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Direct enzymatic acylation of cellulose pretreated in BMIMCl ionic liquid
Bioresource Technology 102 (2011) 1378–1382
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Direct enzymatic acylation of cellulose pretreated in BMIMCl ionic liquid Stavros Gremos, Dimitra Zarafeta, Dimitris Kekos, Fragiskos Kolisis ⇑ Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, Zografou Campus, 15780 Athens, Greece
a r t i c l e
i n f o
Article history: Received 5 July 2010 Received in revised form 3 September 2010 Accepted 6 September 2010 Available online 15 September 2010 Keywords: Enzymatic acylation Cellulose Ionic liquid Solvent free reaction Cellulose ester
a b s t r a c t Cellulose esters are an important class of functional biopolymers with great interest in the chemical industry. In this work the enzymatic acylation of Avicel cellulose with vinyl propionate, vinyl laurate and vinyl stearate, has been performed successfully in a solvent free reaction system. At first cellulose was putted into the ionic liquid BMIMCl (1-n-butyl-3-methylimidazolium chloride) in order to facilitate the unwrap of the structure of the polysaccharide molecule and make it accessible to the enzyme. Thus, after this pretreatment the enzymatic esterification reaction was performed using various hydrolases. The enzymes capable of catalyzing the acylation of cellulose were found to be the immobilized esterase from hog liver and the immobilized cutinase from Fusarium solani, while the lipases used did not show any catalytic activity. Cellulose esters of propionate, laurate and stearate were synthesized with a degree of esterification of 1.9%, 1.3% and 1.0%, respectively. It is the first successful direct enzymatic acylation of cellulose with long chain fatty acids. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Cellulose is the most abundant polysaccharide in nature. It occurs in plants under the form of microfibrils, components of the structurally strong framework of the cell walls. Cellulose is also produced in a highly hydrated form by some bacteria (e.g. Acetobacter xylinum) but it is mostly prepared from wood pulp (Kadla and Gilbert, 2000). It is a linear polymer of b-(1,4)-D-glucopyranose units in C1 conformation. The fully equatorial conformation of b-linked glucopyranose residues stabilizes the chair structure, minimizing its flexibility. Due to the extended intra- and intermolecular hydrogen bonding between the cellulose chains, this biopolymer has a high crystalline structure. As a result it is completely insoluble in water or in common solvents leading to difficulties in chemical manipulation (Kadla and Gilbert, 2000). On the other hand the plenitude, the renewability and the low cost of cellulose make it an ideal feedstock for producing different materials. About a third of the world’s production of purified cellulose is used as the base substance for a number of derivatives with predesigned and wide-ranging properties depending on the groups involved and the degree of derivatization (Lee and Wang, 2006; Siro and Plackett, 2010; Takatani et al., 2008). Cellulose has great potential for the preparation of novel materials (e.g. thermoplastics), exhibiting a number of well-known advantages such as biocompatibility, high stiffness, good mechanical properties and biodegradability. In particular acyl esters of cellulose are ⇑ Corresponding author. Address: Polytechniou 9, Zografou Campus, 15780 Athens, Greece. Tel.: +30 210 7723156. E-mail address: [email protected] (F. Kolisis). 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.09.021
considered to be an important class of polymers used in the production of fibers, plastics, films, cosmetics and drugs (Edgar, 2007; Gradwell, 2004; Wibowo et al., 2006). To date the commercial synthesis of these compounds is achieved in heterogeneous reaction systems containing carboxylic acid anhydrides, tough solvents like N,N-dimethylacetamide and acid catalysts. This process is also limited to the production of short chain esters with a length of four carbon atoms or less. This is not advantageous because the cellulose ester is expected to have better thermoplastic properties when the chain of the ester contains more than six carbons. That happens because the longer chain acts as an internal plasticizer (Yin et al., 2007). In order to overcome the above situation and introduce biocatalysis into the field of cellulose esterification, we designed a system for the enzymatic acylation of this polysaccharide with long chain fatty acids. The realisation that enzymes can function in non-aqueous media has given to their synthetic potential a powerful boost for many processes (Ikeda and Klibanov, 1993; Klibanov, 1989; Patel et al., 1996; Zaks and Klibanov, 1985). Amongst the numerous enzymes studied in such systems, esterases and in particular lipases have been successfully utilized in a wide range of stereoselective and regioselective acylations of various target molecules including sugars (Kirk et al., 1995; Ljunger et al., 1994; Patel et al., 1996; Tsitsimpikou et al., 1998). The first attempt of the enzymatic esterification of cellulose was made a few years ago (Sereti et al., 1998, 2001) by subjecting Avicel cellulose, the fatty acid as substrate and lipase into several organic solvents. The reaction was not successful due to the high crystallinity of cellulose. The macromolecule could not be dissolved and there was no accessibility by the enzyme. Only soluble cellulose derivatives, such as cellulose
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acetate and hydroxypropyl cellulose, were found to be acylated by lipases. In the present work, this problem was tackled by introducing a preliminary step of cellulose treatment, using ionic liquids before the enzymatic reaction in order to make the cellulosic molecules as permeable as possible. Ionic liquids are organic salts whose ions do not pack well and remain liquid in ambient temperatures (Earle and Seddon, 2000). Some studies showed that cellulose can be dissolved in some hydrophilic ionic liquids such as 1-n-butyl3-methylimidazolium chloride (BMIMCl) and 1-allyl-3-methylimidazolium chloride (AMIMCl) (Wu et al., 2004; Zhao et al., 2009; Zhu et al., 2006). Particularly BMIMCl can dissolve cellulose up to 25% (w/w). Cellulose in its BMIMCl solution can be easily precipitated by addition of water, ethanol or acetone. Moreover, the regenerated cellulose has almost the same degree of polymerization (DP) as the initial one (Zhao et al., 2009; Zhu et al., 2006). Furthermore, BMIMCl renders cellulose essentially amorphous, thus its structure may open and the macromolecule can be accessible by the corresponding enzyme (Dadi et al., 2006, 2007). The only restriction is that ionic liquids that dissolve cellulose such as BMIMCl, cause the denaturation of the enzyme molecules; thus the treatment of cellulose with BMIMCl and the enzymatic acylation reaction has to be done separately. This process provides a new platform for the utilization of cellulose resources. Additional, advantages in the use of ionic liquids is the fact that they have low volatility, they are stable in a wide range of temperature, they are not flammable and also they can be easily recycled (Earle and Seddon, 2000; Park, 2003; Vanrantwijk et al., 2003). The above, render the ionic liquids solvents with unique properties, justifying the characterization ‘green solvents’. 2. Methods 2.1. Chemicals Avicel cellulose PH-101 (DP = 225) was purchased from Fluka. 1-n-Butyl-3-methylimidazolium chloride (BMIMCl, 99%) was also obtained from Fluka. Vinyl laurate (99%) was purchased from Fluka, vinyl propionate (98%) and vinyl stearate (>99%) were products of Sigma. Methanol (99.9%) and hexane (99.9%) were obtained from Fisher Scientific. Potassium bromide (KBr, spectroscopic grade) was a product of Merck.
