Table 1.
Origins and binding properties of enzymes in this study.
Table 2.
Properties of polysaccharides used in this study.
Table 3.
Affinity electrophoresis regimes.
Fig 1.
Affinity electrophoresis of cellulases with and without cellobiose.
AE was performed with the polysaccharides barley β-glucan (A; BBG) and hydroxyethyl cellulose (B; HEC) in the presence and absence of the active site inhibitor cellobiose. Lane 1 is a protein ladder (NativeMark from Invitrogen), lane 2 is GH6-1, lane 3 GH8-1 and lane 4 GH48-1.
Fig 2.
SDS-PAGE of fractions from insoluble polysaccharide pulldown assays of the cellulases GH6-1, GH8-1 and GH48-1 with avicel.
For each protein lane 1 is the initial protein sample, lane 2 the fraction bound to avicel in the absence of inhibitors, lane 3 the fraction bound to avicel in the presence of 100 mM cellobiose and lane 4 the fraction bound to avicel in the presence of 4 mM cellopentaose.
Table 4.
Binding affinities and stoichiometries for family GH13 enzymes and cellulases as determined by surface plasmon resonance.
Fig 3.
Conserved surface binding sites in related family members.
The crystal structure is represented as a cartoon in red, with the bound carbohydratesshown in green (carbon) and red (oxygen). Conserved residues of the SBS also found in the enzymes in this studyare shown in yellow (carbon) and red (oxygen), while non-conserved residues are shown in grey. A shows the GH5 exo-β1—3 glucanase from Candida albicans (2PC8), B shows the GH8 xylanase from Pseudoalteromonas haloplanktis (2B4F), C shows the GH10 xylanase from Thermoascus aurantiacus (1GOQ), D shows the GH11 xylanase from Bacillus subtilis (2QZ3), E shows the GH13 (subfamily 5) α-amylase from Bacillus sp. 707, F shows the GH31 α-glucosidase from Ruminococcus obeum (3POC).