title: Development and characterization of a solid-phase biocatalyst based on cyclodextrin glucantransferase reversibly immobilized onto thiolsulfinate-agarose.
abstract: Reduction of disulfide bonds and introduction of "de novo" thiol groups in cyclodextrin glucantransferase from Thermoanaerobacter sp. were assessed in order to perform reversible covalent immobilization onto thiol-reactive supports (thiolsulfinate-agarose). Only the thiolation process dramatically improved the immobilization yield, from 0 % for the native and reduced enzyme, up to nearly 90 % for the thiolated enzyme. The mild conditions of the immobilization process (pH 6.8-7.0 and 22 °C) allowed the achievement of 100 % coupling efficiencies when low loads were applied. Ionic strength was a critical parameter for the immobilization process; for high activity recoveries, 50 mM phosphate buffer supplemented with 0.15 M NaCl was required. The kinetic parameters, pH and thermal stabilities for the immobilized biocatalyst were similar to those for the native enzyme. For β-cyclization activity, optimal pH range and temperature were 4.0-5.4 and 85 °C. The possibility of reusing the support was demonstrated by the reversibility of enzyme-support binding.
authors.affiliation: Departamento de Biociencias, Facultad de Química, Universidad de la República, General Flores, Montevideo, Uruguay.
authors.forename: Santiago Einar
authors.initials: SE
authors.lastname: Viera
authors.forename: Francisco
authors.initials: F
authors.lastname: Batista-Viera
authors.forename: Karen
authors.initials: K
authors.lastname: Ovsejevi
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D055162:Biocatalysis; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism; D005964:Glucosyltransferases / Q000737:chemistry* / Q000378:metabolism; D012685:Sepharose / Q000737:chemistry; D046968:Thermoanaerobacter / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D004800:Enzymes, Immobilized; D012685:Sepharose; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase