Comparative structural analysis of CelE1 (4M1R) with other structurally similar cellulases 5.

<p>(A) Representation of the extended α₈/β₈ loop conserved in thermostable enzymes (BsCel5A, <i>Bacillus subtilis</i>, 3PZU; BaCel5A, <i>Bacillus agaradhaerens</i>, 1QHZ) in comparison to meso- and psychrophilic cellulases (EcCel5, <i>Erwinia chrysanthemi</i>, 1EGZ; Cel5G, <i>Pseudoalteromonas haloplanktis</i>, 1TVN). The helix α1 that makes new interactions with the extended α₈/β₈ loop is colored in light pink. (B) Surface complementarity between the extended α₈/β₈ loop (yellow mesh) and the neighboring structural elements (green) indicating the additional intramolecular contacts favored by this motif. </p

Cleavage pattern of CelE1 on different cellooligosaccharides (cellotetraose (C4), cellopentaose (C5) or cellohexaose (C6)) indicating a classical endo-acting mode.

<p>(A) Capillary-zone-electropherogram of the APTS-labeled-cellohexaose hydrolysis (substrate). (B) Analysis of APTS-labeled products of C4, C5 and C6 hydrolysis.</p

Effect of pH and temperature on the hydrolytic activity of CelE1.

<p>Enzyme was incubated in 200 mM phosphate-citric acid-glycine buffer containing 0.5% (w/v) of CMC as substrate for 20 min and the amount of reducing sugars measured by the 3,5–dinitrosalicylic acid method. (A) Measurements were carried out in pH values ranging from 2 to 12 by incubation at 37 °C. (B) Activity assayed under different temperatures at pH 7. Assays were performed on quadruplicate aliquots. Each experiment was repeated three times.</p

Biophysical characterization of CelE1.

<p>(A) Far-UV CD spectrum of CelE1 with typical profile of α/β proteins. (B) Thermal denaturation profile characterized by a single transition and a melting temperature of 55 °C.</p