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Synthesis of reusable lipases by immobilization on hexagonal mesoporous silica and encapsulation in calcium alginate: Transesterification in non-aqueous medium
Microporous and Mesoporous Materials 86 (2005) 215–222 www.elsevier.com/locate/micromeso
Synthesis of reusable lipases by immobilization on hexagonal mesoporous silica and encapsulation in calcium alginate: Transesterification in non-aqueous medium Ganapati D. Yadav *, Sachin R. Jadhav Department of Chemical Engineering, University Institute of Chemical Technology, University of Mumbai, Matunga, Mumbai 400 019, India Received 8 June 2005; received in revised form 17 July 2005; accepted 17 July 2005 Available online 1 September 2005
Abstract Hexagonal mesoporous silica (HMS) was used for pre-immobilization of Candida antarctica lipase B (CAL B) and the effect of calcium alginate encapsulation was studied. Pseudomonas cepacia lipase (PSL), Candida rugosa lipase (CRL) and porcine pancreatic lipase (PPL) were also studied, amongst which CAL B was found to be the most active for transesterification reaction. All lipases were employed as biocatalysts in four different physical forms, such as unsupported, immobilized on HMS, encapsulated in calcium alginate (CA) and pre-immobilized on HMS and encapsulated using CA. Pre-immobilization of CAL B on hexagonal mesoporous silica (HMS) was carried out by simple physical adsorption with an immobilization yield of 62% and the maximum loading of the enzyme was 100 mg per gram of support. The recovered activity which was retained on the support was found to be 26%. The preimmobilized enzyme when encapsulated in calcium alginate beads yields a highly reusable biocatalyst with no leaching even after the fourth reuse. This novel way of preparation of a biocatalyst overcomes the problem of leaching of the enzyme from mesoporous support by avoiding a direct shear involved within the stirred reactor. The activities of all lipases in different physical forms were evaluated in the transesterification of p-chlorobenzyl alcohol with vinyl acetate to give p-chlorobenzyl acetate at 30 C. A conversion of 68% with 100% selectivity for p-chlorobenzyl acetate was obtained at 30 C in 120 min, with CAL B being pre-immobilized on HMS and encapsulated using CA (CAL B/HMS/Encap) using equimolar concentrations of reactants and 1,4-dioxane as solvent. CAL B/HMS/Encap showed excellent reusability with a decrease of only 4% in the overall conversion of the transesterification reaction even after the fourth reuse. 2005 Elsevier Inc. All rights reserved. Keywords: Pre-immobilization; Candida antarctica lipase B; Hexagonal mesoporous silica; Encapsulation; Transesterification
1. Introduction Lipases (triacylglycerol hydrolase, EC 3.1.1.3) are the most versatile and most efficient biocatalysts employed because of their high activity and stability in non-aqueous systems [1,2]. However, the high cost of the soluble * Corresponding author. Tel.: +91 22 2410 2121; fax: +91 22 2414 5614. E-mail addresses: [email protected], [email protected] (G.D. Yadav).
1387-1811/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.micromeso.2005.07.018
enzymes severely limits their use for commercial purposes. Immobilized lipases are becoming increasingly useful for biotechnological processing. Immobilization ensures reusability of enzymes and minimizes the cost of product isolation, thus overcoming the economic drawback associated with their use. Immobilization also provides operational flexibility and improves enzymesÕ thermal and chemical stability [3]. Amongst the various immobilization techniques employed, adsorption remains the most simple and cost-effective method employed for immobilization. Different supports have
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already been exploited for the immobilization of the enzyme by adsorption such as silica, porous glass beads [4], alumina [5], diatomaceous earth [6], ion exchange resin [7] and celite [8]. Lipases have been immobilized in calcium alginate gel, in inorganic microcapsules of calcium silicate and on macroporous acrylic beads [9]. Recently, various microporous and mesoporous materials have been employed for immobilization. A 1 nm pore size of the microporous solids like zeolites [10] limits their use for immobilization of the enzyme. Delaminated zeolites, however, possess pore size of around 4 nm and hence can be used as better support for immobilization as compared to zeolites [11]. But the pores in case of delaminated zeolites are arranged in a ‘‘house of cards’’ fashion and are more irregular. Moreover, the reusability of delaminated zeolites was found to be surprisingly less as compared to their aluminated counterparts [12]. Mesoporous supports are more promising and have been long sought due to their varied applications. These materials are structured, complex inorganic frameworks which not only offer a large surface area but also possess highly regular pores which are large enough to be used for the immobilization of enzymes. Researchers at Mobil Oil Corporation synthesized the first member of an extensive family of M41S [13]. The process followed a special class of liquid crystal templating (LCT) to achieve the M41S family of materials with highly crystalline and regular arrays of large channels whose diameter range from 1.5 to 10 nm. M41S could be employed for the immobilization of enzymes, as enzymes could be easily accommodated within the mesopores of the support. The mesoporous structure can be controlled by a sophisticated choice of templates (surfactants) and pores of the MCM-41 can be increased adding auxiliary organic chemicals (e.g., mesitylene) or changing reaction parameters (e.g., temperature, compositions) [13]. Hexagonal mesoporous silica (HMS) possesses high mechanical strength; physical, thermal and chemical stability and a blend of hydrophobic and hydrophilic character which makes HMS a good support for immobilization of enzymes. HMS offers a hydroxylated and ordered external surface with a very high surface area over 850 m2/g. Numerous studies on these novel materials have been published since the discovery of MCM-41 [14–17]. However, the previously reported work hardly comments on the aspect of reusability of biocatalyst immobilized on the mesoporous support, which, in fact, is the most critical issue. When employed in stirred tank reactor, leaching of enzyme from mesoporous supports is almost unavoidable. We thought that it would be worthwhile to study the effect of encapsulation on this pre-immobilized biocatalyst. Encapsulation in calcium alginate matrix offers a shield against the shear involved in the stirred reactor and also allows easy passage of reactant and products through the matrix.
The present study exploits this idea with the preimmobilization of Candida antarctica lipase B on hexagonal mesoporous silica (HMS) by adsorption followed by encapsulation in calcium alginate beads to give a highly reusable biocatalyst. The resulting immobilized biocatalyst was employed for the transesterification of p-chlorobenzyl alcohol with vinyl acetate to give p-chlorobenzyl acetate. Vinyl acetate is a very efficient acyl donor and hence was employed for the transesterification reactions involving various important alcohols [18].
