Figure 1.
Effect of pH and temperature on the hydrolytic activity of CelE1.
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.
Figure 2.
Cleavage pattern of CelE1 on different cellooligosaccharides (cellotetraose (C4), cellopentaose (C5) or cellohexaose (C6)) indicating a classical endo-acting mode.
(A) Capillary-zone-electropherogram of the APTS-labeled-cellohexaose hydrolysis (substrate). (B) Analysis of APTS-labeled products of C4, C5 and C6 hydrolysis.
Figure 3.
Biophysical characterization of CelE1.
(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.
Figure 4.
(A) Overall structure of the CelE1 showing a classical (β/α)8-barrel fold with the two catalytic acidic residues depicted. (B) Surface charge distribution with highlight to highly negatively charged active-site pocket that is essential for substrate binding and cleavage. (C) Details of the active site in which catalytically-relevant residues are indicated.
Figure 5.
Comparative structural analysis of CelE1 (4M1R) with other structurally similar cellulases 5.
(A) Representation of the extended α8/β8 loop conserved in thermostable enzymes (BsCel5A, Bacillus subtilis, 3PZU; BaCel5A, Bacillus agaradhaerens, 1QHZ) in comparison to meso- and psychrophilic cellulases (EcCel5, Erwinia chrysanthemi, 1EGZ; Cel5G, Pseudoalteromonas haloplanktis, 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.