Fig 1.
Discovery of the cellulolytic enzyme CelDZ1.
(A) The amino acid sequence of the putative cellulolytic enzyme corresponding to the celDZ1α ORF was analysed against the Pfam-A database. The analysis revealead that the predicted sequence consists of a GH5 catalytic domain (denoted as domain 1), a carbohydrate-binding module (CBM) 17/28 (denoted as domain 2) and a transmembrane anchor (denoted as domain 3). (B) Zymogram analysis for the detection of cellulolytic activity via SDS-PAGE analysis and Congo red staining of a CMC-containing acrylamide gel. M: molecular weight marker; 1: cell lysate producing the target protein 2: cell lysate carrying an empty vector.
Table 1.
Substrate specificity of CelDZ1.
Fig 2.
Effect of pH and temperature on the activity of CelDZ1.
(A) CelDZ1 activity was measured in the standard reaction at 40°C for 5 min at pH values ranging from 4 to 10 and (B) at temperatures between 40 and 90°C for 5 min in a pH 5 buffer. The reported values correspond to the mean value from three independent experiments performed in triplicate and the error bars to one standard deviation from the mean value.
Fig 3.
(A). Catalytic thermostability of CelDZ1 evaluated by measurements of residual CMC-degrading activity after high-temperature exposure at 65, 70 and 75°C for up to 24 h. (B). Thermal denaturation analysis of CelDZ1 using differential scanning fluorimetry with the conformation-sensitive dye SYPRO Orange. The reported values correspond to the mean value from three independent experiments performed in triplicate and the error bars to one standard deviation from the mean value.
Fig 4.
Halostability and halotolerance of CelDZ1.
(A) CelDZ1 was incubated in 5 M NaCl and 4 M KCl for up to 20 days. At different time intervals aliquots were taken and the residual activity of the enzyme was measured in the standard reaction. (B) The activity of CelDZ1 in the presence of different high-salt concentrations was measured in the standard reaction. The reported values correspond to the mean value from three independent experiments performed in triplicate and the error bars to one standard deviation from the mean value.
Table 2.
Effect or metal ions and other denaturing agents on the activity of CelDZ1.
Table 3.
The data processing and refinement statistics for CelDZ1.
Fig 5.
(A) Folding of the CelDZ1α monomer is presented as a cartoon diagram and viewed from the solvent region towards the active site groove formed by the C-terminal ends of the β-strands of the (β/α)8-barrel. The α-helices, β-strands and loops are coloured in turquoise, magenta and pink, respectively. The carbohydrate-binding module (Fig 1), which contains helix α8 at the C terminus, is highlighted in green. The two catalytic residues are shown as stick models and secondary structural elements are labelled. The Met50 indicates the position of the first N-terminal residue defined in the electron density. The image was prepared using PyMol [40]. (B) A stereo representation of the superimposition of the monomers of CelDZ1, CelK and Cel5a displayed as grey carbon traces. The three different insertion regions are highlighted in red for CelDZ1, magenta for CelK and green for Cel5a. The cellobiose ligand bound to CelK is shown as a magenta stick model. The image was prepared using PyMol [40]. (C) The electrostatic potential surface of the CelDZ1 enzyme around the active site groove as viewed from the solvent region. The positive charge is shown in blue and the negative charge is shown in red. The extended active site groove, which crosses the monomer from left to right, is negatively charged disfavoring the binding of halogen ions thereby increasing halotolerance. The image was prepared with ccp4mg [41].