2. Experimental 2.1. Enzymes and chemicals Enzymes were received as gift samples from firms of repute and included crude Pseudomonas cepacia lipase (Amano PS), crude Candida rugosa lipase (Amano AYS) from Amano Pharmaceuticals, Japan; Candida antarctica lipase B (CAL B Liquid) from Novo Nordisk, Denmark; and crude Porcine Pancreatic lipase from Advanced Biochemicals, Thane. All lipases were used as received without further purification. p-Chlorobenzyl alcohol was received as gift sample from Benzochem Industries Limited, Mumbai. Tetraethylorthosilicate (TEOS), hexadecyl amine were purchased from E-merck Ltd. 1,4-dioxane, vinyl acetate and all other chemicals and solvents which were of A.R. grade, were procured from M/s. s.d. Fine Chem Ltd., Mumbai, India. 2.2. Synthesis of support The ordered mesoporous material was prepared in the presence of a primary amine in water with ethanol as a co-solvent. The use of a co-solvent improved the template solubility and product crystallinity. The hexagonal mesoporous silica was prepared using the following procedure: 26 g of hexadecylamine was dissolved in 202 ml of ethanol. To this surfactant solution, 214 ml of deionized water was added drop by drop under vigorous stirring conditions. Any precipitate formed during the addition was solubilized by adding additional ethanol which improved the solubility of the template. Tetraethylorthosilicate (TEOS) (89 ml) was added drop by drop to the above solution under vigorous stirring. The reaction mixture was then stirred for an additional 4 h and was allowed to age for 24 h. The clear liquid above the white colored precipitate was decanted and the precipitate was dried on a glass plate at room temperature. The template was removed by calcining the resulting material at 550 C. The hexagonal mesoporous silica (HMS) thus formed was used for immobilization of Candida antarctica lipase B and other lipases.
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2.3. Characterization of support The structure and crystallinity pattern data were studied using X-ray powder diffraction. XRD data were recorded using a Phillip PW 1729 powder diffractometer ˚ ). Samples were with Cu Ka radiation (k = 1.540562 A step scanned from 1 to 40 in 0.045 steps with a stepping time of 0.5 s. The BET surface area, pore volume and pore size distribution were determined from BJH and multi-point BET methods using a Micromeritics ASAP-2010 surface area analyzer at an adsorption temperature of 77 K, after pretreating the sample under high vacuum at 300 C for 4 h. FT-IR analysis was carried out using a Bruker IFS-66 single channel Fourier transform spectrophotometer. Infrared spectra of the samples pressed in KBr pellets were obtained at a resolution of 2 cm 1 between 4000 and 350 cm 1. Spectra were collected with a Perkin–Elmer instrument and in the each case the sample was referenced against a blank KBr pellet. 2.4. Immobilization of Candida antarctica lipase B on hexagonal mesoporous silica (CAL B/HMS) Immobilization of CAL B on HMS was carried out by a simple adsorption process. The following procedure was adopted: 250 mg of HMS was pre-equilibrated with 10 ml of 0.01 M phosphate buffer (pH 7.0) for an hour. To this mixture, 10 ml of enzyme preparations (ranging form 15–145 Units/ml) was added. The mixture was stirred at room temperature. Aliquots were taken out at 15 min intervals and the stirring was continued until the (low) activity in consecutive supernatant samples remained constant. The supernatant was collected by centrifuging the mixture at 8000 rpm at room temperature for 20 min. The support was washed with a buffer three times and the washings were collected by centrifugation at 8000 rpm at room temperature for 20 min. Percent immobilization was calculated by mass balance of the activity loaded for immobilization and the activity was recovered in the supernatant and washings. The enzyme activity was determined using p-nitrophenyl acetate as substrate whereas the recovered activity on the support was calculated using olive oil as substrate. 2.5. Encapsulation of Candida antarctica lipase B immobilized on HMS in calcium alginate beads (CAL B/HMS/Encap) Candida antarctica lipase B which was pre-immobilized on HMS (CAL B/HMS) by adsorption was encapsulated in calcium alginate beads [19] by the following procedure: 4% sodium alginate solution (5 ml) and 0.1 M calcium chloride solution (10 ml) were prepared in 0.01 M phosphate buffer (pH 7.0). 200 mg of enzyme immobilized HMS was dispersed uniformly in 4%
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sodium alginate solution. The sodium alginate solution containing the enzyme was injected through a syringe to 0.1 M calcium chloride solution from a constant distance. The beads were allowed to harden in calcium chloride solution for an hour. The beads were kept in organic solvent for 2 h, thereafter. 2.6. Enzyme activity 2.6.1. Spectrophotometric method All soluble lipases were assayed for esterase activity using p-nitrophenyl acetate as substrate [20]. 50 mM solution of p-nitrophenyl acetate was prepared in dry acetonitrile. The reaction mixture (3.0 ml) was composed of 2.7 ml of 0.01 M phosphate buffer (pH 6.5) and 0.15 ml of enzyme solution of appropriate dilution where the buffer was used for the blank. The mixture was incubated for 2 min at 37 C. Reactions were initiated by addition of 0.15 ml of p-nitrophenyl acetate solution. The change in the absorbance of solution due to the release of p-nitrophenol was monitored for 2– 5 min at 400 nm (e400nm = 14,200). One unit of esterase activity of enzyme was defined as the amount of enzyme which liberates 1 lmol of p-nitrophenol per min under the assay conditions. 2.6.2. Titrimetric method The hydrolytic activities of various enzyme preparations immobilized on HMS were assayed titrimetrically using olive oil emulsion method [21]. The substrate was prepared by mixing 50 ml of olive oil with 50 ml of arabic gum solution (7% w/v). The reaction mixture consisting of 5 ml of emulsion, 2 ml of 0.1 M sodium phosphate buffer, pH 7.0 and immobilized lipase preparation (100 mg), was incubated for 10 min at 37 C. The reaction was terminated by adding 10 ml of acetone–ethanol solution (1:1). The liberated fatty acid was titrated with 0.025 N potassium hydroxide solution using phenolphthalein as an indicator. One unit (U) of enzyme activity was defined as the amount of enzyme that produced 1 lmol of free fatty acids per min under the assay conditions. 2.7. Experimental set-up for transesterification reaction Reactions were carried out in a 50 ml capacity glass reactor of 4 cm internal diameter, equipped with a close fitted four necked lid, in-built baffles and six-blade pitched turbine impeller. Both reactants in equimolar concentrations (0.01 mol) were dissolved in 1,4-dioxane and placed in the reactor assembly which was then immersed in a thermostatic water bath, whose temperature was maintained within ±1 C of the desired temperature. The reaction mixture was stirred for 15 min at 30 C at a speed of 300 rpm. The reaction was initiated by adding the desired catalyst. Periodic samples were
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removed; the lipase was separated by filtration and returned back to the reactor. 2.8. Method of analysis Analysis was done by gas chromatography using a Chemito 8510 gas chromatograph equipped with flame ionization detector and stainless steel column packed with 5% SE 30 on chromosorb (3.2 mm · 2 m). Quantification of the conversion and reaction rate was done by preparing the synthetic mixture of the reactants and the product and by plotting a calibration curve. The product was confirmed by GCMS (Perkin–Elmer Model Clarus 500) and by comparing its physical properties with the authentic compound. 2.9. Enzyme leakage studies Blank reaction was carried out, using the same experimental set-up and agitation condition as used for transesterification reaction, in 0.01 M phosphate buffer (pH 7.0). The activities of immobilized catalyst before and after the reaction time were assayed and the amount of enzyme leached out of the support was calculated.
Fig. 2. Nitrogen adsorption–desorption isotherm for hexagonal mesoporous silica.