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washed three times with warm deionized water, to remove any traces of BMIMCl and subsequently dried in order for the treated cellulose to be used in the enzymatic reaction.
2.4. Enzymatic esterification Each reaction system was consisted of 10 mL fatty vinyl ester (propionate, laurate, stearate), 100 mg Avicel cellulose treated in BMIMCl and an enzyme loading of 2.5 U/mg cellulose (all enzymes were tested using as substrate the same vinyl ester each time). The reactions were performed in capped round-bottom glass flasks at 50 °C, under stirring (2000 rpm) for 20 h. Samples were taken at different time intervals. The same reactions were also carried out using (i) untreated Avicel cellulose or (ii) treated Avicel cellulose with denaturated enzyme. Furthermore, for the successful reactions, some additional sets of experiments were conducted, at which the enzyme loading varied at 1.0 and 4.0 U/mg cellulose, respectively.
2.5. Purification of the product Reaction samples were firstly centrifuged and the supernatant was removed. Afterwards the remaining solid (cellulosics) was washed twice with methanol and then with hexane to remove the remaining unreacted vinyl ester. The filtrates from the wash out were collected and evaporated in order to detect any trace of cellulose ester which may be soluble in such solvents. All cellulosic material was then dried under vacuum.
2.6. Product identification by Fourier transform infrared spectroscopy (FTIR) The Fourier transform infrared spectroscopy was performed using the Nicolet MAGNA-IR 560 spectrometer. The cellulosic materials studied were milled individually with KBr at 2% (w/w) and samples of 100 mg were used to form the KBr disk for FTIR analysis. The background spectrum of pure KBr was subtracted from that of each sample spectrum and the 400–4000 cm1 region was investigated in order to determine if an ester bond was present.
2.2. Enzymes 2.7. Determination of the ester content (%) Lipase from Candida antarctica (3.8 U/mg), lipase from Candida cylindracea (5.4 U/mg), lipase from Aspergillus niger (3.0 U/mg) and esterase, immobilized on Eupergit, from hog liver (0.9 U/mg) were all purchased from Fluka. Lipase, immobilized on acrylic resin, from C. antarctica (1.8 U/mg) was obtained from Sigma. Cutinase from Fusarium solani pisi was from Unilever and immobilized on Accurel EP100 (1.4 U/mg). One unit corresponds to the amount of enzyme which liberates 1 lmol lauric acid/min at pH 7.5 and 40 °C. The substrate is pNP-laurate in excess.
One gram of esterified cellulose and 40 mL of ethanol (75%) were heated at 55 °C for 30 min. Twenty-five milliliters of 0.5 N NaOH were added. The mixture was heated and left to settle at room temperature for 3 days. The excess NaOH was measured by titration with 0.5 N HCl solution and phenolphthalein as indicator. An excess of 1 mL of the acid was added and the mixture was left to settle for 12 h to avoid any diffusion problems. The excess of added acid was then titrated back with 0.5 N NaOH solution. The ester content (%) was determined as follows (Sereti et al., 2001):
2.3. Pretreatment of cellulose by ionic liquid
Ester content ð%Þ ¼ ½ðA BÞ NB ðC DÞ NA M=10 w A 5% (w/w) mixture of Avicel cellulose in ionic liquid BMIMCl was prepared and placed in a glass vial. The vial was capped and then heated to 120 °C for 30 min under stirring, with the assistance of a magnetic stirrer. It is important to mention that in these conditions cellulose was totally dissolved. After the thermal process cellulose was precipitated by adding warm deionized water (10 folds). The mixture of ionic liquid/water was removed by vacuum filtration and cellulose was collected. Afterwards cellulose was
where A and B are respective volumes of NaOH solution added to sample and blank (mL), NB is the normality of NaOH solution, C and D are the respective volumes of HCl solution added to the sample and blank (mL), NA is the normality of the HCl solution, w is the weight of the sample (g), and M is the molar mass of grafted acyl radical. It must be noted that the ester content (%) corresponds to the weight percentage of the acyl radical on the esterified cellulose.
