title: Heterofunctional Magnetic Metal-Chelate-Epoxy Supports for the Purification and Covalent Immobilization of Benzoylformate Decarboxylase From Pseudomonas Putida and Its Carboligation Reactivity.
abstract: In this study, the combined use of the selectivity of metal chelate affinity chromatography with the capacity of epoxy supports to immobilize poly-His-tagged recombinant benzoylformate decarboxylase from Pseudomonas putida (BFD, E.C. 4.1.1.7) via covalent attachment is shown. This was achieved by designing tailor-made magnetic chelate-epoxy supports. In order to selectively adsorb and then covalently immobilize the poly-His-tagged BFD, the epoxy groups (300 µmol epoxy groups/g support) and a very small density of Co(2+)-chelate groups (38 µmol Co(2+)/g support) was introduced onto magnetic supports. That is, it was possible to accomplish, in a simple manner, the purification and covalent immobilization of a histidine-tagged recombinant BFD. The magnetically responsive biocatalyst was tested to catalyze the carboligation reactions. The benzoin condensation reactions were performed with this simple and convenient heterogeneous biocatalyst and were comparable to that of a free-enzyme-catalyzed reaction. The enantiomeric excess (ee) of (R)-benzoin was obtained at 99 ± 2% for the free enzyme and 96 ± 3% for the immobilized enzyme. To test the stability of the covalently immobilized enzyme, the immobilized enzyme was reused in five reaction cycles for the formation of chiral 2-hydroxypropiophenone (2-HPP) from benzaldehyde and acetaldehyde, and it retained 96% of its original activity after five reaction cycles.
authors.affiliation: Department of Chemistry, Faculty of Education, Dicle University, Diyarbakir, Turkey.
authors.forename: Servet
authors.initials: S
authors.lastname: Tural
authors.affiliation: Department of Chemistry, Faculty of Education, Dicle University, Diyarbakir, Turkey.
authors.forename: Bilsen
authors.initials: B
authors.lastname: Tural
authors.affiliation: Department of Chemistry, Faculty of Arts and Sciences, Middle East Technical University, Ankara, Turkey.
authors.forename: Ayhan S
authors.initials: AS
authors.lastname: Demir
mesh_terms: D000079:Acetaldehyde / Q000737:chemistry; D000096:Acetone / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000737:chemistry; D001547:Benzaldehydes / Q000737:chemistry; D001573:Benzoin / Q000737:chemistry; D055162:Biocatalysis; D002262:Carboxy-Lyases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D002614:Chelating Agents / Q000737:chemistry*; D003035:Cobalt / Q000737:chemistry*; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D004852:Epoxy Compounds / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry; D059346:Magnets / Q000737:chemistry*; D016958:Pseudomonas putida / Q000201:enzymology*; D013237:Stereoisomerism
chemical_list: D001547:Benzaldehydes; D002614:Chelating Agents; D004800:Enzymes, Immobilized; D004852:Epoxy Compounds; D000096:Acetone; C033223:polyhistidine; D003035:Cobalt; D006639:Histidine; C035293:1-hydroxy-1-phenyl-2-propanone; D002262:Carboxy-Lyases; C019778:benzoylformate decarboxylase; D000079:Acetaldehyde; D001573:Benzoin; C032175:benzaldehyde
keywords: covalent attachment; immobilized benzoylformate decarboxylase; metal-affinity magnetic nanoparticles; metal-chelate-epoxy magnetic support; one step purification; selective immobilization