HMS corresponds to a reversible type IV isotherm in the IUPAC classification and no adsorption–desorption hysteresis was observed at the boiling temperature of nitrogen (77 K). 3.2. Immobilization of enzyme
3. Results and discussion 3.1. Characterization of HMS Low angle X-ray diffraction (XRD) was used to elucidate mesoporosity of calcined samples of HMS. HMS shows diffraction patterns with one strong reflection in the region of 1–6 2h (Fig. 1) which is a characteristic of highly ordered hexagonal mesoporous material. The nitrogen adsorption–desorption isotherms for the calcined sample of HMS is presented in Fig. 2. The surface area, pore volume and pore diameter were determined by N2 adsorption. The surface area of HMS was found to be 858.56 m2/g whereas the pore volume and average pore diameter was found to be 0.7885 m3/g and 3.6 nm, respectively. The adsorption–desorption isotherm of
8000 7000
Intensity
6000 5000 4000 3000 2000 1000 0 1
2
3
4
5 6 2θ degree
7
8
9
Fig. 1. XRD pattern for hexagonal mesoporous silica.
10
3.2.1. Adsorption of enzyme on HMS HMS is an inert silica with free hydroxyl groups on its surface which can form hydrogen bonds with the functional groups of the side chains of amino acids (like lysine and histidine) of enzyme. Besides these forces, a weak van der Waals interaction also assists in the enzyme adsorption on the surface of the support. Hydrophobic interactions contribute in adsorption by interaction of hydrophobic patches on the enzyme with the silicon network of support. Unlike other lipases, CAL B has open conformation in organic solvent and interfacial activation was not observed. However, a report had been published where silica was functionalized with aliphatic groups to impart hydrophobic character to the silica surface which allowed strong hydrophobic interactions between the hydrophobic groups surrounding the entrance of the active site and the groups on the surface of support. There is, however, no contribution from covalent bond formation. Moreover, CAL B has molecular dimension of 3.0 · 4.0 · 5.0 nm and hence the enzyme may get plugged in the pores of HMS (3.6 nm) from one side (CAL B/HMS). This could further assist in proper orientation of the enzyme molecule. The percent immobilization of CAL B on HMS was found to be upto 62% at the maximum loading of 100 mg of enzyme per gram of support. However, only 26% of the loaded activity was retained on the support (Table 1). Higher loadings did not result in significant increase in recovered activity on support. This may be
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Table 1 Pre-immobilization of different lipases on hexagonal mesoporous silica (not encapsulated) Enzyme
Enzyme activitya (Units/ml)
Percent immobilization (%)
Recovered activityb on support (%)
Candida antarctica lipase B Pseudomonas cepacia lipase Candida rugosa lipase Porcine pancreatic lipase
362 986 136 49.5
62.2 48.5 54.7 58.7
26 21 36 54
a b
Enzyme activity of soluble lipase was calculated using p-nitrophenyl acetate as substrate. Enzyme activity of immobilized enzyme was calculated using olive oil as substrate.
due to multilayer adsorption of enzyme and formation of enzyme aggregates. As the adsorption is primarily on the surface, diffusion restrictions to reactants would be negligible. However, as the adsorption is basically due to weak forces, the enzyme gets stripped off the support during the reaction due to the direct shear involved between support and the impeller. The leakage of enzyme from support was confirmed by enzyme leakage study. Leaching studies showed that when the immobilized enzyme was employed for blank experiments with distilled water under same agitation conditions, only 60% activity remained after fresh use (Table 2). Further uses showed significant decrease in activity which was in agreement with the reusability studies (Fig. 6). This further established that the loss in the activity was due to the leaching and not due to the deactivation of enzyme. The direct shear can be avoided by further immobilizing the enzyme by encapsulation in calcium alginate beads. Pseudomonas cepacia (PSL) lipase, Candida rugosa lipase (CRL) and porcine pancreatic lipase (PPL) were also immobilized on HMS by the same procedure as that used for CAL B (Table 1). The activity loaded for immobilization was the same as that for CAL B. The same activities were loaded for comparison of transesterification activities of different lipases with CAL B. The hydrolytic activities of all immobilized lipases were calculated using titrimetric assay with olive oil as substrate. The amounts of various immobilized lipases corresponding to same hydrolytic activity were used for encapsulation. This would nullify the differences due to percent immobilization and recovered activity on the support.
3.2.2. Encapsulation of enzyme immobilized HMS in calcium alginate beads (CAL B/HMS/Encap) Encapsulation was originally used for immobilization of cells but is now being continuously sought for the immobilization of enzymes. Encapsulation can be carried out by using natural polymers like alginate [19] and carrageenan [22], synthetic polymers like photocross linkable resins and polyurethane polymers, acrylic polymers like polyacrylamide [23], hydrogel [24], microemulsion based gels and by sol–gel methods [25]. Calcium alginate encapsulation is the most frequently used immobilization method due to the fact that encapsulation can be carried out under very mild conditions [26]. Calcium alginate matrix was exploited for the encapsulation of pre-immobilized enzyme on HMS. Encapsulation of pre-immobilized enzyme avoids, almost completely, leaching of enzyme making the resulting immobilized enzyme a highly reusable biocatalyst. The matrix avoids direct contact of enzyme with impeller and hence acts as a shield against the shear. Even under high agitation conditions, it effectively protects the enzyme. The calcium alginate matrix was sufficiently cross-linked to avoid enzyme leaching of HMS and has macropores to allow easy diffusion of reactants within the matrix. This can be realized from the initial rates of reaction where the reaction started almost instantaneously after the addition of biocatalyst suggesting there was no induction period for diffusion of reactants within the matrix and be accessible to the enzyme active site. The 4% concentration of sodium alginate solution employed for immobilization allows the biocatalyst to be sufficiently sturdy for use in reactions under continuous agitation.
Table 2 Study for leaching of enzyme from HMS in water Number of cycle
Activity of CAL B/HMS (Units/min)a
Percent activity retained on the support (%)
Fresh use First cycle Second cycle Third cycle Fourth cycle
304.5 162.5 124.3 89 64.2
100 53.4 40.8 29.2 21.1
a
Activity assayed using olive oil as substrate.
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3.3. Transesterification of p-chlorobenzyl alcohol with vinyl acetate
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The reaction between the alcohol and vinyl acetate produces ester and acetaldehyde in the presence of enzymes. The reaction did not occur in the absence of enzyme at 30 C. Acetaldehyde can be removed by adding molecular sieves. Vinyl esters are very effective acyl donors for transesterification [18] and offers an effective solution to overcome equilibrium because the enol coproduct is immediately transformed irreversibly into acetaldehyde or acetone [27]. Candida antarctica lipase B (Novozyme CAL B Liquid), Pseudomonas cepacia (PSL) lipase, Candida rugosa lipase (CRL) and porcine pancreatic lipase (PPL) were evaluated for the transesterification of p-chlorobenzyl alcohol with vinyl acetate at 30 C. In the first set of experiments, equal units of all enzymes (homogeneous, unsupported) were employed to check the transesterification activity of different lipases (Fig. 3). In a second set, different lipases pre-immobilized on HMS and encapsulated in calcium alginate beads were employed for the transesterification (Fig. 4). At the same hydrolytic activity, in all studies, using unsupported enzymes, supported on HMS, and pre-immobilized on HMS and encapsulated in CA, CAL B was found to be the most active biocatalyst for the present transesterification system. Although Pseudomonas cepacia lipase showed good activity in crude form, the same enzyme showed less than half of the transesterification activity after adsorption on HMS and encapsulation (PSL/ HMS/Encap) as compared to that shown by CAL B/ HMS/Encap. In case of CAL B/HMS/Encap, 68% conversion was obtained as compared to 30% with PSL/ HMS/Encap. CRL/HMS/Encap (Candida rugosa lipase
60
60
Conversion (%)
50 40 30 20 10 0 0
20
40
60
80
100
120
140
Time (min) Fig. 3. Effect of soluble enzymes on transesterification of vinyl acetate with p-chlorobenzyl alcohol. Reaction conditions: p-chlorobenzyl alcohol, 0.01 mol; vinyl acetate, 0.01 mol, solvent—1,4-dioxane upto 20 ml; catalyst loading, 73 units; speed of agitation, 300 rpm; temperature, 30 C. Key: CAL B, j PSL, m CRL, j PPL.
Conversion (%)
70
50 40 30 20 10 0 0
20
40
60
80
100
120
140
Time (min) Fig. 4. Effect of different enzymes immobilized on HMS and encapsulated in calcium alginate on transesterification of vinyl acetate with p-chlorobenzyl alcohol. Reaction conditions: p-chlorobenzyl alcohol, 0.01 mol; vinyl acetate, 0.01 mol, solvent—1,4-dioxane upto 20 ml; catalyst loading, 200 mg; speed of agitation, 300 rpm; temperature, 30 C. Key: CAL B, j PSL, m CRL, –PPL.
pre-immobilized on HMS and encapsulated in calcium alginate beads) showed little activity whereas PPL/ HMS/Encap (porcine pancreatic lipase pre-immobilized on HMS and encapsulated in calcium alginate beads) showed no activity even after 180 min from the start of the reaction. The acetaldehyde liberated in the reaction deactivates some enzymes like Candida rugosa lipase and porcine pancreatic lipase through formation of Schiff base with lysine residue of enzyme. CAL B/ HMS/Encap was stable in the presence of acetaldehyde and retains almost total activity. 3.4. Reusability studies In a stirred tank reactor, the shear involved do not allow good reusability of enzymes immobilized on mesoporous supports due to leakage of enzymes from the surface of the support. Encapsulation of enzyme immobilized on HMS in calcium alginate beads avoids direct shear and overcomes the problem of leaching which results in the higher reusability of the biocatalyst. Reusability of CAL B was tested in three different forms to identify the effects of heterogenization of the enzyme; soluble CAL B encapsulated in calcium alginate, CAL B/HMS (CAL B immobilized on HMS) and CAL B/ HMS/Encap (Fig. 5). After the reaction, the biocatalyst was filtered off, washed with 1,4-dioxane and reused. Encapsulated CAL B and CAL B/HMS showed 50% decrease in the transesterification activity in first use itself whereas CAL B/HMS/Encap retained almost full activity even after fourth reuse (Fig. 6). Leakage studies showed that the loss in activity of CAL B/HMS was due to leaching from support and not due to the inactivation of the enzyme. Encapsulation prevents direct
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4. Conclusion
Percent activity retained (%)
120
100
80
60
40
20
0 1
2
3
4
5
6
Number of use Fig. 5. Reusability of CAL B in different immobilized forms for the transesterification of vinyl acetate with p-chlorobenzyl alcohol. Reaction conditions: p-chlorobenzyl alcohol, 0.01 mol; vinyl acetate, 0.01 mol, solvent—1,4-dioxane upto 20 ml; speed of agitation, 300 rpm; temperature, 30 C. Key: CAL B/HMS, j CAL B/ HMS/Encap, m CAL B/Encap.
80 70 60
Conversion (%)
221
50 40 30
Hexagonal mesoporous silica (HMS) was used to immobilize a variety of lipases such as Candida antarctica lipase B (CAL B), Pseudomonas cepacia lipase (PSL), Candida rugosa lipase (CRL) and porcine pancreatic lipase (PPL). The activity of the catalyst was evaluated in four different physical forms, such as unsupported, immobilized on HMS, encapsulated in calcium alginate (CA) and pre-immobilized on HMS and encapsulated using CA. In all cases, at equal hydrolytic activity of different enzymes, CAL B was found to be the most active enzyme for present transesterification reaction. Pre-immobilization of CAL B on hexagonal mesoporous silica (HMS) was carried out by a simple physical adsorption with an immobilization yield of 62% and maximum enzyme loading of 100 mg per gram of support. The recovered activity which was retained on support was found to be 26%. The pre-immobilized lipase was encapsulated in calcium alginate beads to give a highly reusable biocatalyst. Encapsulation overcomes the leaching of enzyme from mesoporous support by avoiding direct shear involved within the stirred reactor. The activity of these lipases was evaluated in the transesterification of p-chlorobenzyl alcohol with vinyl acetate to give p-chlorobenzyl acetate at 30 C. A conversion of 68% with 100% selectivity for p-chlorobenzyl acetate was obtained at 30 C in 120 min, with CAL B/HMS/ Encap using equimolar concentrations of reactants and 1,4-dioxane as solvent. CAL B/HMS/Encap showed excellent reusability with a decrease of only 4% in the overall conversion of the transesterification reaction even after the fourth reuse.
20
Acknowledgements
10 0 0
20
40
60
80
100 120 140
160
180 200
Time (min) Fig. 6. Reusability of CAL B pre-immobilized on HMS and encapsulated in calcium alginate for the transesterification of vinyl acetate with p-chlorobenzyl alcohol. Reaction conditions: p-chlorobenzyl alcohol, 0.01 mol; vinyl acetate, 0.01 mol, solvent 1,4-dioxane upto 20 ml; catalyst CAL B/HMS/Encap, 200 mg; speed of agitation, 300 rpm; temperature, 30 C. Key: fresh use, j first use, m second use, j third use, –fourth use.
contact between the support and the impeller and provides a suitable cage around the pre-immobilized enzyme avoiding the leaching of enzyme from the support. Thus, encapsulation ensures the reusability of biocatalyst and allows the biocatalyst to be used in a stirred tank reactor. Reusability of the biocatalyst also confirmed that acetaldehyde did not deactivate the enzyme.
G.D. Yadav acknowledges support from the Darbari Seth Professorship Endowment. S.R. Jadhav acknowledges the Department of Biotechnology, Government of India, for the award of JRF, which enabled the present work to be carried out.
