title: Fusion to a pull-down domain: a novel approach of producing Trigonopsis variabilisD-amino acid oxidase as insoluble enzyme aggregates.
abstract: Insoluble protein particles showing high specific enzyme activity are potentially useful biocatalysts. The commercialized crosslinked enzyme crystals and aggregates have the disadvantage that their preparation requires isolation of the protein before the critical precipitation step. We introduce a novel concept of controlled precipitation in vivo in which the target enzyme is fused to the cellulose-binding domain (CBD) of Clostridium cellulovorans, and expression in Escherichia coli is performed under conditions that induce selective pull down of the folded chimeric protein via intermolecular self-aggregation of the CBD. The case of D-amino acid oxidase from Trigonopsis variabilis shows that upon fusion of the CBD to its N-terminus, the otherwise mainly soluble recombinant enzyme was quantitatively precipitated in protein particles, which displayed 40% of the specific activity of the highly purified oxidase. By contrast, inclusion bodies derived from an enzyme chimera, which harbored a C-terminal peptide tag, showed only little oxidase activity (<or= 10%). The aggregated CBD retained the ability to bind microcrystalline cellulose and flocculated polysaccharide particles upon attachment to them. The cellulose-bound oxidase was stabilized about 36 times against inactivation of the soluble enzyme during conversion of D-methionine and bubble aeration.
authors.affiliation: Research Centre Applied Biocatalysis, c/o Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12, A-8010 Graz, Austria.
authors.forename: Jozef
authors.initials: J
authors.lastname: Nahalka
authors.forename: Bernd
authors.initials: B
authors.lastname: Nidetzky
mesh_terms: D001203:Ascomycota / Q000201:enzymology*; D001665:Binding Sites; D001709:Biotechnology / Q000379:methods; D002482:Cellulose / Q000378:metabolism*; D003605:D-Amino-Acid Oxidase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D010316:Particle Size; D012995:Solubility
chemical_list: D002482:Cellulose; D003605:D-Amino-Acid Oxidase