Entrapment of enzymes into cellulose–biopolymer composite hydrogel beads using biocompatible ionic liquid

https://doi.org/10.1016/j.molcatb.2011.11.011Get rights and content

Abstract

For the first time, lipase from Candida rugosa was successfully entrapped into various cellulose–biopolymer composite hydrogels by using a biocompatible ionic liquid, 1-ethyl-3-methylimidazolium acetate ([Emim][Ac]). Lipase-entrapped cellulose and cellulose–biopolymer composite hydrogel beads were simply prepared by co-dissolution of biopolymers in [Emim][Ac] and dispersion of lipase in biopolymer solution followed by formation of biopolymer hydrogel using distilled water. Immobilization yields (specific activity ratio of entrapped lipase to free lipase) of cellulose, cellulose–carrageenan, cellulose–chitosan, cellulose–agarose, and cellulose–agar bead were 35.0, 9.6, 39.7, 41.4, and 52.6%, respectively. Cellulose–biopolymer composite hydrogels proved to be good supports for entrapment of enzymes and have many potential applications, including drug delivery, biosensors, biofuel cells, and tissue engineering due to their inherent excellent biocompatibility and biodegradability.

Highlights

► For the first time, enzyme was successfully entrapped into unmodified cellulose–biopolymer composite hydrogels. ► Lipase could be entrapped into various cellulose–biopolymer composite hydrogels with higher immobilization yields than cellulose beads. ► Enzymes entrapped in cellulose–biopolymer composites can be used in various biomedical fields.

Introduction

Gels are defined as three-dimensional polymer networks swollen by large amounts of solvent. Hydrogels are usually structures formed from natural or synthetic polymers, and contain large amounts of trapped water [1]. Recently, biopolymer-based hydrogels have received considerable attention for applications in biomedical fields, including tissue engineering, drug delivery systems, contact lenses, and biosensors, because of their inherent biocompatibility and biodegradability [2]. Various hydrogels from biopolymers have been fabricated by using hyaluronate, alginate, agarose, starch, gelatin, cellulose, chitosan, and their derivatives.

Cellulose is the most abundant renewable biopolymer. It has excellent thermal and mechanical properties and biocompatibility, and for economic and scientific reasons, is a promising material for biochemical engineering [3], [4]. Cellulose hydrogel can be prepared from a cellulose solution through physical cross-linking, because cellulose has abundant hydroxyl groups which can form hydrogen bonds. However, the development of cellulose hydrogel has been hampered by the difficulty of dissolving cellulose, because cellulose is highly crystalline. Recently, ionic liquids (ILs) have been developed to dissolve cellulose, providing great opportunities for the preparation of cellulose hydrogels. ILs are organic salts that usually melt at temperatures <100 °C. Interest in ILs stems from their potential applications as ‘green solvents’. ILs are good solvents for polar organic, nonpolar organic, inorganic, and polymeric compounds [5]. Cellulose hydrogels have been prepared by regenerating a cellulose solution in 1-allyl-3-methylimidazolium chloride ([Amim][Cl]) using deionized water as a coagulant [6]. Additionally, biopolymers such as chitin, chitosan, silk, and DNA can be fabricated from ILs to produce films, membranes, fibers, spheres, and molded shapes [4]. Therefore, various biopolymer composite hydrogels can also be prepared by co-dissolution of two or more biopolymers into ILs. Recently, Sun et al. [7] prepared cellulose–chitosan composite beads using 1-butyl-3-methylimidazolium chloride ([Bmim][Cl]) for heavy metal ion adsorption. Blending of different biopolymers is an extremely attractive inexpensive and advantageous method to obtain new structural materials. Cellulose-based composite hydrogels blended with various biopolymers will create novel materials for special applications [1], [8].

Enzymes have been recognized as efficient and environmentally friendly catalysts because of their high specificity and catalytic activity under mild conditions. However, the industrial applications of enzymes have been limited due to their low stability, and difficult recovery for subsequent use. Enzyme immobilization is the most commonly used strategy to overcome these drawbacks [9]. 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 [9]. Various polysaccharide hydrogels such as alginate, chitosan, agarose, and carrageenan have been employed for the entrapment of a number of enzymes such as lipases, lactases, invertase, endo-β-glucanase, and peroxidase [10], [11], [12], [13], [14]. However, at this time, there are few reports on the entrapment of enzymes into non-derivatized cellulose. Even though microbial cells, not isolated enzymes, were entrapped within a cellulose fiber and beads with a mixture of N-ethylpyridinium chloride and dimethylformamide [15], [16], the resulting fiber was generally brittle due to insufficient gelation [17]. Recently, Turner et al. [18] attempted to use ionic liquid [Bmim][Cl] to entrap laccase into a cellulose membrane. The enzyme was entrapped into cellulose but showed low residual activity because of IL-induced denaturation. It was known that ILs capable of dissolving cellulose also have a denaturing effect on enzymes. It is expected that the activity of the entrapped enzyme could be enhanced by using ILs, which cannot only dissolve cellulose but also do little harm to enzymes.

In this study, for the enzyme entrapment we used 1-ethyl-3-methylimidazolium acetate ([Emim][Ac]), which is known to be one of the best solvents for lignocellulosic materials among the ILs [19], [20]. In addition, [Emim][Ac] is one of the most promising candidates for industrial applications due to its low viscosity, low melting point, non-toxicity, and biodegradability [19], [21]. Lipase from Candida rugosa, which has more industrial applications than any other enzymes: pharmaceutical, cosmetics, food, perfumery, and bioremediation [22], was entrapped into cellulose hydrogel beads with high residual activity. Moreover, the lipase was successfully immobilized in various cellulose composite hydrogel beads formed with agarose, chitosan, carrageenan, and agar as a counter biopolymer. To the best of our knowledge, this is the first report concerning the successful entrapment of enzyme into non-derivatized cellulose–biopolymer composite hydrogels.

Section snippets

Materials

Cellulose (microcrystalline), chitosan (high molecular weight, deacetylation degree of 75%), carrageenan (Type I, predominantly κ and lesser amounts of λ carrageenan), 1-ethyl-3-methylimidazolim acetate ([Emim][Ac]), p-nitrophenyl butyrate, p-nitrophenol, isopropanol, and lipase from C. rugosa were purchased from Sigma–Aldrich (St. Louis, MO, USA). Agarose was purchased from Biopure (Ontario, Canada). Agar (gel strength 500–1000 g/cm2) and sodium alginate were purchased from Samchun Pure

Entrapment of lipase in cellulose hydrogel beads

Preparation of enzyme-entrapped cellulose hydrogel has been limited by difficulty in selecting a suitable solvent to dissolve cellulose without inactivating enzyme. Although [Bmim][Cl] was used to entrap laccase, the residual activity was only ∼18%, due to enzyme denaturing conditions by [Bmim][Cl] containing a high concentration of [Cl] [18], [24]. In this study, [Emim][Ac], which was used as an enzyme-friendly co-solvent for resolution of amino acids [25], was first employed to dissolve

Conclusions

We have achieved entrapment of lipase into cellulose hydrogel beads with high immobilization yields by using [Emim][Ac] as a dissolving solvent of cellulose. The lipase could be also entrapped into various cellulose–biopolymer composite hydrogels with higher immobilization yields than cellulose beads. Our procedure for enzyme entrapment in biopolymer hydrogels was simple and mild: dissolution of biopolymers in [Emim][Ac], dispersion of enzyme in the biopolymer solution, followed by

Acknowledgement

This work was supported by the Faculty Research Fund of Konkuk University in 2009.

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