Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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Record number 449970
Title Bifunctional immobilization of a hyperthermostable endo ß 1,3 glucanase
Author(s) Przybysz, A.; Volmer, A.A.; Westphal, A.H.; Berkel, W.J.H. van
Source Applied Microbiology and Biotechnology 98 (2014)3. - ISSN 0175-7598 - p. 1155 - 1163.
DOI https://doi.org/10.1007/s00253-013-4953-3
Department(s) Biochemistry
VLAG
Publication type Refereed Article in a scientific journal
Publication year 2014
Keyword(s) pyrococcus-furiosus - enzyme immobilization - epoxy supports - proteins - stabilization - adsorption - reagent - acid
Abstract Laminarinase A (LamA) from Pyrococcus furiosus is a hyperthermostable endo-ß-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.
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