## 4 Discussion
In GH26 \(\beta\)-mannanases, the residues binding the substrate at the \(-\)1 subsite show strict conservative feature, such as His114, His182, Glu183 and Glu282 in Man26E have structural equivalents in CjMan26C and CjMan26A, which correspond to His156 and His143, His182 His220 and His211, Glu221 and Glu212, Glu338 and Glu320, respectively [46]. At the \(+\)1 subsite, Glu183 and Asp234 of Man26E formed limited polar interactions with the sugar, whereas in CjMan26C and CjMan26A, the equivalents to Asp234 of Man26E are Asp266 and Asn257, respectively. The \(+\)2 site is quite different among Man26E, CjMan26C and CjMan26A. In Man26E, Asp234 and Arg237 produced polar connections with the sugar. For CjMan26C, Ser268 and Arg269 generates interactions with the sugar, however, the equivalent loop in CjMan26A does not exist any residues near \(+\)2 subsite. The activity (497.9 U/mg) of the recombinant Man26E was higher than those of \(\beta\)-mannanases from _Bacillus circulans_ K-1 (481.55 U/mg) [47] and _Bacillus sp_. JAMB-750 (36.3 U/mg) [48], however, lower than those of \(\beta\)-mannanase from _Clostridium thermocellum_ (520 U/mg) [49] and _Pantoea agglomerans_ A021 (514 U/mg). The molecular mass of Man26E was predicted to be 38.61 kDa and the purified enzyme was about 40 kDa evaluated by SDS-PAGE. It has been reported that the molecular weights of \(\beta\)-mannanases from a variety of sources are in the range of 30-80 kDa [50]. Exceptionally, a GH26 \(\beta\)-mannanase from _Clostridium thermocellum_ shows low molecular weight of 15 kDa [51], and the other \(\beta\)-mannanase from _Thermoanaerobacterium polysaccharide_ has a high molecular weight of 120 kDa [52]. The optimal pH value (6.5) of the recombinant Man26E is comparable to those of \(\beta\)-mannanases from _Bacillus subtilis_ WL7 [53], _Pantoea agglomerans_ A021, _Bacillus subtilis_ WL-3 [54], _Bacillus_ sp. SWU60 [55] and _Bacillus subtilis_ (TBS2) [56]. Most bacterial \(\beta\)-mannanases have the highest activity from neutral to alkaline pH. For instance, \(\beta\)-mannanases (GH 26) from _Bacillus_ sp. CFR1601 [57], _Bacillus subtilis_ BCC41051 [58], _Bacillus licheniformis_[59] and _Bacillus subtilis_ B23 [20] are optimally active at pH 7. \(\beta\)-mannanases (GH 26) from _Bacillus_ N16-5 (GH 26) [60], _Bacillus licheniformis_ THCM 3.1 [61] and _Bacillus sp. strain_ JAMB-750 [62] are optimally active at pH 9.6, 9.0 and 10.0, respectively. Bacterial \(\beta\)-mannanases show remarkable stability in the range of pH 6-9 [50]. Exceptionally, the activity of \(\beta\)-mannanase from _Acinetobacter_ sp. ST 1-1 is maintained over 80% in the range of pH 3-10 [63]. The optimal temperature (55 \({}^{\circ}\)C) of Man26E is similar to \(\beta\)-mannanases from _Bacillus_ sp. CFR1601, _Pantoea agglomerans_ A021, _Erwinia carotorova_ CXJZ95-198 [64] and _Bacillus subtilis_ WL7. However, \(\beta\)-mannanases from _Bacillus_ N16-5 (GH 26) and _Caldicellustivuptor_ Rt 8B.4 (GH 26) [6] show the highest activity above 70 \({}^{\circ}\)C. Interestingly, \(\beta\)-mannanase from _Thermotoga neapolitana_ 5068 exhibits maximum activity at 92 \({}^{\circ}\)C [65]. The recombinant Man26E is less stable than those of \(\beta\)-mannanases from _Pantoea agglomerans_ A021, _Bacillus subtilis_ B23, _Bacillus_ sp. SWU60 and _Enterobacter_ sp. N18. However, the thermostability of Man26E is better than those of \(\beta\)-mannanases from _Cellulosinicrobium_ sp. strain HY-13 [66] and _Spingomonas_ sp. ERB1-3 [67]. Similar results to Man26E have been reported that Co\({}^{2+}\) shows positive action on the activities of the recombinant \(\beta\)-mannanases from _Enterobacter_ sp strain N18 and _Bacillus subtilis_ WY34 [68], and Mn\({}^{2+}\) performs positive effect on the \(\beta\)-mannanases from _Bacillus_ sp. N16-5 and _Bacillus subtilis_ WY34. The activation of Fe\({}^{2+}\) on the recombinant Man26E is different from several published \(\beta\)-mannanases.