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[17] H. Takahashi, B. Li, T. Sasaki, C. Miyazaki, T. Kajino, S. Inagaki, Micropor. Mesopor. Mater. 44–45 (2001) 755. [18] G.D. Yadav, A.H. Trivedi, Enzyme Microb. Technol. 32 (2003) 783. [19] S. Hertzberg, K. Lise, T. Anthonsen, S. Gudmund, Enzyme Microb. Technol. 14 (1992) 42. [20] F. Thara, Y. Kageyama, M. Hirata, T. Nihira, Y. Yamada, J. Biol. Chem. 266 (1991) 18135. [21] C.M.F. Soares, M.H.A. Santana, G.M. Zanin, H.F. de Castro, Biotech. Prog. 19 (2003) 803. [22] G. Para, J. Barrati, Enzyme Microb. Technol. 10 (1988) 729. [23] S.F. DÕSouza, G.B. Nadkarni, Enzyme Microb. Technol. 2 (1980) 217. ¨ ztop, E. Karadagˇ, Y. Isßikver, D. Saraydin, [24] H.N. Oztop, A.Y. O Enzyme Microb. Technol. 32 (2003) 114. [25] Gill, A. Ballesteros, Trends Biotechnol. 18 (2000) 282. [26] B. Berger, K. Faber, J. Chem. Soc. Chem. Commun. (1991) 1198. [27] A. Blandino, M. Macıas, D. Cantero, Process Biochem. 36 (2001) 601.
Synthesis of reusable lipases by immobilization on hexagonal mesoporous silica and encapsulation in calcium alginate: Transesterification in non-aqueous medium Ganapati D. Yadav *, Sachin R. Jadhav Department of Chemical Engineering, University Institute of Chemical Technology, University of Mumbai, Matunga, Mumbai 400 019, India Received 8 June 2005; received in revised form 17 July 2005; accepted 17 July 2005 Available online 1 September 2005
Abstract Hexagonal mesoporous silica (HMS) was used for pre-immobilization of Candida antarctica lipase B (CAL B) and the effect of calcium alginate encapsulation was studied. Pseudomonas cepacia lipase (PSL), Candida rugosa lipase (CRL) and porcine pancreatic lipase (PPL) were also studied, amongst which CAL B was found to be the most active for transesterification reaction. All lipases were employed as biocatalysts in four different physical forms, such as unsupported, immobilized on HMS, encapsulated in calcium alginate (CA) and pre-immobilized on HMS and encapsulated using CA. Pre-immobilization of CAL B on hexagonal mesoporous silica (HMS) was carried out by simple physical adsorption with an immobilization yield of 62% and the maximum loading of the enzyme was 100 mg per gram of support. The recovered activity which was retained on the support was found to be 26%. The preimmobilized enzyme when encapsulated in calcium alginate beads yields a highly reusable biocatalyst with no leaching even after the fourth reuse. This novel way of preparation of a biocatalyst overcomes the problem of leaching of the enzyme from mesoporous support by avoiding a direct shear involved within the stirred reactor. The activities of all lipases in different physical forms were evaluated in the transesterification of p-chlorobenzyl alcohol with vinyl acetate to give p-chlorobenzyl acetate at 30 C. A conversion of 68% with 100% selectivity for p-chlorobenzyl acetate was obtained at 30 C in 120 min, with CAL B being pre-immobilized on HMS and encapsulated using CA (CAL B/HMS/Encap) using equimolar concentrations of reactants and 1,4-dioxane as solvent. CAL B/HMS/Encap showed excellent reusability with a decrease of only 4% in the overall conversion of the transesterification reaction even after the fourth reuse. 2005 Elsevier Inc. All rights reserved. Keywords: Pre-immobilization; Candida antarctica lipase B; Hexagonal mesoporous silica; Encapsulation; Transesterification
1. Introduction Lipases (triacylglycerol hydrolase, EC 3.1.1.3) are the most versatile and most efficient biocatalysts employed because of their high activity and stability in non-aqueous systems [1,2]. However, the high cost of the soluble * Corresponding author. Tel.: +91 22 2410 2121; fax: +91 22 2414 5614. E-mail addresses: [email protected], [email protected] (G.D. Yadav).
1387-1811/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.micromeso.2005.07.018
enzymes severely limits their use for commercial purposes. Immobilized lipases are becoming increasingly useful for biotechnological processing. Immobilization ensures reusability of enzymes and minimizes the cost of product isolation, thus overcoming the economic drawback associated with their use. Immobilization also provides operational flexibility and improves enzymesÕ thermal and chemical stability [3]. Amongst the various immobilization techniques employed, adsorption remains the most simple and cost-effective method employed for immobilization. Different supports have
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already been exploited for the immobilization of the enzyme by adsorption such as silica, porous glass beads [4], alumina [5], diatomaceous earth [6], ion exchange resin [7] and celite [8]. Lipases have been immobilized in calcium alginate gel, in inorganic microcapsules of calcium silicate and on macroporous acrylic beads [9]. Recently, various microporous and mesoporous materials have been employed for immobilization. A 1 nm pore size of the microporous solids like zeolites [10] limits their use for immobilization of the enzyme. Delaminated zeolites, however, possess pore size of around 4 nm and hence can be used as better support for immobilization as compared to zeolites [11]. But the pores in case of delaminated zeolites are arranged in a ‘‘house of cards’’ fashion and are more irregular. Moreover, the reusability of delaminated zeolites was found to be surprisingly less as compared to their aluminated counterparts [12]. Mesoporous supports are more promising and have been long sought due to their varied applications. These materials are structured, complex inorganic frameworks which not only offer a large surface area but also possess highly regular pores which are large enough to be used for the immobilization of enzymes. Researchers at Mobil Oil Corporation synthesized the first member of an extensive family of M41S [13]. The process followed a special class of liquid crystal templating (LCT) to achieve the M41S family of materials with highly crystalline and regular arrays of large channels whose diameter range from 1.5 to 10 nm. M41S could be employed for the immobilization of enzymes, as enzymes could be easily accommodated within the mesopores of the support. The mesoporous structure can be controlled by a sophisticated choice of templates (surfactants) and pores of the MCM-41 can be increased adding auxiliary organic chemicals (e.g., mesitylene) or changing reaction parameters (e.g., temperature, compositions) [13]. Hexagonal mesoporous silica (HMS) possesses high mechanical strength; physical, thermal and chemical stability and a blend of hydrophobic and hydrophilic character which makes HMS a good support for immobilization of enzymes. HMS offers a hydroxylated and ordered external surface with a very high surface area over 850 m2/g. Numerous studies on these novel materials have been published since the discovery of MCM-41 [14–17]. However, the previously reported work hardly comments on the aspect of reusability of biocatalyst immobilized on the mesoporous support, which, in fact, is the most critical issue. When employed in stirred tank reactor, leaching of enzyme from mesoporous supports is almost unavoidable. We thought that it would be worthwhile to study the effect of encapsulation on this pre-immobilized biocatalyst. Encapsulation in calcium alginate matrix offers a shield against the shear involved in the stirred reactor and also allows easy passage of reactant and products through the matrix.
The present study exploits this idea with the preimmobilization of Candida antarctica lipase B on hexagonal mesoporous silica (HMS) by adsorption followed by encapsulation in calcium alginate beads to give a highly reusable biocatalyst. The resulting immobilized biocatalyst was employed for the transesterification of p-chlorobenzyl alcohol with vinyl acetate to give p-chlorobenzyl acetate. Vinyl acetate is a very efficient acyl donor and hence was employed for the transesterification reactions involving various important alcohols [18].
