported in Fig. 2 are the average from at least three independent experiments. The small error bars indicate the high reproducibility of the enzymatic activity in the CTAB organogel. We next decided to compare the yield of ester synthesis catalyzed by free lipase in hexane to that obtained from the enzyme trapped in the CTAB MBGs system. As depicted in Fig. 3, notwithstanding the same amount of enzyme utilized, the kinetics of pentyl caprylate production catalyzed by the unprotected lipase is slower than that obtained with the organogel entrapped enzyme. This result strongly suggests that the lipase entrapped in the CTAB MBGs retains its native structure. Another likely explanation could be that the water produced in the ester synthesis is highly bound to the gelatin and not available for the hydrolysis reaction. Most probably, both the two factors concur to the improvement of the yield and rate with respect to the free enzymes in solvent.
Scheme 1.
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Fig. 3. Yields of pentyl caprylate at 30 8C in hexane as function of time using C. rugosa lipase immobilized in the CTAB microemulsion based organogel (closed circles) and free lipase (closed triangles). Experiments were performed with the same amount of enzyme and reagents (0.33 mg lipase dissolved in 1.5 ml of gel, concentrations of caprylic acid and pentanol were 5 mM). For the free lipase reaction, lipase was dissolved directly in the reaction bulk. The composition of the CTAB MBG gel was: CTAB/buffer solution/hexane/pentanol microemulsion at W0 = 65, P0 = 4.8, [CTAB] = 0.1 M, and gelatin 7%. Lines reported in figure are mere guides for eyes.
3.3. Reutilization of enzymes As already stated, another important aspect of MBGs use for the related biotechnological implications concerns the possibility of enzyme recycling. To investigate in this direction, we analyzed the enzymatic activity of lipase in CTAB MBGs in successive cycles. As indicated in Fig. 4, after the first cycle (which took several days), a negligible amount of the enzymatic activity was lost.
Fig. 4. Yields of pentyl caprylate at 30 8C in hexane as function of time using C. rugosa lipase immobilazed in the CTAB microemulsion based organogel after washing of the pulverized gel. Circles, triangles and squares represent cycle I, cycle II, cycle IV, respectively. Experiment were performed with the same amount of enzyme and reagents (0.33 mg lipase dissolved in 1.5 ml of gel, concentrations of caprylic acid and pentanol were 5 mM). The composition of the CTAB MBG gel was: CTAB/buffer solution/hexane/pentanol microemulsion at W0 = 65, P0 = 4.8, [CTAB] = 0.1 M, and gelatin 7%. Lines reported in figure are mere guides for eyes.
Fig. 5. Yields of pentyl caprylate at 30 8C in hexane after 215 h reaction as function of run number and days passed from the sample preparation of C. rugosa lipase immobilized in the CTAB microemulsion based organogel.
This result indicates that after the reaction and the washing steps, the ‘‘pulverized’’ gel in the second and in the third runs almost completely retained its integrity and the yield of the products seems to remain almost stable after 215 h. Only in the fourth cycle, the reaction rate slowed down to some extent, most likely because of the loss of material caused by the repeated washing steps and/or the accumulation of water as a product of the reaction. As far as the enzymatic activity is concerned, the data collected indicate that lipase remains stable for at least 2 months even at 30 8C. This evidence is clearly illustrated in Fig. 5, where we try to further discuss the data in terms of enzyme stability in function of the time passed since the lipase containing MBGs preparation. In details, the histogram reports the yield of pentyl caprylate production after 215 h as a function of number of cycles and, in the same time, of number of days. The time 0 corresponds to the day of the lipase containing organogel preparation (see Section 2). From this representation, it becomes apparent that even after 20 days (the end of the second cycle), the enzyme entrapped in CTAB MBG retains almost completely its activity. It should be noticed here that in our procedure, the MBGs containing lipase were kept in hexane at 30 8C in between the reaction cycles. Remarkably, the esterification yield (the end of the third cycle) slightly but not significantly decreased after 45 days and an appreciable drop of the yield (to 60%) was observed only after 71 days. Noticeably, the same amount of ester obtained in our conditions in approximately 200 h in the first cycle can be obtained after 2 months. This information could be in our opinion highly valuable for industrial applications, since it precisely indicates the time-range of the maximal products yield after the enzymes encapsulation in these experimental conditions.
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4. Conclusions In this work, the enzymatic activity of lipase from C. rugosa immobilized on a novel cationic MBG was studied. The data presented indicate that lipase entrapped in CTAB based organogel is able to catalyze a quantitative conversion (94%) of caprylic acid and pentanol in pentyl caprylate after 215 h. Furthermore, the recycling of the cationic organogel was shown to be possible for several runs without loosing significant amount of lipase activity. As a whole, the data collected suggest that the pulverized form of these MBGs could be a useful tool for a number of biotechnological applications. Based on the results here presented, it should be possible to design continuous systems, i.e. column reactors, that could allow an appreciable reduction of the esterification process costs. Furthermore, the novel organogel based on cationic surfactant here used as reactor for lipase catalyzed esterification could represent, in our opinion, an example for the improvement of bioconversion processes in organic solvents. By using these kind of cationic based MBGs, it would be in fact possible to encapsulate negatively charged hosts like nucleic acids and different kind of proteins. Immobilization in this matrix of different enzymes like lactamase, better solubilized in cationic surfactants [25], is our next research challenge.
Acknowledgements This work was supported by the MIUR of Italy (PRIN 2003 Nanoscienze Per Lo Sviluppo Di Nuove Tecnologie) and by Consorzio Interuniversitario per lo sviluppo dei Sistemi a Grande Interfase (CSGI-Firenze). We wish to thank Dr. Gerardo Palazzo for useful discussion.
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