title: Bifunctional immobilization of a hyperthermostable endo-β-1,3-glucanase.
abstract: Laminarinase A (LamA) from Pyrococcus furiosus is a hyperthermostable endo-β-1,3-glucanase (EC 3.2.1.39) belonging to the glycosyl hydrolase family GH16. Here, we report the two-step immobilization of LamA on macroporous acrylic epoxy beads, extra-functionalized with disulfide groups. To facilitate initial immobilization via thiol-disulfide exchange, we introduced, by site-directed mutagenesis, a superficial cysteine residue near the protein C-terminal end. The thus-obtained S296C variant showed similar catalytic properties as native LamA. The activity of immobilized S296C displayed an inverse relationship with particle size. Use of conventional beads (150-300 μm in diameter) obstructed the catalytic efficiency due to pore diffusion limitation of the polysaccharide substrate. Bifunctional attachment to milled beads (20-40 μm) resulted in high enzyme load and outstanding catalytic features. Bifunctional immobilized S296C showed extreme pH stability and could be repeatedly used at 60 °C without significant activity loss.
authors.affiliation: Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA, Wageningen, The Netherlands.
authors.forename: Agata
authors.initials: A
authors.lastname: Przybysz
authors.forename: Astrid A
authors.initials: AA
authors.lastname: Volmer
authors.forename: Adrie H
authors.initials: AH
authors.lastname: Westphal
authors.forename: Willem J H
authors.initials: WJ
authors.lastname: van Berkel
mesh_terms: D000180:Acrylic Resins; D044602:Cellulases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004693:Glucan Endo-1,3-beta-D-Glucosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008863:Microspheres; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020027:Pyrococcus furiosus / Q000201:enzymology*
chemical_list: D000180:Acrylic Resins; D004800:Enzymes, Immobilized; D050505:Mutant Proteins; D044602:Cellulases; D004693:Glucan Endo-1,3-beta-D-Glucosidase