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Table 1 Collective results of the enzymatically catalyzed acylation of the pretreated in BMIMCl Avicel cellulose with various fatty acid vinyl esters. Fatty substrate
Enzyme
Product
Vinyl propionate Immobilized lipase Candida antarctica Lipase Candida antarctica Lipase Candida cylindracea Lipase Aspergillus niger Immobilized esterase from hog liver
– – – – Cellulose propionate Immobilized cutinase Fusarium solani Cellulose propionate
Vinyl laurate
Immobilized lipase Candida antarctica Lipase Candida antarctica Lipase Candida cylindracea Lipase Aspergillus niger Immobilized esterase from hog liver Immobilized cutinase Fusarium solani
– – – – Cellulose laurate Cellulose laurate
Vinyl stearate
Immobilized lipase Candida antarctica Lipase Candida antarctica Lipase Candida cylindracea Lipase Aspergillus niger Immobilized esterase from hog liver Immobilized cutinase Fusarium solani
– – – – Cellulose stearate Cellulose stearate
3. Results and discussion FTIR analysis of the purified products reveals the formation of the ester bonds on cellulose. The region between 1800 and 1500 cm1 is of special interest, because it permits observation of infrared absorption by the carboxylic esters of the cellulose
molecule. As it was found through the FTIR analysis, the immobilized esterase from hog liver and the immobilized cutinase F. solani can catalyze successfully the acylation of treated in BMIMCl Avicel cellulose with the corresponding fatty substrates. On the other hand, the lipases that were used could not catalyze this reaction (Table 1). At this point it must be also noted that all the control systems containing (i) untreated cellulose or (ii) treated cellulose with denaturated enzyme showed no reaction. These facts indicate that (i) cellulose pretreatment into the ionic liquid BMIMCl is essential for the reaction that follows and (ii) the acylation of pretreated Avicel cellulose is controlled by the corresponding biocatalyst. Pretreatment of Avicel cellulose into the BMIMCl is a novel approach by which microcrystalline cellulose can be converted to amorphous one (Dadi et al., 2007; Zhao et al., 2009). Thus the structure of cellulose is opened and the macromolecule is becoming accessible to the enzymes. This process can be attributed to the nature of the imidazolium cation and the relatively strong electronegativity and small size of the chloride anion. BMIMCl has high hydrogen bond basicity and the anion attacks the free hydroxyl groups and deprotonates the cellulose. Additionally, the imidazolium cation with its electron rich aromatic p system interacts with cellulose hydroxyl oxygen atoms through nonbonding electrons and prevents cross linking of cellulose molecules. The resultant cellulose which is referred as ‘regenerated cellulose’ is amorphous and it can be recovered by precipitation. Concerning the inability of lipases to catalyze the acylation of the pretreated cellulose as opposed to the successful action of esterase and cutinase, we can be led to a possible assumption that would be of great importance. Namely, lipases having a hydrophobic lid which covers the catalytic site of the enzyme are not able to
Fig. 1. Reaction course of the enzymatic acylation of pretreated in BMIMCl Avicel cellulose with vinyl propionate (A); vinyl laurate (B); vinyl stearate (C). The biocatalyst was immobilized esterase from hog liver (N) and immobilized cutinase Fusarium solani (j). Reaction temperature was 50 °C, with stirring at 2000 rpm and an enzyme loading of 2.5 U/mg cellulose.
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Table 2 Initial rates of the acylation reactions of pretreated Avicel cellulose in BMIMCl, with the corresponding vinyl ester. Each reaction system was consisted of 10 mL vinyl ester (solvent free system), 100 mg cellulose and an enzyme loading of 2.5 U/mg cellulose. Reaction temperature was 50 °C, with stirring at 2000 rpm. Fatty substrate
Enzyme
Initial rate (% ester content/mL/h)
Vinyl propionate
Immobilized esterase from hog liver Immobilized cutinase Fusarium solani
0.067 0.089
Vinyl laurate
Immobilized esterase from hog liver Immobilized cutinase Fusarium solani
0.021 0.046
Vinyl stearate
Immobilized esterase from hog liver Immobilized cutinase Fusarium solani
0.018 0.041
function, in contrast to esterase and cutinase, which do not have this hydrophobic part and thus providing a more hydrophilic behaviour (Beer et al., 1996; Garcia et al., 2004; Guebitz and Cavacopaulo, 2008; Gundlach, 1973; Micaelo, 2005; Silva et al., 2005; Thamwiriyasati et al., 2010; Yamada et al., 1994). Besides, cellulose is by definition extremely hydrophilic because it is replete with hydroxyl groups. Thus there is a preference of hydrophilic esterase and cutinase to approach cellulose and interact with it instead of the more hydrophobic lipases (hydrophilic enzymes have relatively better affinity with the hydrophilic polysaccharide). In addition, unlike the lipases, the catalytic serine of esterase and cutinase is not buried under surface loops, but is accessible to the external environment. Fig. 1 depicts the formation of the ester bonds of pretreated Avicel cellulose of the successful acylation reactions catalyzed by the immobilized esterase from hog liver and the immobilized cutinase from F. solani, as a function of time. The reaction progress is expressed as % ester content. As shown in Fig. 1(A) the acylation of cellulose with vinyl propionate in the reaction conditions used, reaches an ester content of 1.5% when the biocatalyst is the immobilized esterase from hog liver, while when the biocatalyst is the immobilized cutinase F. solani, the ester content is 1.9%. Also, the initial rate of the reaction with the immobilized esterase from hog liver is slightly lower than the initial rate of the reaction at which the enzyme is the immobilized cutinase F. solani. This comparative eminence of the F. solani cutinase over the hog liver esterase is possibly due to the preference that cutinases show for macromolecules (Guebitz and Cavacopaulo, 2008; Hunsen et al., 2007; Lee et al., 2009). The natural substrate of cutinases is cutin, which consists of omega hydroxy acids and their derivatives which are interlinked via ester bonds, forming a polyester polymer. Specifically, cutinases are capable of degrading cutin by braking down the ester bonds. Similar results are observed in the acylation
Fig. 2. Role of cutinase Fusarium solani concentration on the reaction rate of the acylation of pretreated in BMIMCl Avicel cellulose with vinyl stearate [1.0 U/mg cellulose (), 2.5 U/mg cellulose (j) and 4.0 U/mg cellulose (N)]. Reaction temperature was 50 °C, with stirring at 2000 rpm.
