Production of Bioactive Cellulose Films Reconstituted from Ionic Liquids Megan B. Turner, Scott K. Spear, John D. Holbrey, and Robin D. Rogers* Center for Green Manufacturing and Department of Chemistry, The University of Alabama, Tuscaloosa, Alabama 35487 Received April 27, 2004 A new method for introducing enzymes into cellulosic matrixes which can be formed into membranes, films, or beads has been developed using a cellulose-in-ionic-liquid dissolution and regeneration process. Initial results on the formation of thin cellulose films incorporating dispersed laccase indicate that active enzyme-encapsulated films can be prepared using this methodology and that precoating the enzyme with a second, hydrophobic ionic liquid prior to dispersion in the cellulose/ionic liquid solution can provide an increase in enzyme activity relative to that of untreated films, presumably by providing a stabilizing microenvironment for the enzyme. Introduction Cellulose appears to be an ideal support material for many enzyme systems, being both biosourced and biologically compatible. Many examples of cellulose-supported enzymes are known; in most cases, these supported-enzyme systems are prepared with cellulose derivatives and/or covalent binding, via functional linkers, to attach the enzymes on the surfaces of beads or membranes. 1,2 Entrapment or encapsula- tion of an enzyme or protein using other procedures, especially physical entrapment of the biomolecule, without recourse to chemical attachment is certainly desirable, if it can be achieved. The first organic molten salt system investigated as a cellulose solvent was N-ethylpyridinium chloride (mp 118- 120 °C), in 1934; 3 no commercial use of this solvent system seems to have been developed, possibly due to the high melting point of this salt. Husemann and Seifert 4 found that N-ethylpyridinium chloride mixed with 50% dimethylfor- mamide or dimethyl sulfoxide has a lower melting point (around 77 °C) and that this mixture dissolves cellulose well. In the late 1970s and early 1980s, researchers at the Helsinki University of Technology began experimenting with the N-ethylpyridinium chloride mixtures for immobilization of enzymes within cellulose fibers, 5 microbial cells within cellulose beads, 6 and whole cell yeast -galactosidase within cellulose, 7 largely without success. The investigation into the use of ionic liquids (ILs) as alternative solvents has been steadily increasing over the past 5 years. 8,9 Properties of existing ILs, including low melting points, wide liquid ranges, and lack of vapor pressure, have encouraged researchers to explore known chemical reactions and processes using ILs in place of volatile organic sol- vents. 8,9 Our interests in ILs lie in the utilization of their unique solvent properties for process applications, for example, in liquid-liquid extraction and separation pro- cesses 10 based on the consideration and matching of solvent and solute solubility parameters. 11,12 For example, we have demonstrated that the IL 1-butyl-3-methylimidazolium chlo- ride ([C 4 mim]Cl) can be used to readily dissolve cellulose without derivatization (Figure 1). 13,14 More recently, Wu and co-workers have shown that the IL 1-allyl-3-methylimida- zolium chloride also can be used in a similar manner, as a solvent to dissolve 15 or derivatize cellulose. 16 To determine whether the ready dissolution of cellulose in molten [C 4 mim]Cl could be used as a processing route for encapsulation of enzymatic species in biocompatible cellulosic membranes or beads, for reactive and sensing applications, two questions needed to be answered. First, can cellulose materials containing encapsulated enzymes be prepared using the [C 4 mim]Cl dissolution process, and if so, do the enzymes supported in such a cellulosic matrix remain active? As a model system, we chose to investigate the entrapment of laccase from Rhus Vernificera (E.C. 1.10.3.2) within reconstituted cellulosic films that were made using the [C 4 mim]Cl-cellulose dissolution and regeneration process. 13 Here we demonstrate the proof of concept, focusing on a single cellulose solvent, [C 4 mim]Cl, and its use to incorporate actiVe biomolecules directly into underivatized cellulose resulting in the formation of biologically active membranes. Experimental Section Preparation of ILs. The ILs, [C 4 mim]Cl and 1-(2- hydroxypropyl)-3-methylimidazolium chloride, were pre- * To whom correspondence should be addressed. E-mail: rdrogers@ bama.ua.edu. Figure 1. Chemical structure of 1-butyl-3-methylimidazolium chloride ([C4mim]Cl). 1379 Biomacromolecules 2004, 5, 1379-1384 10.1021/bm049748q CCC: $27.50 © 2004 American Chemical Society Published on Web 06/22/2004