Application of cellulose/lignin hydrogel beads as novel supports for immobilizing lipase
Graphical abstract
Introduction
Hydrogels are defined as three-dimensional network structures formed by natural or synthetic polymers swollen in a large amount of water [1]. Recently, biopolymer-based hydrogels have received considerable attention for use in biomedical and biotechnological applications, including enzyme immobilization, tissue engineering, drug delivery systems, and biosensors, due to their inherent biocompatibility and biodegradability [2]. In the field of enzyme immobilization, polysaccharide hydrogels such as alginate, chitosan, starch, and agarose have been employed for entrapping a large number of enzymes such as lipase, lactase, invertase, endo-β-glucanase, and peroxidase [3], [4], [5], [6], [7]. Entrapment, one of the immobilization techniques, can be defined as physical restriction of an enzyme within a confined polymer network and, unlike support binding, requires the synthesis of a polymeric network in the presence of enzymes [8]. In this regard, biopolymers are efficient candidates for immobilizing various enzymes due to their biocompatibility.
Lignocellulosic biomass, including agricultural residues, forestry wastes, waste paper, and energy crops, has long been recognized as a potential sustainable source of biopolymers [9], [10]. Lignocellulose consists of three major biopolymers, namely cellulose, hemicellulose, and lignin, which show distinct chemical, physical, and structural properties. Cellulose, a linear polysaccharide of d-glucose residues linked by β-(1→4)-glycosidic bonds, is the most abundant renewable biopolymer on earth. It shows excellent thermal and mechanical properties and high biocompatibility [11], [12]. Cellulose hydrogels can be prepared from a cellulose solution through physical cross-linking, because cellulose contains abundant hydroxyl groups that can form hydrogen bonds. However, the development of cellulose hydrogels has been severely hampered by the difficulty of dissolving cellulose, because of its high crystallinity [13]. Recently, ionic liquids (ILs) have been developed for dissolving cellulose, and this method provides great opportunities for preparing cellulose hydrogels. ILs are organic salts that usually melt at temperatures lower than 100 °C, and they are good solvents for various biopolymers and synthetic polymers [14]. Therefore, cellulose and various cellulose/biopolymer composite hydrogels have been prepared by co-dissolution of cellulose and biopolymers in ILs and then regeneration using an anti-solvent [15].
Cellulose hydrogels have been used for immobilizing various enzymes such as laccase, pectinase, lipase, peroxidase, phosphatase, and glucoamylase [16], [17], [18], [19]. Although cellulose hydrogels have been widely used to immobilize enzymes by physical adsorption and covalent binding, only a few studies have been performed on the entrapment of enzymes into non-derivatized cellulose hydrogels. Generally, enzymes are fully inactivated during the gelation process of cellulose, because enzymes cannot retain their activity in the solvents to dissolve cellulose such as N-methylmorpholine-N-oxide (NMMO), CdO/ethylenediamine, and LiCl/N,N-dimethylacetamide (DMAc). Recently, Turner et al. attempted to use [Bmim][Cl] to entrap laccase in a cellulose hydrogel membrane [20]. More recently, we reported the successful entrapment of lipase in a cellulose hydrogel by using biocompatible [Emim][Ac]. In particular, cellulose/biopolymer composite hydrogels such as cellulose/chitosan, cellulose/agarose, and cellulose/carrageenan have been proved to be good supports for the entrapment of lipase [13]. The preparation of various cellulose/biopolymer composite hydrogels relies on the high biopolymer-dissolving capability of ILs. The combination of different biopolymers is an inexpensive, extremely attractive, and advantageous method to produce new structural materials. Therefore, cellulose/biopolymer composite hydrogels have attracted considerable interests in recent years. Various cellulose/biopolymer composite hydrogels prepared by ILs were used for heavy metal adsorption and enzyme immobilization. For example, Wang et al. prepared cellulose/collagen hydrogel beads for Cu(II) adsorption [21]. Liu et al. reported the immobilization of glucose oxidase on cellulose/chitosan hydrogel microspheres prepared by using [Bmim][Cl] [22]. In addition, Peng et al. reported the immobilization of laccase on cellulose/chitosan beads [23]. These indicate that cellulose-based hydrogels blended with various biopolymers can be potentially used as novel materials for enzyme immobilization.