reactions with vinyl laurate and vinyl stearate (Fig. 1(B) and (C)). The ester content of acylated cellulose with vinyl laurate is 0.9% with immobilized esterase from hog liver and 1.3% with immobilized cutinase F. solani as the biocatalyst. When the acylation is occurred with vinyl stearate as substrate, the ester content of cellulose is 0.7% with immobilized esterase from hog liver and 0.9% with immobilized cutinase F. solani. Also the initial rates of the reactions at which the enzyme is the immobilized esterase from hog liver are slightly lower than those at which the enzyme used is the immobilized cutinase F. solani (Table 2). Another useful remark is that the values of the degree of esterification (% ester content) are relatively low; the ester content is up to 1.9%, 1.3% and 0.9% when the acylation is conducted with vinyl propionate, vinyl laurate and vinyl stearate, respectively. This observation in combination with the fact that pretreated cellulose was not soluble in the solvent free reaction system may lead to the conclusion that the esterification of cellulose has occurred on the surface of the molecule. For the successful reactions, some additional sets of experiments at which the enzyme loading varied were also conducted. The initial enzyme loading was 2.5 U/mg cellulose while the additional enzyme loadings were 1.0 and 4.0 U/mg cellulose, respectively. Fig. 2 shows the profile of the reaction rate in the representative case of cellulose acylation with vinyl stearate by immobilized cutinase from F. solani at different enzyme loadings. It is clear that by increasing the enzymatic activity on the reaction system, the initial rate is increasing relatively, supporting that the studied acylation reaction is enzymatically controlled. The same
Fig. 3. Initial rates of the acylation reactions of pretreated Avicel cellulose in BMIMCl, with vinyl propionate (A); vinyl laurate (B); vinyl stearate (C). Immobilized esterase from hog liver and immobilized cutinase from Fusarium solani were used as biocatalysts at different concentrations. Each reaction system was consisted of 10 mL vinyl ester (solvent free system), 100 mg cellulose and an enzyme loading of 1.0, 2.5 or 4.0 U/mg cellulose, respectively. Reaction temperature was 50 °C, with stirring at 2000 rpm.
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observation is also reported for the other acylation reactions of cellulose with vinyl propionate and vinyl laurate, using both the immobilized esterase from hog liver and the immobilized cutinase F. solani as biocatalysts (Fig. 3). At this point it must be mentioned that the direct enzymatic acylation of Avicel cellulose with vinyl propionate, vinyl laurate and vinyl stearate is accomplished successfully for first time in the literature. 4. Conclusions In this work the enzymatic acylation of Avicel cellulose with vinyl propionate, vinyl laurate and vinyl stearate has been achieved. A pretreatment of cellulose into the ionic liquid BMIMCl was necessary before the enzymatic reaction in order to open the structure of the polysaccharide and make it accessible to the enzyme molecule. Immobilized esterase from hog liver and immobilized cutinase F. solani were capable of catalyzing this acylation reaction in contrast with four common lipases (immobilized lipase C. antarctica, lipase C. antarctica, lipase C. cylindracea, lipase A. niger) that could not esterify the cellulosic substrate. As it was found, the ester content of the products was 1.9% for cellulose propionate, 1.3% for cellulose laurate and 1.0% for cellulose stearate. Acknowledgement Stavros Gremos would like to thank the State Scholarships Foundation (Greece) for a grant. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.biortech.2010.09.021. References Beer, H.D., Wohlfahrt, G., McCarthy, J.E.G., Schomburg, D., Schmid, R.D., 1996. Analysis of the catalytic mechanism of a fungal lipase using computer-aided design and structural mutants. Protein Engineering 9 (6), 507–517. Dadi, A.P., Varanasi, S., Schall, C.A., 2006. Enhancement of cellulose saccharification kinetics using an ionic liquid pretreatment step. Biotechnology and Bioengineering 95 (5), 904–910. Dadi, A.P., Schall, C.A., Varanasi, S., 2007. Mitigation of cellulose recalcitrance to enzymatic hydrolysis by ionic liquid pretreatment. Applied Biochemistry and Biotechnology 137, 407–421. Earle, M.J., Seddon, K.R., 2000. Ionic liquids. Green solvents for the future. Pure and Applied Chemistry 72 (7), 1391–1398. Edgar, K.J., 2007. Cellulose esters in drug delivery. Cellulose 14 (1), 49–64. Garcia, S., Lourenço, N.M.T., Lousa, D., Sequeira, A.F., Mimoso, P., Cabral, J.M.S., Afonso, C.A.M., Barreiros, S., 2004. A comparative study of biocatalysis in nonconventional solvents: ionic liquids, supercritical fluids and organic media. Green Chemistry 6 (9), 466. Gradwell, S., 2004. Surface modification of cellulose fibers: towards wood composites by biomimetics? Comptes Rendus Biologies 327 (9–10), 945–953. Guebitz, G., Cavacopaulo, A., 2008. Enzymes go big: surface hydrolysis and functionalisation of synthetic polymers. Trends in Biotechnology 26 (1), 32–38. Gundlach, G., 1973. Mechanism for esterase-activity. Hoppe-Seylers Zeitschrift Fur Physiologische Chemie 354 (1), 7–8. Hunsen, M., Azim, A., Mang, H., Wallner, S.R., Ronkvist, A., Xie, W.C., Gross, R.A., 2007. A cutinase with polyester synthesis activity. Macromolecules 40 (2), 148– 150.
