## 4 Discussion
For instance, Fe\({}^{2+}\) inhibits the activity of \(\beta\)-mannanase from _Paenibacillus_ cookie [69], however, Fe\({}^{2+}\) makes no difference in \(\beta\)-mannanases from _Bacillus subtilis_ WY34 and _Cellulosinicrobium_ sp. strain HY-13. The inhibition of Mg\({}^{2+}\) and Cu\({}^{2+}\) on the recombinant Man26E is in consistent with several reported \(\beta\)-mannanases [38; 70; 71]. The substrate specificity of the recombinant Man26E is the same as those of _Bacillus circulans_ NT 6.7 [72], _Bacillus subtilis_ WY34, _Bacillus_ sp. N16-5, _Bacillus stearothermophilus_[73], _Bacillus circulans_ CGMCC 1416 [74] and _Bacillus subtilis_ WL-3, which show higher activity toward LBG than toward KGM. By comparison, the substrate specificity of Man26E is different from those of \(\beta\)-mannanases from _B. licheniformis_ mannanase [48], _Bacillus_ sp. SWU60, _Bacillus subtilis_ WD23 [75] and _Paenibacillus_ cookie, which show more activity against glucomannan than against galactomannan. It has been reported that the \(K_{m}\) values of \(\beta\)-mannanase are between 0.0095 and 27.4 mg mL\({}^{-1}\), and the V\({}_{\text{max}}\) values are ranging from 29 to 16,666.7 umol mL\({}^{-1}\) or mg min\({}^{-1}\)[50]. The \(K_{m}\) value (7.16 mg mL\({}^{-1}\)) of the recombinant Man26E for LBG is similar to the values (7.22 mg mL\({}^{-1}\)) reported for a \(\beta\)-mannanase from _Bacillus nealsonii_ PN-11 [76]. The \(K_{m}\) value of the recombinant Man26E is significantly lower than those of BCman from _Bacillus subtilis_ Z-2 (10.2 mg mL\({}^{-1}\)) and _Bacillus subtilis_ YH12 (30 mg mL\({}^{-1}\)) [77]. Distinguishingly, the Km value of the \(\beta\)-mannanase from _Paenibacillus_ cookie is 0.0095 mg mL\({}^{-1}\) for LBG. Man26E displayed many excellent features, for example, acid and alkali tolerance, high stability over a wide range of neutral-to-alkaline pH, good thermostability, high stability in the presence of various metal ions. These characteristics suggested that Man26E could have great potential for commercial application in the biofuels, the food/feed and paper/pulp and other industries. In addition, the simulation of the three-dimensional structure and the analysis of the active domain of this mannanase will help to understand the structure- function relationship, and lay a foundation for the molecular modification of the enzyme to improve its enzymatic properties. This paper was accomplished based on collaborative work of the authors. Conceptualization, T.C. and H.L. ; methodology, T.C. ; software, T.C. ; validation, H.L., J.L. and T.C. ; formal analysis, H.L. ; investigation, H.L. ; resources, J.L. ; data curation, H.L. and J.L. ; writing--original draft preparation, H.L. ; writing--review and editing, T.C. ; visualization, T.C. ; supervision, T.C. ; project administration, T.C. All authors have read and agreed to the published version of the manuscript. This research received no external funding. No grants in this work. The authors thank [PERSON] from School of Biological Science and Bioengineering, South China University of Technology because he useful suggestions and language editing help us greatly improve the quality of our manuscript. We declare that we have no conflict of interest.