2. Experimental 2.1. Enzymes and chemicals Enzymes were received as gift samples from firms of repute and included crude Pseudomonas cepacia lipase (Amano PS), crude Candida rugosa lipase (Amano AYS) from Amano Pharmaceuticals, Japan; Candida antarctica lipase B (CAL B Liquid) from Novo Nordisk, Denmark; and crude Porcine Pancreatic lipase from Advanced Biochemicals, Thane. All lipases were used as received without further purification. p-Chlorobenzyl alcohol was received as gift sample from Benzochem Industries Limited, Mumbai. Tetraethylorthosilicate (TEOS), hexadecyl amine were purchased from E-merck Ltd. 1,4-dioxane, vinyl acetate and all other chemicals and solvents which were of A.R. grade, were procured from M/s. s.d. Fine Chem Ltd., Mumbai, India. 2.2. Synthesis of support The ordered mesoporous material was prepared in the presence of a primary amine in water with ethanol as a co-solvent. The use of a co-solvent improved the template solubility and product crystallinity. The hexagonal mesoporous silica was prepared using the following procedure: 26 g of hexadecylamine was dissolved in 202 ml of ethanol. To this surfactant solution, 214 ml of deionized water was added drop by drop under vigorous stirring conditions. Any precipitate formed during the addition was solubilized by adding additional ethanol which improved the solubility of the template. Tetraethylorthosilicate (TEOS) (89 ml) was added drop by drop to the above solution under vigorous stirring. The reaction mixture was then stirred for an additional 4 h and was allowed to age for 24 h. The clear liquid above the white colored precipitate was decanted and the precipitate was dried on a glass plate at room temperature. The template was removed by calcining the resulting material at 550 C. The hexagonal mesoporous silica (HMS) thus formed was used for immobilization of Candida antarctica lipase B and other lipases.
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2.3. Characterization of support The structure and crystallinity pattern data were studied using X-ray powder diffraction. XRD data were recorded using a Phillip PW 1729 powder diffractometer ˚ ). Samples were with Cu Ka radiation (k = 1.540562 A step scanned from 1 to 40 in 0.045 steps with a stepping time of 0.5 s. The BET surface area, pore volume and pore size distribution were determined from BJH and multi-point BET methods using a Micromeritics ASAP-2010 surface area analyzer at an adsorption temperature of 77 K, after pretreating the sample under high vacuum at 300 C for 4 h. FT-IR analysis was carried out using a Bruker IFS-66 single channel Fourier transform spectrophotometer. Infrared spectra of the samples pressed in KBr pellets were obtained at a resolution of 2 cm 1 between 4000 and 350 cm 1. Spectra were collected with a Perkin–Elmer instrument and in the each case the sample was referenced against a blank KBr pellet. 2.4. Immobilization of Candida antarctica lipase B on hexagonal mesoporous silica (CAL B/HMS) Immobilization of CAL B on HMS was carried out by a simple adsorption process. The following procedure was adopted: 250 mg of HMS was pre-equilibrated with 10 ml of 0.01 M phosphate buffer (pH 7.0) for an hour. To this mixture, 10 ml of enzyme preparations (ranging form 15–145 Units/ml) was added. The mixture was stirred at room temperature. Aliquots were taken out at 15 min intervals and the stirring was continued until the (low) activity in consecutive supernatant samples remained constant. The supernatant was collected by centrifuging the mixture at 8000 rpm at room temperature for 20 min. The support was washed with a buffer three times and the washings were collected by centrifugation at 8000 rpm at room temperature for 20 min. Percent immobilization was calculated by mass balance of the activity loaded for immobilization and the activity was recovered in the supernatant and washings. The enzyme activity was determined using p-nitrophenyl acetate as substrate whereas the recovered activity on the support was calculated using olive oil as substrate. 2.5. Encapsulation of Candida antarctica lipase B immobilized on HMS in calcium alginate beads (CAL B/HMS/Encap) Candida antarctica lipase B which was pre-immobilized on HMS (CAL B/HMS) by adsorption was encapsulated in calcium alginate beads [19] by the following procedure: 4% sodium alginate solution (5 ml) and 0.1 M calcium chloride solution (10 ml) were prepared in 0.01 M phosphate buffer (pH 7.0). 200 mg of enzyme immobilized HMS was dispersed uniformly in 4%
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sodium alginate solution. The sodium alginate solution containing the enzyme was injected through a syringe to 0.1 M calcium chloride solution from a constant distance. The beads were allowed to harden in calcium chloride solution for an hour. The beads were kept in organic solvent for 2 h, thereafter. 2.6. Enzyme activity 2.6.1. Spectrophotometric method All soluble lipases were assayed for esterase activity using p-nitrophenyl acetate as substrate [20]. 50 mM solution of p-nitrophenyl acetate was prepared in dry acetonitrile. The reaction mixture (3.0 ml) was composed of 2.7 ml of 0.01 M phosphate buffer (pH 6.5) and 0.15 ml of enzyme solution of appropriate dilution where the buffer was used for the blank. The mixture was incubated for 2 min at 37 C. Reactions were initiated by addition of 0.15 ml of p-nitrophenyl acetate solution. The change in the absorbance of solution due to the release of p-nitrophenol was monitored for 2– 5 min at 400 nm (e400nm = 14,200). One unit of esterase activity of enzyme was defined as the amount of enzyme which liberates 1 lmol of p-nitrophenol per min under the assay conditions. 2.6.2. Titrimetric method The hydrolytic activities of various enzyme preparations immobilized on HMS were assayed titrimetrically using olive oil emulsion method [21]. The substrate was prepared by mixing 50 ml of olive oil with 50 ml of arabic gum solution (7% w/v). The reaction mixture consisting of 5 ml of emulsion, 2 ml of 0.1 M sodium phosphate buffer, pH 7.0 and immobilized lipase preparation (100 mg), was incubated for 10 min at 37 C. The reaction was terminated by adding 10 ml of acetone–ethanol solution (1:1). The liberated fatty acid was titrated with 0.025 N potassium hydroxide solution using phenolphthalein as an indicator. One unit (U) of enzyme activity was defined as the amount of enzyme that produced 1 lmol of free fatty acids per min under the assay conditions. 2.7. Experimental set-up for transesterification reaction Reactions were carried out in a 50 ml capacity glass reactor of 4 cm internal diameter, equipped with a close fitted four necked lid, in-built baffles and six-blade pitched turbine impeller. Both reactants in equimolar concentrations (0.01 mol) were dissolved in 1,4-dioxane and placed in the reactor assembly which was then immersed in a thermostatic water bath, whose temperature was maintained within ±1 C of the desired temperature. The reaction mixture was stirred for 15 min at 30 C at a speed of 300 rpm. The reaction was initiated by adding the desired catalyst. Periodic samples were
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removed; the lipase was separated by filtration and returned back to the reactor. 2.8. Method of analysis Analysis was done by gas chromatography using a Chemito 8510 gas chromatograph equipped with flame ionization detector and stainless steel column packed with 5% SE 30 on chromosorb (3.2 mm · 2 m). Quantification of the conversion and reaction rate was done by preparing the synthetic mixture of the reactants and the product and by plotting a calibration curve. The product was confirmed by GCMS (Perkin–Elmer Model Clarus 500) and by comparing its physical properties with the authentic compound. 2.9. Enzyme leakage studies Blank reaction was carried out, using the same experimental set-up and agitation condition as used for transesterification reaction, in 0.01 M phosphate buffer (pH 7.0). The activities of immobilized catalyst before and after the reaction time were assayed and the amount of enzyme leached out of the support was calculated.