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Direct enzymatic acylation of cellulose pretreated in BMIMCl ionic liquid Stavros Gremos, Dimitra Zarafeta, Dimitris Kekos, Fragiskos Kolisis ⇑ Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, Zografou Campus, 15780 Athens, Greece
a r t i c l e
i n f o
Article history: Received 5 July 2010 Received in revised form 3 September 2010 Accepted 6 September 2010 Available online 15 September 2010 Keywords: Enzymatic acylation Cellulose Ionic liquid Solvent free reaction Cellulose ester
a b s t r a c t Cellulose esters are an important class of functional biopolymers with great interest in the chemical industry. In this work the enzymatic acylation of Avicel cellulose with vinyl propionate, vinyl laurate and vinyl stearate, has been performed successfully in a solvent free reaction system. At first cellulose was putted into the ionic liquid BMIMCl (1-n-butyl-3-methylimidazolium chloride) in order to facilitate the unwrap of the structure of the polysaccharide molecule and make it accessible to the enzyme. Thus, after this pretreatment the enzymatic esterification reaction was performed using various hydrolases. The enzymes capable of catalyzing the acylation of cellulose were found to be the immobilized esterase from hog liver and the immobilized cutinase from Fusarium solani, while the lipases used did not show any catalytic activity. Cellulose esters of propionate, laurate and stearate were synthesized with a degree of esterification of 1.9%, 1.3% and 1.0%, respectively. It is the first successful direct enzymatic acylation of cellulose with long chain fatty acids. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Cellulose is the most abundant polysaccharide in nature. It occurs in plants under the form of microfibrils, components of the structurally strong framework of the cell walls. Cellulose is also produced in a highly hydrated form by some bacteria (e.g. Acetobacter xylinum) but it is mostly prepared from wood pulp (Kadla and Gilbert, 2000). It is a linear polymer of b-(1,4)-D-glucopyranose units in C1 conformation. The fully equatorial conformation of b-linked glucopyranose residues stabilizes the chair structure, minimizing its flexibility. Due to the extended intra- and intermolecular hydrogen bonding between the cellulose chains, this biopolymer has a high crystalline structure. As a result it is completely insoluble in water or in common solvents leading to difficulties in chemical manipulation (Kadla and Gilbert, 2000). On the other hand the plenitude, the renewability and the low cost of cellulose make it an ideal feedstock for producing different materials. About a third of the world’s production of purified cellulose is used as the base substance for a number of derivatives with predesigned and wide-ranging properties depending on the groups involved and the degree of derivatization (Lee and Wang, 2006; Siro and Plackett, 2010; Takatani et al., 2008). Cellulose has great potential for the preparation of novel materials (e.g. thermoplastics), exhibiting a number of well-known advantages such as biocompatibility, high stiffness, good mechanical properties and biodegradability. In particular acyl esters of cellulose are ⇑ Corresponding author. Address: Polytechniou 9, Zografou Campus, 15780 Athens, Greece. Tel.: +30 210 7723156. E-mail address: [email protected] (F. Kolisis). 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.09.021
considered to be an important class of polymers used in the production of fibers, plastics, films, cosmetics and drugs (Edgar, 2007; Gradwell, 2004; Wibowo et al., 2006). To date the commercial synthesis of these compounds is achieved in heterogeneous reaction systems containing carboxylic acid anhydrides, tough solvents like N,N-dimethylacetamide and acid catalysts. This process is also limited to the production of short chain esters with a length of four carbon atoms or less. This is not advantageous because the cellulose ester is expected to have better thermoplastic properties when the chain of the ester contains more than six carbons. That happens because the longer chain acts as an internal plasticizer (Yin et al., 2007). In order to overcome the above situation and introduce biocatalysis into the field of cellulose esterification, we designed a system for the enzymatic acylation of this polysaccharide with long chain fatty acids. The realisation that enzymes can function in non-aqueous media has given to their synthetic potential a powerful boost for many processes (Ikeda and Klibanov, 1993; Klibanov, 1989; Patel et al., 1996; Zaks and Klibanov, 1985). Amongst the numerous enzymes studied in such systems, esterases and in particular lipases have been successfully utilized in a wide range of stereoselective and regioselective acylations of various target molecules including sugars (Kirk et al., 1995; Ljunger et al., 1994; Patel et al., 1996; Tsitsimpikou et al., 1998). The first attempt of the enzymatic esterification of cellulose was made a few years ago (Sereti et al., 1998, 2001) by subjecting Avicel cellulose, the fatty acid as substrate and lipase into several organic solvents. The reaction was not successful due to the high crystallinity of cellulose. The macromolecule could not be dissolved and there was no accessibility by the enzyme. Only soluble cellulose derivatives, such as cellulose
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acetate and hydroxypropyl cellulose, were found to be acylated by lipases. In the present work, this problem was tackled by introducing a preliminary step of cellulose treatment, using ionic liquids before the enzymatic reaction in order to make the cellulosic molecules as permeable as possible. Ionic liquids are organic salts whose ions do not pack well and remain liquid in ambient temperatures (Earle and Seddon, 2000). Some studies showed that cellulose can be dissolved in some hydrophilic ionic liquids such as 1-n-butyl3-methylimidazolium chloride (BMIMCl) and 1-allyl-3-methylimidazolium chloride (AMIMCl) (Wu et al., 2004; Zhao et al., 2009; Zhu et al., 2006). Particularly BMIMCl can dissolve cellulose up to 25% (w/w). Cellulose in its BMIMCl solution can be easily precipitated by addition of water, ethanol or acetone. Moreover, the regenerated cellulose has almost the same degree of polymerization (DP) as the initial one (Zhao et al., 2009; Zhu et al., 2006). Furthermore, BMIMCl renders cellulose essentially amorphous, thus its structure may open and the macromolecule can be accessible by the corresponding enzyme (Dadi et al., 2006, 2007). The only restriction is that ionic liquids that dissolve cellulose such as BMIMCl, cause the denaturation of the enzyme molecules; thus the treatment of cellulose with BMIMCl and the enzymatic acylation reaction has to be done separately. This process provides a new platform for the utilization of cellulose resources. Additional, advantages in the use of ionic liquids is the fact that they have low volatility, they are stable in a wide range of temperature, they are not flammable and also they can be easily recycled (Earle and Seddon, 2000; Park, 2003; Vanrantwijk et al., 2003). The above, render the ionic liquids solvents with unique properties, justifying the characterization ‘green solvents’. 2. Methods 2.1. Chemicals Avicel cellulose PH-101 (DP = 225) was purchased from Fluka. 1-n-Butyl-3-methylimidazolium chloride (BMIMCl, 99%) was also obtained from Fluka. Vinyl laurate (99%) was purchased from Fluka, vinyl propionate (98%) and vinyl stearate (>99%) were products of Sigma. Methanol (99.9%) and hexane (99.9%) were obtained from Fisher Scientific. Potassium bromide (KBr, spectroscopic grade) was a product of Merck.
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washed three times with warm deionized water, to remove any traces of BMIMCl and subsequently dried in order for the treated cellulose to be used in the enzymatic reaction.
2.4. Enzymatic esterification Each reaction system was consisted of 10 mL fatty vinyl ester (propionate, laurate, stearate), 100 mg Avicel cellulose treated in BMIMCl and an enzyme loading of 2.5 U/mg cellulose (all enzymes were tested using as substrate the same vinyl ester each time). The reactions were performed in capped round-bottom glass flasks at 50 °C, under stirring (2000 rpm) for 20 h. Samples were taken at different time intervals. The same reactions were also carried out using (i) untreated Avicel cellulose or (ii) treated Avicel cellulose with denaturated enzyme. Furthermore, for the successful reactions, some additional sets of experiments were conducted, at which the enzyme loading varied at 1.0 and 4.0 U/mg cellulose, respectively.
2.5. Purification of the product Reaction samples were firstly centrifuged and the supernatant was removed. Afterwards the remaining solid (cellulosics) was washed twice with methanol and then with hexane to remove the remaining unreacted vinyl ester. The filtrates from the wash out were collected and evaporated in order to detect any trace of cellulose ester which may be soluble in such solvents. All cellulosic material was then dried under vacuum.
2.6. Product identification by Fourier transform infrared spectroscopy (FTIR) The Fourier transform infrared spectroscopy was performed using the Nicolet MAGNA-IR 560 spectrometer. The cellulosic materials studied were milled individually with KBr at 2% (w/w) and samples of 100 mg were used to form the KBr disk for FTIR analysis. The background spectrum of pure KBr was subtracted from that of each sample spectrum and the 400–4000 cm1 region was investigated in order to determine if an ester bond was present.
2.2. Enzymes 2.7. Determination of the ester content (%) Lipase from Candida antarctica (3.8 U/mg), lipase from Candida cylindracea (5.4 U/mg), lipase from Aspergillus niger (3.0 U/mg) and esterase, immobilized on Eupergit, from hog liver (0.9 U/mg) were all purchased from Fluka. Lipase, immobilized on acrylic resin, from C. antarctica (1.8 U/mg) was obtained from Sigma. Cutinase from Fusarium solani pisi was from Unilever and immobilized on Accurel EP100 (1.4 U/mg). One unit corresponds to the amount of enzyme which liberates 1 lmol lauric acid/min at pH 7.5 and 40 °C. The substrate is pNP-laurate in excess.
One gram of esterified cellulose and 40 mL of ethanol (75%) were heated at 55 °C for 30 min. Twenty-five milliliters of 0.5 N NaOH were added. The mixture was heated and left to settle at room temperature for 3 days. The excess NaOH was measured by titration with 0.5 N HCl solution and phenolphthalein as indicator. An excess of 1 mL of the acid was added and the mixture was left to settle for 12 h to avoid any diffusion problems. The excess of added acid was then titrated back with 0.5 N NaOH solution. The ester content (%) was determined as follows (Sereti et al., 2001):
2.3. Pretreatment of cellulose by ionic liquid
Ester content ð%Þ ¼ ½ðA BÞ NB ðC DÞ NA M=10 w A 5% (w/w) mixture of Avicel cellulose in ionic liquid BMIMCl was prepared and placed in a glass vial. The vial was capped and then heated to 120 °C for 30 min under stirring, with the assistance of a magnetic stirrer. It is important to mention that in these conditions cellulose was totally dissolved. After the thermal process cellulose was precipitated by adding warm deionized water (10 folds). The mixture of ionic liquid/water was removed by vacuum filtration and cellulose was collected. Afterwards cellulose was
where A and B are respective volumes of NaOH solution added to sample and blank (mL), NB is the normality of NaOH solution, C and D are the respective volumes of HCl solution added to the sample and blank (mL), NA is the normality of the HCl solution, w is the weight of the sample (g), and M is the molar mass of grafted acyl radical. It must be noted that the ester content (%) corresponds to the weight percentage of the acyl radical on the esterified cellulose.
