## 3 Results
### The Structural Prediction and the Catalytic Region Analysis  
The amino acid sequence of Man26E was assayed with NCBI CDD program ([[https://www.ncbi.nlm.nih.gov/cdd](https://www.ncbi.nlm.nih.gov/cdd)]([https://www.ncbi.nlm.nih.gov/cdd](https://www.ncbi.nlm.nih.gov/cdd))) and only one CDD matches obtained in Man26E was Glycosl hydrolase family 26 (9 aa-340 aa). The result showed that Man26E belonged to GH26 family with a (\(\beta\)/\(\alpha\))8 TIM barrel domain. The secondary structure, which was analyzed with SOPMA tool ([[https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html](https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html)]([https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html](https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html))), included 29.89% alpha-helix (104 aa), 17.53% extended strand (61 aa), 45.40% random coil (158 aa) and 7.18% beta-turn (25 aa). \(\beta\)-mannanase from _Bacillus subtilis_ str. 168 (PDB ID. 2 WHK-A) has 47.97% similarity (>30%) in the amino acid sequence with Man26E, so it can be used as the template for homology modelling. The final structural model covering residue 5 to 348 of Man26E was constructed with SWISS-MODEL tool, based on the structure of _B. subtilis_\(\beta\)-mannanase (PDB ID. 2 WHK-A). The rationality of the structure model of Man26E was evaluated with SAVES v5.0 tool, the Ramachandran plot was drawn using the PROCHECK program (Figure 2b). Three-dimensional structure of Man26E indicated that it had a saddle-shaped active center (Figure 3a). In terms of the three-dimensional map (Figure 2a), six key loops in the active region of Man26E participated in binding in the saddle -shaped active site. CjMan26C (PDB ID. 2 VX7) from _Cellvibrio japonicus_, a homologous \(\beta\)-mannanase (GH26) crystallographic structure in complex with mannose residues in PDB, was chosen as the template of substrate docking for Man26E. Man26E had 30.45% sequence homology (comparatively high in PDB) with CjMan26C, and the conserved active center of Man26E showed high similarity to that of CjMan26C. The substrate docking for Man26E and CjMan26C were shown in Figure 3b,c. The result indicated that all the residues located in the loops around the active region, except for Glu282 and Asp256, could form potential hydrogen bonds with the substrate. These residues included Trp318, Asp317, Asn316, Asn315, Trp314, Glu282, Tyr258, Asp256, Arg237, Asn236, Ala235, Asp234, Phe189, Trp188, Glu187, Glu183, His182, Arg179, Lys133, His114, Trp79, Asp74, Tyr43 and Phe40 (Figure 3d). The acid-base catalytic glutamate (Glu183) was at the loop between \(\beta\)-strand 4 and \(\alpha\)- helix 6, and the nucleophilic glutamate (Glu282) was at the N-terminal end of \(\beta\)-strand 8. The binding of Man26E to mannan was mainly mediated by a hydrophobic plane composed of a strip of solvent-exposed aromatic rings of tryptophans, which included Trp318, Trp314,  
Figure 2: Homology modeling of \(\beta\)-mannanase of Man26E. (**a**) The predicted 3D-structure of Man26E based on _B. subtilis_\(\beta\)-mannanase (2 WHK_A). The figure was generated using the program SWISS-MODEL and the software PyMol 2.3.2; C-terminus (bright _blue_), N-terminus (_red_); (**b**) Ramachandran plot of the homology-modeled structure of Man1E based on _B. subtilis_\(\beta\)-mannanase; (**c**) the overlap between Man26E (represented in gray) and BCman (represented in blue); (**d**) the overlap between Man26E (represented in gray) and BsMan26A (represented in yellow). Trp188, and Trp79. Moreover, many hydrophilic and charged residues, such as Asp317, Asn316, Asn315, Tyr258, Asp256, Arg237, Asn236, Asp234, Glu187, His182, Arg179, Lys133, His114, Asp74 and Tyr43, surrounded the four aromatic rings in the binding site.