Fig. 2. Nitrogen adsorption–desorption isotherm for hexagonal mesoporous silica.
HMS corresponds to a reversible type IV isotherm in the IUPAC classification and no adsorption–desorption hysteresis was observed at the boiling temperature of nitrogen (77 K). 3.2. Immobilization of enzyme
3. Results and discussion 3.1. Characterization of HMS Low angle X-ray diffraction (XRD) was used to elucidate mesoporosity of calcined samples of HMS. HMS shows diffraction patterns with one strong reflection in the region of 1–6 2h (Fig. 1) which is a characteristic of highly ordered hexagonal mesoporous material. The nitrogen adsorption–desorption isotherms for the calcined sample of HMS is presented in Fig. 2. The surface area, pore volume and pore diameter were determined by N2 adsorption. The surface area of HMS was found to be 858.56 m2/g whereas the pore volume and average pore diameter was found to be 0.7885 m3/g and 3.6 nm, respectively. The adsorption–desorption isotherm of
8000 7000
Intensity
6000 5000 4000 3000 2000 1000 0 1
2
3
4
5 6 2θ degree
7
8
9
Fig. 1. XRD pattern for hexagonal mesoporous silica.
10
3.2.1. Adsorption of enzyme on HMS HMS is an inert silica with free hydroxyl groups on its surface which can form hydrogen bonds with the functional groups of the side chains of amino acids (like lysine and histidine) of enzyme. Besides these forces, a weak van der Waals interaction also assists in the enzyme adsorption on the surface of the support. Hydrophobic interactions contribute in adsorption by interaction of hydrophobic patches on the enzyme with the silicon network of support. Unlike other lipases, CAL B has open conformation in organic solvent and interfacial activation was not observed. However, a report had been published where silica was functionalized with aliphatic groups to impart hydrophobic character to the silica surface which allowed strong hydrophobic interactions between the hydrophobic groups surrounding the entrance of the active site and the groups on the surface of support. There is, however, no contribution from covalent bond formation. Moreover, CAL B has molecular dimension of 3.0 · 4.0 · 5.0 nm and hence the enzyme may get plugged in the pores of HMS (3.6 nm) from one side (CAL B/HMS). This could further assist in proper orientation of the enzyme molecule. The percent immobilization of CAL B on HMS was found to be upto 62% at the maximum loading of 100 mg of enzyme per gram of support. However, only 26% of the loaded activity was retained on the support (Table 1). Higher loadings did not result in significant increase in recovered activity on support. This may be
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Table 1 Pre-immobilization of different lipases on hexagonal mesoporous silica (not encapsulated) Enzyme
Enzyme activitya (Units/ml)
Percent immobilization (%)
Recovered activityb on support (%)
Candida antarctica lipase B Pseudomonas cepacia lipase Candida rugosa lipase Porcine pancreatic lipase
362 986 136 49.5
62.2 48.5 54.7 58.7
26 21 36 54
a b
Enzyme activity of soluble lipase was calculated using p-nitrophenyl acetate as substrate. Enzyme activity of immobilized enzyme was calculated using olive oil as substrate.
due to multilayer adsorption of enzyme and formation of enzyme aggregates. As the adsorption is primarily on the surface, diffusion restrictions to reactants would be negligible. However, as the adsorption is basically due to weak forces, the enzyme gets stripped off the support during the reaction due to the direct shear involved between support and the impeller. The leakage of enzyme from support was confirmed by enzyme leakage study. Leaching studies showed that when the immobilized enzyme was employed for blank experiments with distilled water under same agitation conditions, only 60% activity remained after fresh use (Table 2). Further uses showed significant decrease in activity which was in agreement with the reusability studies (Fig. 6). This further established that the loss in the activity was due to the leaching and not due to the deactivation of enzyme. The direct shear can be avoided by further immobilizing the enzyme by encapsulation in calcium alginate beads. Pseudomonas cepacia (PSL) lipase, Candida rugosa lipase (CRL) and porcine pancreatic lipase (PPL) were also immobilized on HMS by the same procedure as that used for CAL B (Table 1). The activity loaded for immobilization was the same as that for CAL B. The same activities were loaded for comparison of transesterification activities of different lipases with CAL B. The hydrolytic activities of all immobilized lipases were calculated using titrimetric assay with olive oil as substrate. The amounts of various immobilized lipases corresponding to same hydrolytic activity were used for encapsulation. This would nullify the differences due to percent immobilization and recovered activity on the support.
3.2.2. Encapsulation of enzyme immobilized HMS in calcium alginate beads (CAL B/HMS/Encap) Encapsulation was originally used for immobilization of cells but is now being continuously sought for the immobilization of enzymes. Encapsulation can be carried out by using natural polymers like alginate [19] and carrageenan [22], synthetic polymers like photocross linkable resins and polyurethane polymers, acrylic polymers like polyacrylamide [23], hydrogel [24], microemulsion based gels and by sol–gel methods [25]. Calcium alginate encapsulation is the most frequently used immobilization method due to the fact that encapsulation can be carried out under very mild conditions [26]. Calcium alginate matrix was exploited for the encapsulation of pre-immobilized enzyme on HMS. Encapsulation of pre-immobilized enzyme avoids, almost completely, leaching of enzyme making the resulting immobilized enzyme a highly reusable biocatalyst. The matrix avoids direct contact of enzyme with impeller and hence acts as a shield against the shear. Even under high agitation conditions, it effectively protects the enzyme. The calcium alginate matrix was sufficiently cross-linked to avoid enzyme leaching of HMS and has macropores to allow easy diffusion of reactants within the matrix. This can be realized from the initial rates of reaction where the reaction started almost instantaneously after the addition of biocatalyst suggesting there was no induction period for diffusion of reactants within the matrix and be accessible to the enzyme active site. The 4% concentration of sodium alginate solution employed for immobilization allows the biocatalyst to be sufficiently sturdy for use in reactions under continuous agitation.
Table 2 Study for leaching of enzyme from HMS in water Number of cycle
Activity of CAL B/HMS (Units/min)a
Percent activity retained on the support (%)
Fresh use First cycle Second cycle Third cycle Fourth cycle
304.5 162.5 124.3 89 64.2
100 53.4 40.8 29.2 21.1
a
Activity assayed using olive oil as substrate.
