Structure and function of a novel cellulase 5 from sugarcane soil metagenome

Abstract

Cellulases play a key role in enzymatic routes for degradation of plant cell-wall polysaccharides into simple and economically-relevant sugars. However, their low performance on complex substrates and reduced stability under industrial conditions remain the main obstacle for the large-scale production of cellulose-derived products and biofuels. Thus, in this study a novel cellulase with unusual catalytic properties from sugarcane soil metagenome (CelE1) was isolated and characterized. The polypeptide deduced from the celE1 gene encodes a unique glycoside hydrolase domain belonging to GH5 family. The recombinant enzyme was active on both carboxymethyl cellulose and β-glucan with an endo-acting mode according to capillary electrophoretic analysis of cleavage products. CelE1 showed optimum hydrolytic activity at pH 7.0 and 50 °C with remarkable activity at alkaline conditions that is attractive for industrial applications in which conventional acidic cellulases are not suitable. Moreover, its three-dimensional structure was determined at 1.8 Å resolution that allowed the identification of an insertion of eight residues in the β8-α8 loop of the catalytic domain of CelE1, which is not conserved in its psychrophilic orthologs. This 8-residue-long segment is a prominent and distinguishing feature of thermotolerant cellulases 5 suggesting that it might be involved with thermal stability. Based on its unconventional characteristics, CelE1 could be potentially employed in biotechnological processes that require thermotolerant and alkaline cellulases.

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. Structural studies of CelE1.

(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.