Biomacromolecules 2005, 6, 2497-2502
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Ionic Liquid-Reconstituted Cellulose Composites as Solid Support Matrices for Biocatalyst Immobilization Megan B. Turner,† Scott K. Spear,† John D. Holbrey,†,§ Daniel T. Daly,‡ and Robin D. Rogers*,† Center for Green Manufacturing, Department of Chemistry, and the Alabama Institute for Manufacturing Excellence, The University of Alabama, Tuscaloosa, Alabama 35487 Received March 14, 2005; Revised Manuscript Received June 10, 2005
Preparation of cellulose-polyamine composite films and beads, which provide high loading of primary amines on the surface allowing direct one-step bioconjugation of active species, is reported using an ionic liquid (IL) dissolution and regeneration process. Films and bead architectures were prepared and used as immobilization supports for laccase as a model system demonstrating the applicability of this approach. Performance of these materials, compared to commercially available products, has been assessed using millimeter-sized beads of the composites and the lipase-catalyzed transesterification of ethyl butyrate. Introduction The attachment of enzymes to solid support materials has been studied extensively as a simple means of protein stabilization as well as catalyst separation and recovery from reaction systems.1,2 Typical materials used for these purposes include silica, polyanaline, acrylics, chitin, and cellulose.1,3 Cellulose provides unique inherent qualities that make it a desirable material to use as a biological support; it is hydrophilic and wettable, which can help create a compatible environment for proteins compared to more hydrophobic materials such as silica.4,5 In addition, cellulose is robust, chemically inert under physiological conditions, and nontoxic, all of which are important for protein survival and advantageous for industrial processing. However, the use of cellulose is generally associated with a number of drawbacks, most notably the need for extensive chemical activation and functionalization necessary to attach biomolecules to the surface.6-8 The development of methodologies which can reduce, or simplify, the functionalization process while allowing for the utilization of cellulosic materials is highly desirable. We have explored the use of ionic liquids, particularly 1-butyl-3-methylimidazolium chloride ([C4mim]Cl), as novel solvents for nonderivatizing cellulose dissolution and regeneration.9 Wu10 et al. have shown how related 1-alkyl-3allylimidazolium chloride ionic liquids (ILs) could also be used as solvents for homogeneous derivatization of cellulose, and Liebert and Heinze11 recently described using a novel alkylammonium fluoride/dimethyl sulfoxide solvent system for similar derivatizations of cellulose. In contrast, we have focused on using the dissolution characteristics of the ILs * To whom correspondence should be addressed. E-mail: rdrogers@ bama.ua.edu. † Department of Chemistry. ‡ The Alabama Institute for Manufacturing Excellence. § Current address: The Quill Research Center, Queen’s University of Belfast, Belfast BT9 5AG, Northern Ireland, U.K.
for cellulose to enable physical encapsulation of macromolecules such as Rhus Vernificera laccase (EC No. 1.10.3.2) in cellulose films using this approach and have demonstrated the compatibility of this particular enzyme with IL-cellulose environments.12 Enzymatically active membranes were prepared; however, significant loss in activity of the entrapped laccase, compared to the enzyme in an aqueous environment, was observed. This loss of activity, relative to that of the free enzyme, can likely be attributed to either decreased conformational flexibility of the enzyme when constrained within the support matrix and/or decreased diffusion limitations for the transport of substrates and products in to and out of the films. To improve biocatalytic activity by alleviating these problems, surface immobilization rather than bulk encapsulation is a preferred immobilization approach.13 Both physical and chemical attachment of catalysts to support materials is possible and has been widely demonstrated.1,14 Physical attachment is achieved through simple adsorption or weak ionic interaction between the enzyme and the surface of the support material; however, this type of attachment is easily reversible and generally associated with enzyme leaching. Chemical attachment through covalent bonds on the other hand leads to typically more stable, nonreversible binding and is the preferred mechanism for this application due to its increased stability. As a means of increasing the stability of the attachment bonds between the enzyme and the support, surface active conditions are necessary. We describe here the development of a process, using the [C4mim]Cl dissolution and regeneration procedure,9 to obtain cellulose-based composite materials formed as both transparent thin films and beads containing pendant primary amine functions providing the reactive surface coating necessary for bioconjugation. Further, use of the composites as immobilization materials has been demonstrated using a laccase-catalyzed reaction as a model
10.1021/bm050199d CCC: $30.25 © 2005 American Chemical Society Published on Web 07/29/2005
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Biomacromolecules, Vol. 6, No. 5, 2005
Turner et al.
Table 1. Secondary Polymers Incorporated into Cellulose Composite Films secondary polymer
MW
1° amine concn (mmol g-1)
solubility in H2O
appearance of film
A, polylysine hydrobromide B, bovine serum albumin C, Jeffamine D-230 D, Jeffamine T-403 E, Jeffamine D-2000 F, Jeffamine T-5000 G, poly(ethyleneimine) (linear) H, poly(ethyleneimine) (branched)
∼48 500 ∼66 50018 230 403 2 000 5 000 18 250 25 000
∼4.4 ∼0.5 8.3 6.0 1.0 0.5 5.2 0.1
50 mg mL-1 40 mg mL-1 >10% >10% 0.1-1%