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3.3. Transesterification of p-chlorobenzyl alcohol with vinyl acetate
80
The reaction between the alcohol and vinyl acetate produces ester and acetaldehyde in the presence of enzymes. The reaction did not occur in the absence of enzyme at 30 C. Acetaldehyde can be removed by adding molecular sieves. Vinyl esters are very effective acyl donors for transesterification [18] and offers an effective solution to overcome equilibrium because the enol coproduct is immediately transformed irreversibly into acetaldehyde or acetone [27]. Candida antarctica lipase B (Novozyme CAL B Liquid), Pseudomonas cepacia (PSL) lipase, Candida rugosa lipase (CRL) and porcine pancreatic lipase (PPL) were evaluated for the transesterification of p-chlorobenzyl alcohol with vinyl acetate at 30 C. In the first set of experiments, equal units of all enzymes (homogeneous, unsupported) were employed to check the transesterification activity of different lipases (Fig. 3). In a second set, different lipases pre-immobilized on HMS and encapsulated in calcium alginate beads were employed for the transesterification (Fig. 4). At the same hydrolytic activity, in all studies, using unsupported enzymes, supported on HMS, and pre-immobilized on HMS and encapsulated in CA, CAL B was found to be the most active biocatalyst for the present transesterification system. Although Pseudomonas cepacia lipase showed good activity in crude form, the same enzyme showed less than half of the transesterification activity after adsorption on HMS and encapsulation (PSL/ HMS/Encap) as compared to that shown by CAL B/ HMS/Encap. In case of CAL B/HMS/Encap, 68% conversion was obtained as compared to 30% with PSL/ HMS/Encap. CRL/HMS/Encap (Candida rugosa lipase
60
60
Conversion (%)
50 40 30 20 10 0 0
20
40
60
80
100
120
140
Time (min) Fig. 3. Effect of soluble enzymes on transesterification of vinyl acetate with p-chlorobenzyl alcohol. Reaction conditions: p-chlorobenzyl alcohol, 0.01 mol; vinyl acetate, 0.01 mol, solvent—1,4-dioxane upto 20 ml; catalyst loading, 73 units; speed of agitation, 300 rpm; temperature, 30 C. Key: CAL B, j PSL, m CRL, j PPL.
Conversion (%)
70
50 40 30 20 10 0 0
20
40
60
80
100
120
140
Time (min) Fig. 4. Effect of different enzymes immobilized on HMS and encapsulated in calcium alginate on transesterification of vinyl acetate with p-chlorobenzyl alcohol. Reaction conditions: p-chlorobenzyl alcohol, 0.01 mol; vinyl acetate, 0.01 mol, solvent—1,4-dioxane upto 20 ml; catalyst loading, 200 mg; speed of agitation, 300 rpm; temperature, 30 C. Key: CAL B, j PSL, m CRL, –PPL.
pre-immobilized on HMS and encapsulated in calcium alginate beads) showed little activity whereas PPL/ HMS/Encap (porcine pancreatic lipase pre-immobilized on HMS and encapsulated in calcium alginate beads) showed no activity even after 180 min from the start of the reaction. The acetaldehyde liberated in the reaction deactivates some enzymes like Candida rugosa lipase and porcine pancreatic lipase through formation of Schiff base with lysine residue of enzyme. CAL B/ HMS/Encap was stable in the presence of acetaldehyde and retains almost total activity. 3.4. Reusability studies In a stirred tank reactor, the shear involved do not allow good reusability of enzymes immobilized on mesoporous supports due to leakage of enzymes from the surface of the support. Encapsulation of enzyme immobilized on HMS in calcium alginate beads avoids direct shear and overcomes the problem of leaching which results in the higher reusability of the biocatalyst. Reusability of CAL B was tested in three different forms to identify the effects of heterogenization of the enzyme; soluble CAL B encapsulated in calcium alginate, CAL B/HMS (CAL B immobilized on HMS) and CAL B/ HMS/Encap (Fig. 5). After the reaction, the biocatalyst was filtered off, washed with 1,4-dioxane and reused. Encapsulated CAL B and CAL B/HMS showed 50% decrease in the transesterification activity in first use itself whereas CAL B/HMS/Encap retained almost full activity even after fourth reuse (Fig. 6). Leakage studies showed that the loss in activity of CAL B/HMS was due to leaching from support and not due to the inactivation of the enzyme. Encapsulation prevents direct
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4. Conclusion
Percent activity retained (%)
120
100
80
60
40
20
0 1
2
3
4
5
6
Number of use Fig. 5. Reusability of CAL B in different immobilized forms for the transesterification of vinyl acetate with p-chlorobenzyl alcohol. Reaction conditions: p-chlorobenzyl alcohol, 0.01 mol; vinyl acetate, 0.01 mol, solvent—1,4-dioxane upto 20 ml; speed of agitation, 300 rpm; temperature, 30 C. Key: CAL B/HMS, j CAL B/ HMS/Encap, m CAL B/Encap.
80 70 60
Conversion (%)
221
50 40 30
Hexagonal mesoporous silica (HMS) was used to immobilize a variety of lipases such as Candida antarctica lipase B (CAL B), Pseudomonas cepacia lipase (PSL), Candida rugosa lipase (CRL) and porcine pancreatic lipase (PPL). The activity of the catalyst was evaluated in four different physical forms, such as unsupported, immobilized on HMS, encapsulated in calcium alginate (CA) and pre-immobilized on HMS and encapsulated using CA. In all cases, at equal hydrolytic activity of different enzymes, CAL B was found to be the most active enzyme for present transesterification reaction. Pre-immobilization of CAL B on hexagonal mesoporous silica (HMS) was carried out by a simple physical adsorption with an immobilization yield of 62% and maximum enzyme loading of 100 mg per gram of support. The recovered activity which was retained on support was found to be 26%. The pre-immobilized lipase was encapsulated in calcium alginate beads to give a highly reusable biocatalyst. Encapsulation overcomes the leaching of enzyme from mesoporous support by avoiding direct shear involved within the stirred reactor. The activity of these lipases was evaluated in the transesterification of p-chlorobenzyl alcohol with vinyl acetate to give p-chlorobenzyl acetate at 30 C. A conversion of 68% with 100% selectivity for p-chlorobenzyl acetate was obtained at 30 C in 120 min, with CAL B/HMS/ Encap using equimolar concentrations of reactants and 1,4-dioxane as solvent. CAL B/HMS/Encap showed excellent reusability with a decrease of only 4% in the overall conversion of the transesterification reaction even after the fourth reuse.
20
Acknowledgements
10 0 0
20
40
60
80
100 120 140
160
180 200
Time (min) Fig. 6. Reusability of CAL B pre-immobilized on HMS and encapsulated in calcium alginate for the transesterification of vinyl acetate with p-chlorobenzyl alcohol. Reaction conditions: p-chlorobenzyl alcohol, 0.01 mol; vinyl acetate, 0.01 mol, solvent 1,4-dioxane upto 20 ml; catalyst CAL B/HMS/Encap, 200 mg; speed of agitation, 300 rpm; temperature, 30 C. Key: fresh use, j first use, m second use, j third use, –fourth use.
contact between the support and the impeller and provides a suitable cage around the pre-immobilized enzyme avoiding the leaching of enzyme from the support. Thus, encapsulation ensures the reusability of biocatalyst and allows the biocatalyst to be used in a stirred tank reactor. Reusability of the biocatalyst also confirmed that acetaldehyde did not deactivate the enzyme.
G.D. Yadav acknowledges support from the Darbari Seth Professorship Endowment. S.R. Jadhav acknowledges the Department of Biotechnology, Government of India, for the award of JRF, which enabled the present work to be carried out.
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