**4. Conclusions**

The full-length endo-α-(1→6)-D-mannanase gene from *B. circulans* TN-31 was successfully cloned for the first time and its glycosyl hydrolase domain expressed and purified in active and stable form from *E. coli*. The analysis of the digestion pattern of synthetic mannosides, PIM and LM provided further insights into the substrate specificity of the endomannanase domain of the enzyme. The availability of this enzyme, together with a better understanding of its catalytic activity, should greatly facilitate the structural analysis of α-(1→6)-D-mannan-containing polysaccharides, including mycobacterial LM and LAM.

#### **Supplementary Materials:** Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/24/ 6244/s1.

**Author Contributions:** Funding acquisition, M.J.; Conceptualization, M.J., M.R.M.; Investigation, S.K.A., W.L., Z.P., L.Z.; Data analysis, S.K.A., W.L., and M.R.M.; Writing—S.K.A., W.L., M.J., M.R.M.; Writing—reviewing & editing, M.J., M.R.M., and T.L.L.

**Acknowledgments:** This work was supported by the National Institute of Allergy and Infectious Diseases (NIAID)/National Institutes of Health (NIH) grant AI064798 (to MJ), and the Alberta Glycomics Centre. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. We thank Claudia M. Boot and Zachery C. Moen from CSU's Central Instrumentation Facility for their assistance with LC/MS analyses. The following reagents were obtained through BEI Resources, NIAID, NIH: *M. tuberculosis*, Strain H37Rv, Purified Phophatidylinositol Mannoside 6 (PIM6) NR-14847 (lot 70009918), and *M. smegmatis*, lipoarabinomannan (LAM), NR-14849 (lot 61699475).

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**


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