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Öğe Immobilization of glucoamylase onto spacer-arm attached magnetic poly(methylmethacrylate) microspheres: characterization and application to a continuous flow reactor(Elsevier Science Bv, 2000) Arica, MY; Yavuz, H; Patir, S; Denizli, AMagnetic poly(methylmethacrylate) microspheres (MPMMA) were prepared by the solvent evaporation method and a 6-carbon spacer-arm (i.e. hexamethylene diamine, HMDA) was covalently attached by the reaction of carbonyl groups of poly(methylmethacrylate). Glucoamylase was then covalently immobilized through the spacer-arm of the MPMMA microspheres by using either carbodiimide (CDI) or cyanogen bromide (CNBr) as a coupling agent. The activity yield of the immobilized glucoamylase was 57% for CDI coupling and 73% for CNBr coupling. Kinetic parameters were determined for both immobilized glucoamylase preparations as well as for the free enzyme. The K-m values for immobilized glucoamylases CDI coupling (12.5 g l(-1) dextrin) and CNBr coupling (9.3 g l(-1) dextrin) were higher than that of the free enzyme (2.1 g l(-1) dextrin) whereas V-max values were smaller for the immobilized glucoamylase preparations. The optimum operational temperature was 5 degreesC higher for both immobilized preparations than that of the free enzyme. Operational, thermal and storage stabilities were found to be increased with immobilization. (C) 2000 Elsevier Science B.V. All rights reserved.Öğe Monosize and non-porous p(HEMA-co-MMA) microparticles designed as dye- and metal-chelate affinity sorbents(Elsevier Science Bv, 2000) Denizli, A; Yavuz, H; Arica, YCongo red was immobilised onto monosize and non-porous poly(2-hydroxyethylmethacrylate-co-methylmethacrylate) [p(HEMA-co-MMA)] copolymer microparticles (4.0 mu m in diameter). Then Fe(III) ions were complexed by chelation with the immobilised congo red molecules. Different amounts of Fe(III) ions were loaded on the dye-derived microparticles by changing the concentration of Fe(III) ions and pH of the reaction medium. Congo red-derived and Fe(III)-complexed microparticles were used in the adsorption of glucose oxidase, catalase, lysozyme and bovine serum albumin. The maximum adsorption capacities of these microparticles were determined by changing pH and the concentration of the proteins in the adsorption medium. Their adsorption behavior can be described at least approximately with the Langmuir equation. Glucose oxidase, catalase, lysozyme and bovine serum albumin adsorption capacities of the Fe(III) complexed microparticles (165.1, 135.2, 67.6 and 44.5 mg g(-1)) were higher than those of the congo red-immobilised microparticles (125.9, 94.2, 35.8 and 21.2 mg g(-1), respectively). The non-specific adsorption of the proteins on the p(HEMA-co-MMA) microparticles was negligible. The resulting dye- and metal-chelate affinity microparticles have excellent reusability and long term storage stability. (C) 2000 Elsevier Science B.V. All rights reserved.Öğe Nonporous monosize polymeric sorbents: Dye and metal chelate affinity separation of lysozyme(John Wiley & Sons Inc, 2000) Denizli, A; Yavuz, H; Garipcan, B; Arica, MYLysozyme adsorption onto dye-attached nonporous monosize poly(2-hydroxyethyl-methacrylate-methylmethacrylate) [poly(HEMA-MMA)] microspheres was investigated. Poly(HEMA-MNA) microspheres were prepared by dispersion polymerization. The monochloro-triazine dye, Cibacron Blue F3GA, was immobilized covalently as dye-ligand. These dye-affinity microspheres were used in the lysozyme adsorption-desorption studies. The effect of initial concentration of lysozyme and medium pH on the adsorption efficiency of dye-attached and metal-chelated microspheres were studied in a batch reactor. Effect of Cu(II) chelation on lysozyme adsorption was also studied. The nonspecific adsorption of lysozyme on the poly(HEMA-MMA) microspheres was 3.6 mg/g. Cibacron Blue F3GA attachment significantly increased the lysozyme adsorption up to 247.8 mg/g. Lysozyme adsorption capacity of the Cu(II) incorporated microspheres (318.9 mg/g) was greater than that of the Cibacron Blue F3GA-attached microspheres. Significant amount of the adsorbed lysozyme (up to 97%) was desorbed in 1 h in the desorption medium containing 1.0M NaSCN at pH 8.0 and 25 mM EDTA at pH 4.9. In order to examine the effects of separation conditions on possible conformational changes of lysozyme structure, fluorescence spectrophotometry was employed. We conclude that dye-attached and metal-chelate affinity chromatography with poly(HEMA-MMA) microspheres can be applied for lysozyme separation without causing any significant changes and denaturation. Repeated adsorption/desorption processes showed that these novel dye-attached monosize microspheres are suitable for lysozyme adsorption. (C) 2000 John Wiley & Sons, Inc.Öğe Therapeutic affinity adsorption of iron(III) with dye- and ferritin-immobilized pHEMA adsorbent(John Wiley & Sons Inc, 2001) Yavuz, H; Arica, Y; Denizli, AMicroporous poly(2-hydroxyethylmethacrylate) (pHEMA) films carrying cibacron blue F3GA, Congo red, and ferritin were prepared and used for iron(III) removal from human plasma. pHEMA films were produced by a photopolymerization of 2-hydroxyethylmethacrylate in the presence of azobisisobutyronitrile. The reactive dye ligands cibacron blue F3GA, Congo red, and bioligand ferritin were then covalently attached to the pHEMA films. The maximum dye loadings were 1.07 and 0.80 mu mol/cm(2) for cibacron blue and Congo red, respectively. The maximum amount of ferritin attached was 1.04 x 10(-3) mu mol/cm(2). Characterizations of the films were achieved by contact-angle, water-uptake, and scanning electron microscopy studies as well as atomic force microscopy images. The aqueous water-uptake properties and contact angles lair underwater of the pHEMA films did not change after derivatization with cibacron blue F3GA, Congo red, and ferritin. These hydrophilic films (contact angle = 45.3 degrees), having a swelling ratio of 58% (w/w) and carrying cibacron blue F3GA, Congo red, and ferritin, were used in Fe(III) removal studies. The maximum amounts of Fe(III) removed from human plasma by cibacron blue F3GA-, Congo red-, and ferritin-attached pHEMA films were 3.80 mug/cm(2) for cibacron blue F3GA, 4.41 mug/cm(2) for Congo red, and 8.1 mug/cm(2) for ferritin-attached films. Fe(III) ions could be repeatedly adsorbed and desorbed with these affinity pHEMA films without a noticeable loss in their Fe(III) adsorption capacity. (C) 2001 John Wiley & Sons, Inc.
