title: Site directed immobilization of glucose-6-phosphate dehydrogenase via thiol-disulfide interchange: influence on catalytic activity of cysteines introduced at different positions.
abstract: This study shows the effect of site-directed enzyme immobilization upon the enzyme activity of covalently bound glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides. Immobilization points were introduced at sterically accessible sites in order to control the protein's orientation and twice as much activity was recovered in comparison to conventionally immobilized enzyme. Immobilization of G6PDH via genetically engineered cysteine provided a simple, but effective method to control the immobilization process. G6PDH variants with cysteine close to the active center (L218C), close to the dimer interface (D205C) as well as far from the active center (D453C) showed changes in activity and the efficacy of immobilization.
authors.affiliation: Institute of Technical Biocatalysis, Hamburg University of Technology, Denickestr. 15, 21073 Hamburg, Germany.
authors.forename: J R
authors.initials: JR
authors.lastname: Simons
authors.forename: M
authors.initials: M
authors.lastname: Mosisch
authors.forename: A E
authors.initials: AE
authors.lastname: Torda
authors.forename: L
authors.initials: L
authors.lastname: Hilterhaus
mesh_terms: D002384:Catalysis; D004220:Disulfides / Q000737:chemistry; D004800:Enzymes, Immobilized / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics; D005954:Glucosephosphate Dehydrogenase / Q000737:chemistry* / Q000235:genetics; D007934:Leuconostoc / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000737:chemistry*
chemical_list: D004220:Disulfides; D004800:Enzymes, Immobilized; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D005954:Glucosephosphate Dehydrogenase
keywords: 2-PDS; 2-pyridyldisulfide; A(343); DTT; G6P; G6PDH; Glucose-6-phosphate dehydrogenase; IPTG; LB; Leuconostoc mesenteroides; NADP(+); Ni-TED; OD; OPA; SDS-PAGE; Site directed enzyme immobilization; Site directed mutagenesis; Thiol disulfide interchange; Wt; absorption at 343nm; dithiothreitol; glucose-6-phosphate; glucose-6-phosphate dehydrogenase; isopropyl-β-d-thiogalactopyranosid; lysogenic broth; nickel-Tris(carboxymethyl)ethylene diamine; nicotinamide adenine dinucleotide phosphate; o-phthalaldehyde; optical density; sodium dodecyl sulfate polyacrylamide gel electrophoresis; wildtype