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Table 1 Collective results of the enzymatically catalyzed acylation of the pretreated in BMIMCl Avicel cellulose with various fatty acid vinyl esters. Fatty substrate
Enzyme
Product
Vinyl propionate Immobilized lipase Candida antarctica Lipase Candida antarctica Lipase Candida cylindracea Lipase Aspergillus niger Immobilized esterase from hog liver
– – – – Cellulose propionate Immobilized cutinase Fusarium solani Cellulose propionate
Vinyl laurate
Immobilized lipase Candida antarctica Lipase Candida antarctica Lipase Candida cylindracea Lipase Aspergillus niger Immobilized esterase from hog liver Immobilized cutinase Fusarium solani
– – – – Cellulose laurate Cellulose laurate
Vinyl stearate
Immobilized lipase Candida antarctica Lipase Candida antarctica Lipase Candida cylindracea Lipase Aspergillus niger Immobilized esterase from hog liver Immobilized cutinase Fusarium solani
– – – – Cellulose stearate Cellulose stearate
3. Results and discussion FTIR analysis of the purified products reveals the formation of the ester bonds on cellulose. The region between 1800 and 1500 cm1 is of special interest, because it permits observation of infrared absorption by the carboxylic esters of the cellulose
molecule. As it was found through the FTIR analysis, the immobilized esterase from hog liver and the immobilized cutinase F. solani can catalyze successfully the acylation of treated in BMIMCl Avicel cellulose with the corresponding fatty substrates. On the other hand, the lipases that were used could not catalyze this reaction (Table 1). At this point it must be also noted that all the control systems containing (i) untreated cellulose or (ii) treated cellulose with denaturated enzyme showed no reaction. These facts indicate that (i) cellulose pretreatment into the ionic liquid BMIMCl is essential for the reaction that follows and (ii) the acylation of pretreated Avicel cellulose is controlled by the corresponding biocatalyst. Pretreatment of Avicel cellulose into the BMIMCl is a novel approach by which microcrystalline cellulose can be converted to amorphous one (Dadi et al., 2007; Zhao et al., 2009). Thus the structure of cellulose is opened and the macromolecule is becoming accessible to the enzymes. This process can be attributed to the nature of the imidazolium cation and the relatively strong electronegativity and small size of the chloride anion. BMIMCl has high hydrogen bond basicity and the anion attacks the free hydroxyl groups and deprotonates the cellulose. Additionally, the imidazolium cation with its electron rich aromatic p system interacts with cellulose hydroxyl oxygen atoms through nonbonding electrons and prevents cross linking of cellulose molecules. The resultant cellulose which is referred as ‘regenerated cellulose’ is amorphous and it can be recovered by precipitation. Concerning the inability of lipases to catalyze the acylation of the pretreated cellulose as opposed to the successful action of esterase and cutinase, we can be led to a possible assumption that would be of great importance. Namely, lipases having a hydrophobic lid which covers the catalytic site of the enzyme are not able to
Fig. 1. Reaction course of the enzymatic acylation of pretreated in BMIMCl Avicel cellulose with vinyl propionate (A); vinyl laurate (B); vinyl stearate (C). The biocatalyst was immobilized esterase from hog liver (N) and immobilized cutinase Fusarium solani (j). Reaction temperature was 50 °C, with stirring at 2000 rpm and an enzyme loading of 2.5 U/mg cellulose.
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Table 2 Initial rates of the acylation reactions of pretreated Avicel cellulose in BMIMCl, with the corresponding vinyl ester. Each reaction system was consisted of 10 mL vinyl ester (solvent free system), 100 mg cellulose and an enzyme loading of 2.5 U/mg cellulose. Reaction temperature was 50 °C, with stirring at 2000 rpm. Fatty substrate
Enzyme
Initial rate (% ester content/mL/h)
Vinyl propionate
Immobilized esterase from hog liver Immobilized cutinase Fusarium solani
0.067 0.089
Vinyl laurate
Immobilized esterase from hog liver Immobilized cutinase Fusarium solani
0.021 0.046
Vinyl stearate
Immobilized esterase from hog liver Immobilized cutinase Fusarium solani
0.018 0.041
function, in contrast to esterase and cutinase, which do not have this hydrophobic part and thus providing a more hydrophilic behaviour (Beer et al., 1996; Garcia et al., 2004; Guebitz and Cavacopaulo, 2008; Gundlach, 1973; Micaelo, 2005; Silva et al., 2005; Thamwiriyasati et al., 2010; Yamada et al., 1994). Besides, cellulose is by definition extremely hydrophilic because it is replete with hydroxyl groups. Thus there is a preference of hydrophilic esterase and cutinase to approach cellulose and interact with it instead of the more hydrophobic lipases (hydrophilic enzymes have relatively better affinity with the hydrophilic polysaccharide). In addition, unlike the lipases, the catalytic serine of esterase and cutinase is not buried under surface loops, but is accessible to the external environment. Fig. 1 depicts the formation of the ester bonds of pretreated Avicel cellulose of the successful acylation reactions catalyzed by the immobilized esterase from hog liver and the immobilized cutinase from F. solani, as a function of time. The reaction progress is expressed as % ester content. As shown in Fig. 1(A) the acylation of cellulose with vinyl propionate in the reaction conditions used, reaches an ester content of 1.5% when the biocatalyst is the immobilized esterase from hog liver, while when the biocatalyst is the immobilized cutinase F. solani, the ester content is 1.9%. Also, the initial rate of the reaction with the immobilized esterase from hog liver is slightly lower than the initial rate of the reaction at which the enzyme is the immobilized cutinase F. solani. This comparative eminence of the F. solani cutinase over the hog liver esterase is possibly due to the preference that cutinases show for macromolecules (Guebitz and Cavacopaulo, 2008; Hunsen et al., 2007; Lee et al., 2009). The natural substrate of cutinases is cutin, which consists of omega hydroxy acids and their derivatives which are interlinked via ester bonds, forming a polyester polymer. Specifically, cutinases are capable of degrading cutin by braking down the ester bonds. Similar results are observed in the acylation
Fig. 2. Role of cutinase Fusarium solani concentration on the reaction rate of the acylation of pretreated in BMIMCl Avicel cellulose with vinyl stearate [1.0 U/mg cellulose (), 2.5 U/mg cellulose (j) and 4.0 U/mg cellulose (N)]. Reaction temperature was 50 °C, with stirring at 2000 rpm.
