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.
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