Protease-Containing Silicates as Active Antifouling Materials Jungbae Kim, † Ray Delio, and Jonathan S. Dordick* Department of Chemical Engineering, Rensselaer Polytechnic Institute, Troy, New York 12180 Biocatalytic silicates, composite materials composed of R-chymotrypsin and a silicate prepolymer, were prepared via a two-step polymerization process following solubili- zation of the enzyme in the polymerization media. This new approach resulted in active and stable composites, and a calculated half-life of over 350 days in aqueous buffer at 30 °C. The high stability and activity of this biocatalytic silicate was likely due to the covalent attachment between R-chymotrypsin and the silicate matrix. The protease- containing silicate was resistant to fouling by nonselective protein binding, as demonstrated by the dramatically reduced binding of human serum albumin to the silicate material when compared to that of a silicate containing pre-inactivated R-chymotrypsin. Introduction Silicates have been used as immobilization matrices for enzymes, antibodies, cells, inorganic catalysts, and dyes (Ellerby et al., 1992; Bhatia et al., 1998 and 2000; Gill and Ballesteros, 1998; Bergogne et al., 2000; Blum et al., 1999; Avnir, 1995) and in biosensors (Lin and Brown, 1997). Such broad appeal is a result of their chemical inertness, mechanical strength, thermal stabil- ity, optical transparency, biocompatibility, and relatively low cost. The entrapment of enzymes in silica-based materials typically has involved conventional sol-gel processing (Ellerby et al., 1992), consisting of discrete hydrolysis and condensation steps (Bergogne et al., 2000). Tetramethyl orthosilicate (TMOS) or tetraethyl ortho- silicate (TEOS) is hydrolyzed in an acidic medium while enzymes are dissolved in a second buffer, which facili- tates gel formation as soon as it contacts the hydrolyzed TMOS (or TEOS). Such an abrupt contact may result in the propensity of entrapped enzymes to leach out from the gel, thereby limiting the biocatalytic lifetime of the sol-gel preparations. We have developed new approaches for synthesis of composite materials of enzymes with polymers, wherein acryloylated enzymes are copolymerized in the presence of vinyl monomers (e.g., methyl methacrylate, styrene, and vinyl acetate, among others) using standard free radical initiation (Wang et al., 1997; Novick and Dordick, 2000). These “biocatalytic plastics” display high catalytic activity, particularly in organic solvents, and high stabil- ity in aqueous and organic media and can be prepared using a wide range of enzymes. In the present work, we have extended the biocatalytic plastic methodology to the formation of R-chymotrypsin-containing (CT) biocatalytic silicates. Acryloylated CT is copolymerized in the pres- ence of a vinyl silicate monomer to yield biocatalytic silicate matrices with high activity and stability. The presence of a protease contained within the silicate polymer network results in surfaces that resist nonselec- tive protein binding. Therefore, these materials may be useful as broad-spectrum antifouling materials. Materials and Methods Materials. CT, human serum albumin (HSA), aerosol dioctylsodium sulfosuccinate (AOT), o-phthaldialdehyde (OPA), N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide (TP), and N-acetyl-Phe ethyl ester (APEE) were purchased from Sigma Chemical (St. Louis, MO). All other reagents and solvents were purchased from Aldrich (Milwaukee, WI) and were of the highest grade commercially avail- able. The solvents were dried over 3 Å molecular sieves for 24 h prior to use, and the water content was less than 0.005% (v/v) as determined by Karl Fischer titration. Enzyme Modification and Solubilization. Enzyme modification and solubilization were performed by fol- lowing the procedure of Wang et al. (1997). CT (80 mg) was added to 10 mL of phosphate buffer (0.2 M potassium phosphate, pH 8.0). The enzyme solution was cooled to 4 °C, and 40 μL of acryloyl chloride was gradually added to the solution over 10 min. The acryloylated enzyme was recovered by gel filtration chromatography (Sephadex G-25 gel, 100-300 μM). Ten milliliters of an aqueous enzyme solution (containing 1 mg/mL acryloylated R-chy- motrypsin, 1% (v/v) 2-propanol, and 2 mM CaCl 2 dis- solved in 10 mM Bis-Tris buffer, pH 7.8) was contacted with an equal volume of hexane containing 2 mM AOT. The two-phase mixture was stirred vigorously at 22 °C and 300 rpm for 5 min and centrifuged at 7000 × g for 10 min. Upon separation of the organic phase from the aqueous solution, the enzyme-surfactant complex was dried by evaporating hexane under vacuum and then reconstituted into the solvent of choice. The concentration of protein in the organic phase was determined by UV absorption at 280 nm. Two-Step Polymerization. Vinyltrimethoxysilane (VTMS) and a cross-linker (trimethylpropane trimethacry- late) were added at a molar ratio of 1:0.05 in hexane containing solubilized enzyme (2 and 20 mg/mL acryloy- lated CT in hexane for 1% and 10% initial CT loading, respectively). The volume excess of hexane to all mono- mers (VTMS, acryloylated CT, and cross-linker) was * To whom correspondence should be addressed. Ph: 518 276- 2899. Fax: 518 276-2207. Email: dordick@rpi.edu. † Current address: Environmental Molecular Science Labora- tory, Pacific Northwest National Laboratory, P.O. Box 999, Rich- land, WA 99352. 551 Biotechnol. Prog. 2002, 18, 551-555 10.1021/bp020036q CCC: $22.00 © 2002 American Chemical Society and American Institute of Chemical Engineers Published on Web 04/25/2002