Effects of Macroporous Resin Size on Candida antarctica Lipase B Adsorption, Fraction of Active Molecules, and Catalytic Activity for Polyester Synthesis Bo Chen, † Elizabeth M. Miller, ‡ Lisa Miller, § John J. Maikner, ‡ and Richard A. Gross* ,† NSF I/UCRC for Biocatalysis and Bioprocessing of Macromolecules, Polytechnic UniVersity, 6 Metrotech Center, Brooklyn, New York 11201, Rohm and Haas Co., P.O. Box 904, Spring House, PennsylVania 19477, and National Synchrotron Light Source, BrookhaVen National Laboratory, Upton, New York 11973 ReceiVed July 31, 2006. In Final Form: October 7, 2006 Methyl methacrylate resins with identical average pore diameter (250 Å) and surface area (500 m 2 /g) but with varied particle size (35 to 560-710 µm) were employed to study how immobilization resin particle size influences Candida antarctica Lipase B (CALB) loading, fraction of active sites, and catalytic properties for polyester synthesis. CALB adsorbed more rapidly on smaller beads. Saturation occurred in less than 30 s and 48 h for beads with diameters 35 and 560-710 µm, respectively. Linearization of adsorption isotherm data by the Scatchard analysis showed for the 35 µm resin that: (i) CALB loading at saturation was well below that required to form a monolayer and fully cover the support surface and (ii) CALB has a high affinity for this resin surface. Infrared microspectroscopy showed that CALB forms protein loading fronts for resins with particle sizes 560-710 and 120 µm. In contrast, CALB appears evenly distributed throughout 35 µm resins. By titration with p-nitrophenyl n-hexyl phosphate (MNPHP), the fraction of active CALB molecules adsorbed onto resins was <50% which was not influenced by particle size. The fraction of active CALB molecules on the 35 µm support increased from 30 to 43% as enzyme loading was increased from 0.9 to 5.7% (w/w) leading to increased activity for ǫ-caprolactone (ǫ-CL) ring-opening polymerization. At about 5% w/w CALB loading, by decreasing the immobilization support diameter from 560-710 to 120, 75, and 35 µm, conversion of ǫ-CL % to polyester increased (20 to 36, 42, and 61%, respectively, at 80 min). Similar trends were observed for condensation polymerizations between 1,8-octanediol and adipic acid. Introduction Application of immobilized enzymes in biocatalytic practice offers unique advantages over soluble enzymes, such as enhanced activity, increased selectivity, improved stability, and reusability. Adsorption is a simple and straightforward route for biomolecule immobilization. By this method, sufficient quantities of active enzyme have been immobilized and used for industrial processes. For example, Assemblase, the commercial name of immobilized pencillin-G acylase from Escherichia coli, has been used by industry for manufacture of the semi-synthetic -lactam antibiotic cephalexin. 1,2 Candida antarctica Lipase B (CALB), due to its unique properties, is attracting increased attention as a biocatalyst for the synthesis of low molar mass and polymeric molecules. 3-6 Almost all publications on immobilized CALB use the com- mercially available catalyst Novozyme 435, which consists of CALB physically adsorbed onto a macroporous acrylic polymer resin (Lewatit VP OC 1600, Bayer). Primarily, commercial uses of CALB are limited to production of high-priced specialty chemicals 7-9 because of the high cost of commercially available CALB preparations: Novozyme 435 (Novozymes A/S) and Chirazyme (Roche Molecular Biochemicals). It is urgent for CALB and other enzymes of commercial importance to focus attention on studies to better correlate enzyme activity to support parameters. 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