reactions with vinyl laurate and vinyl stearate (Fig. 1(B) and (C)). The ester content of acylated cellulose with vinyl laurate is 0.9% with immobilized esterase from hog liver and 1.3% with immobilized cutinase F. solani as the biocatalyst. When the acylation is occurred with vinyl stearate as substrate, the ester content of cellulose is 0.7% with immobilized esterase from hog liver and 0.9% with immobilized cutinase F. solani. Also the initial rates of the reactions at which the enzyme is the immobilized esterase from hog liver are slightly lower than those at which the enzyme used is the immobilized cutinase F. solani (Table 2). Another useful remark is that the values of the degree of esterification (% ester content) are relatively low; the ester content is up to 1.9%, 1.3% and 0.9% when the acylation is conducted with vinyl propionate, vinyl laurate and vinyl stearate, respectively. This observation in combination with the fact that pretreated cellulose was not soluble in the solvent free reaction system may lead to the conclusion that the esterification of cellulose has occurred on the surface of the molecule. For the successful reactions, some additional sets of experiments at which the enzyme loading varied were also conducted. The initial enzyme loading was 2.5 U/mg cellulose while the additional enzyme loadings were 1.0 and 4.0 U/mg cellulose, respectively. Fig. 2 shows the profile of the reaction rate in the representative case of cellulose acylation with vinyl stearate by immobilized cutinase from F. solani at different enzyme loadings. It is clear that by increasing the enzymatic activity on the reaction system, the initial rate is increasing relatively, supporting that the studied acylation reaction is enzymatically controlled. The same
Fig. 3. Initial rates of the acylation reactions of pretreated Avicel cellulose in BMIMCl, with vinyl propionate (A); vinyl laurate (B); vinyl stearate (C). Immobilized esterase from hog liver and immobilized cutinase from Fusarium solani were used as biocatalysts at different concentrations. Each reaction system was consisted of 10 mL vinyl ester (solvent free system), 100 mg cellulose and an enzyme loading of 1.0, 2.5 or 4.0 U/mg cellulose, respectively. Reaction temperature was 50 °C, with stirring at 2000 rpm.
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observation is also reported for the other acylation reactions of cellulose with vinyl propionate and vinyl laurate, using both the immobilized esterase from hog liver and the immobilized cutinase F. solani as biocatalysts (Fig. 3). At this point it must be mentioned that the direct enzymatic acylation of Avicel cellulose with vinyl propionate, vinyl laurate and vinyl stearate is accomplished successfully for first time in the literature. 4. Conclusions In this work the enzymatic acylation of Avicel cellulose with vinyl propionate, vinyl laurate and vinyl stearate has been achieved. A pretreatment of cellulose into the ionic liquid BMIMCl was necessary before the enzymatic reaction in order to open the structure of the polysaccharide and make it accessible to the enzyme molecule. Immobilized esterase from hog liver and immobilized cutinase F. solani were capable of catalyzing this acylation reaction in contrast with four common lipases (immobilized lipase C. antarctica, lipase C. antarctica, lipase C. cylindracea, lipase A. niger) that could not esterify the cellulosic substrate. As it was found, the ester content of the products was 1.9% for cellulose propionate, 1.3% for cellulose laurate and 1.0% for cellulose stearate. Acknowledgement Stavros Gremos would like to thank the State Scholarships Foundation (Greece) for a grant. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.biortech.2010.09.021. References Beer, H.D., Wohlfahrt, G., McCarthy, J.E.G., Schomburg, D., Schmid, R.D., 1996. Analysis of the catalytic mechanism of a fungal lipase using computer-aided design and structural mutants. Protein Engineering 9 (6), 507–517. Dadi, A.P., Varanasi, S., Schall, C.A., 2006. Enhancement of cellulose saccharification kinetics using an ionic liquid pretreatment step. Biotechnology and Bioengineering 95 (5), 904–910. Dadi, A.P., Schall, C.A., Varanasi, S., 2007. Mitigation of cellulose recalcitrance to enzymatic hydrolysis by ionic liquid pretreatment. Applied Biochemistry and Biotechnology 137, 407–421. Earle, M.J., Seddon, K.R., 2000. Ionic liquids. Green solvents for the future. Pure and Applied Chemistry 72 (7), 1391–1398. Edgar, K.J., 2007. Cellulose esters in drug delivery. Cellulose 14 (1), 49–64. Garcia, S., Lourenço, N.M.T., Lousa, D., Sequeira, A.F., Mimoso, P., Cabral, J.M.S., Afonso, C.A.M., Barreiros, S., 2004. A comparative study of biocatalysis in nonconventional solvents: ionic liquids, supercritical fluids and organic media. Green Chemistry 6 (9), 466. Gradwell, S., 2004. Surface modification of cellulose fibers: towards wood composites by biomimetics? Comptes Rendus Biologies 327 (9–10), 945–953. Guebitz, G., Cavacopaulo, A., 2008. Enzymes go big: surface hydrolysis and functionalisation of synthetic polymers. Trends in Biotechnology 26 (1), 32–38. Gundlach, G., 1973. Mechanism for esterase-activity. Hoppe-Seylers Zeitschrift Fur Physiologische Chemie 354 (1), 7–8. Hunsen, M., Azim, A., Mang, H., Wallner, S.R., Ronkvist, A., Xie, W.C., Gross, R.A., 2007. A cutinase with polyester synthesis activity. Macromolecules 40 (2), 148– 150.
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