Lignin is an aromatic network polymer composed of phenylpropanoid units. It is helpful for binding cellulose and hemicelluloses together in lignocelluloses. Its content in lignocelluloses normally ranges from 18% to 35% [24]. The hydrophobicity of lignin is higher than that of cellulose and hemicelluloses, and its properties depend on the applied extraction methods. Although lignin is usually regarded as a waste material, many studies on the application of lignin have been conducted in recent years [25]. Sakagami et al. reported the pharmacological activities of lignin, such as antitumor, antimicrobial, and antioxidant activities [26]. Dhingra et al. found that lignin showed various therapeutic functions such as the abilities for reducing the risk of heart disease and lowering the variance in blood sugar level [27]. Zhang et al. revealed that lignin could be used as an activator to increase the activity of α-amylase and lipase [28]. Our recent studies showed that cellulose/lignin-based composites could be easily prepared by using [Emim][Ac]. Cellulose, lignin, and xylan were fully dissolved in [Emim][Ac] and successfully reconstituted into various gel forms such as molded shape, thin film, and microfiber [29], [30], [31]. In addition, cellulose/lignin/starch film and lignocellulose aerogel were successfully prepared by using ILs [32], [33]. The development of cellulose/lignin-based composites implies great opportunities to develop environmentally friendly and biocompatible materials, because of their high biocompatibility and biodegradability.
In this study, cellulose/lignin composite hydrogels in various cellulose to lignin ratios were prepared and used for immobilizing lipase from Candida rugosa. The effects of lignin content in the cellulose/lignin hydrogels on the protein loading, activity, and stability of immobilized lipase were investigated by systematically changing the hydrogel compositions. The controllability and predictability of the properties of hydrogel beads and the activity of immobilized lipase were also determined by statistical analysis.
Section snippets
Materials
Cellulose (cotton linter with long fiber form), alkali lignin, [Emim][Ac], p-nitrophenyl butyrate, sodium phosphates monobasic, sodium phosphate dibasic, and lipase from C. rugosa (1176 U/mg) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Isopropanol, acetic acid, sodium acetate trihydrate, and acetonitrile were purchased from Samchun Pure Chemical (Gyeonggi-do, South Korea). p-Nitrophenol was obtained from Kanto Chemical (Tokyo, Japan). All other chemicals used in this study were of
Characteristics of cellulose/lignin hydrogel beads
Fig. 1 shows photographic and SEM images of cellulose and cellulose/lignin hydrogel beads. The addition of lignin to the cellulose solution changed the color of hydrogel beads to dark brown. Although cellulose and cellulose/lignin hydrogel beads showed almost equivalent wet bead sizes, the cellulose hydrogel beads shrank more than cellulose/lignin hydrogel beads did during the freeze-drying process, and the latter retained the original spherical shape after free-drying. This may be caused by
Conclusions
In this study, lipase from C. rugosa was immobilized on various cellulose/lignin hydrogel beads prepared by using [Emim][Ac]. The loaded content, activity, and stability of immobilized lipase were greatly enhanced by increasing the lignin content in the gel-forming solution containing cellulose and lignin. The hydrophobic nature of lignin may induce the interfacial activation of lipase and increase the interaction between lipase and hydrogel beads. In particular, the stability of lipase
Acknowledgements
This study was supported by the Korea Ministry of Environment through Converging Technology Project (2012-000620001) and The EI Project 2012 (405-112-038). This work was also supported by the Rural Development Administration through the Cooperative Research Program for Agriculture Science & Technology Development (010205022014), the Korea CCS R&D Center (KCRC) grant (2013M1A8A1038187), and partially supported by the Energy Efficiency & Resources of the Korea Institute of Energy Technology
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