## 4 Discussion  
Hemicellulose biomass is considered as a renewable source of sugar next to cellulose. In order to make better use of hemicellulose, more attention should be paid to hemicellulose producing bacteria. In recent years, culturable \(\beta\)-mannanase producing bacteria have been isolated from many sources. _Enterobacter aerogenes_, which is a gram-negative bacillary microorganism from _Enterobacteriaceae_, is widely found in water, soil and other environments. _Enterobacter aerogenes_ is one of the members of the normal intestinal flora, but also an important conditional pathogenic bacteria. _Enterobacter aerogenes_ can ferment a variety of sugars to produce clean and regenerated energy, such as ethanol [26], hydrogen [28], butanediol [35]. Here, we first reported a \(\beta\)-mannanase (Man26E) from _Enterobacter aerogenes_. Although a large number of highly homologous sequences with Man26E are found in NCBI library. However, at present, there are only a few reports about \(\beta\)-mannanase from _Enterobacteriaceae_ strains, such as _Klebsiella pneumoniae_ SS11 [36], _Klebsiella oxytoca_ KUB-CW2-3 [37], _Enterobacter_ sp. N18 [38], _Enterobacter ludwigii_ MY271 [39], _Enterobacter asburiae_ SD26 [40] and _Pantea agglomerans_ A021 [41]. Man26E belonged to GH26 family whose overall structure of domain possessed (\(\beta\)/\(\alpha\))8 TIM barrel domain [42]. The modeled 3D structure of Man26E was close to the standard for a good quality model which is 90% of total residues in the most favoured regions [43], indicating that it was reasonable and reliable. The modeled structure of Man26E is similar to those of BCman (PDB ID. 2 QHA) [44] and BsMan26A (PDB ID. 3 CBW) [45]. A major difference among the three structures lies in the number and the position of \(\alpha\)- helix, \(\beta\)-strand and loop. In addition, no disulfide bond was found in Man26E,  
Figure 8: Substrate specificity of the recombinant Man26E. LBG: locust bean gum; KP: konjac powder; MOS: manno-oligosaccharides; pNPM: \(p\)-nitrobenzene-p-pyranoside. however, a disulfide bond exists between Cys75 at \(\beta\)-strand 2 and Cys95 at \(\alpha\)- helix 4 in BsMan26A, between Cys66 at \(\beta\)-strand 2 and Cys 86 at \(\alpha\)- helix 4 in BCman, respectively. The conserved active center of Man26E showed high similarity to that of CjMan26C (PDB ID. 2 VX7) from _Cellvibrio japonicus_, suggesting that the similar binding patterns of mannose are present in their active centers. The acid-base catalytic residue and the nucleophilic residue in GH26 family \(\beta\)-mannanases are highly conserved, and are all glutamate. For instance, they are Glu183 and Glu282 in Man26E, Glu221 and Glu338 in CjMan26C [46], Glu176 and Glu275 in BsMan26A, respectively. The four enzymes contain a similar platform composed of four solvent-exposed tryptophan rings, which are Trp318, Trp314, Trp188 and Trp79 in Man26E, Trp302, Trp298, Trp172 and Trp72 in BCman, Trp373, Trp298, Trp226 and Trp167 in CjMan26C, Trp328, Trp324, Trp198, Trp98 in BsMan26A. Two of the four aromatic residues exhibit highly conserved properties in GH26 enzymes, they are Trp172 and Trp298 in BCman, Trp198 and Trp324 in BsMan26A, Trp373 and Trp226 in CjMan26C, which are equivalent to Trp188 and Trp314 in Man26E, respectively. The platform is involved in the binding of the active domain to the substrate. [PERSON] et al. [43] have demonstrated that Trp298 and Trp172 are very important for binding and catalysis. We speculated that Trp188 and Trp314 may have similar functions. The Man26E- Gal1 Man4 and CjMan26C- Gal1 Man4 complexes display the interactions between Gal1 Man4 and the \(-\)1, \(-\)2, \(+\)1 and \(+\)2 subsites in the active region. However, the positions and quantities of hydrogen bond between amino acid residues and sugars are different. For example, at the \(-\)2 subsite, only Trp314 and Tyr43 in Man26E made limited interactions with the mannose, by comparison, an amount of interactions are found, involving Arg374, Gln385, Trp373 and Asp130 in CjMan26C.