Enzyme Immobilization on Ultrafine Cellulose Fibers via Poly(acrylic acid) Electrolyte Grafts Hong Chen, You-Lo Hsieh Fiber and Polymer Science, University of California, Davis, California 95616; telephone: 530-752-7364; e-mail: ylhsieh @ucdavis.edu Received 12 April 2004; accepted 26 August 2004 Published online 6 April 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.20324 Abstract: Ultrafine cellulose fiber (diameter 200 – 400 nm) surfaces were grafted with polyacrylic acid (PAA) via either ceric ion initiated polymerization or methacrylation of cel- lulose with methacrylate chloride (MACl) and subsequent free-radical polymerization of acrylic acid. PAA grafts by ceric ion initiated polymerization increased with increasing reaction time (2 – 24 h), monomer (0.3 – 2.4 M), and initi- ator (1 – 10 mM) concentrations, and spanned a broad range from 5.5 – 850%. PAA grafts on the methacrylated cellulose fibers also increased with increasing molar ratios of MACl to cellulosic hydroxyl groups (MACl/OH, 2 – 6.4) and monomer acrylic acid (AA) to initiator potassium per- sulfate (KPS) ratios ([AA]/[KPS], 1.5 – 6), and were in a much narrower range between 12.8% and 29.4%. The ad- sorption of lipase (at 1 mg/ml lipase and pH 7) and the activity of adsorbed lipase (pH 8.5, 30jC), in both cases decreased with increasing PAA grafts. The highest adsorp- tion and activity of the lipase on the ceric ion initiated grafted fibers were 1.28 g/g PAA and 4.3 U/mg lipase, respectively, at the lowest grafting level of 5.5% PAA, whereas they were 0.33 g/g PAA and 7.1 U/mg lipase, respectively, at 12.8% PAA grafts on the methacrylated and grafted fibers. The properties of the grafted fibers and the absorption behavior and activity of lipase suggest that the PAA grafts are gel-like by ceric-initiated reaction and brush-like by methacrylation and polymerization. The adsorbed lipase on the ceric ion-initiated grafted surface possessed greatly improved organic solvent stability over the crude lipase. The adsorbed lipases exhibited 0.5 and 0.3 of the initial activity in the second and third assay cy- cles, respectively. B 2004 Wiley Periodicals, Inc. Keywords: fiber; cellulose; poly(acrylic acid); lipase; adsorption INTRODUCTION Enzymes are natural catalysts with many advantageous characteristics, such as high specificity, mild reaction con- ditions, water solubility, biodegradability, and nontoxicity, over inorganic catalysts. Enzymatic biocatalysts function in homogeneous solutions and are most commonly applied in single use batch fashion. Reusing the enzymes requires separation and purification processes which are often com- plicated and costly and may cause contamination and loss of activity. Therefore, improving efficiency of enzyme re- covery would widen their applications in industry. Immo- bilization of the enzymes onto solid supports enables direct retrieval and is a reasonable approach to achieve this goal. Various chemical and physical methods have been re- ported to immobilize enzymes on a variety of polymeric solids (Jia et al., 2002; Kulik et al., 1993; Michael et al., 2001; Moeschel et al., 2003; Piletsky et al., 2003). Adding functionalized matrixes capable of immobilizing enzymes to polymer surfaces has been achieved by either chemical conversion or grafting of the polymer surfaces. Surface grafting is a desirable approach because it imposes less effect on the bulk properties, such as mechanical strength, of the materials, while offering wider options of chemically reactive groups and functional structures for immobilizing different enzymes. Fibrous materials are among the most suitable solid supports for immobilization of proteins and enzymes due to their intrinsically high specific surfaces and porous structures, providing the quantity and the accessibility of the active sites necessary for high reaction rates and con- versions. Moreover, the superior mechanical and handling properties of fibrous supports give them major advantages over loose particulates. Little, however, has been reported on fibrous supports in contrast to the abundant work on other forms of polymers. Polyethylene hollow fibers that were surface grafted with glycidyl methacrylate (GMA) and subsequently reacted with diethylamine were found to adsorb 490 mg of bovine serum albumin (BSA) on 1 g of modified fibers (Tsuneda et al., 1995). Grafting of poly- propylene fabrics with 2-vinyl-4,4-dimethylazlactone (VDM) by electron-beam initiation generated electrophilic azlactone groups that could covalently bond sericin up to 10% mass uptake (Fontaine et al., 2002). Cellulose fibers modified with 10–60 wt% of polyethyleneimine (PEI) in the presence of glutaraldehyde could adsorb up to 10 mg endotoxin per g of modified fibers (Morimoto et al., 1995). Trypsin was physically adsorbed on PAA grafted (via ozone) B 2004 Wiley Periodicals, Inc. Correspondence to: Y-L. Hsieh