*Article* **Cloning and Partial Characterization of an Endo-**α**-(1**→**6)-**d**-Mannanase Gene from** *Bacillus circulans*

**Shiva kumar Angala 1,\*,**†**, Wei Li 1,**†**, Zuzana Palˇceková 1, Lu Zou 2, Todd L. Lowary 2, Michael R. McNeil <sup>1</sup> and Mary Jackson 1,\***


Received: 22 November 2019; Accepted: 6 December 2019; Published: 11 December 2019

**Abstract:** Mycobacteria produce two major lipoglycans, lipomannan (LM) and lipoarabinomannan (LAM), whose broad array of biological activities are tightly related to the fine details of their structure. However, the heterogeneity of these molecules in terms of internal and terminal covalent modifications and complex internal branching patterns represent significant obstacles to their structural characterization. Previously, an endo-α-(1→6)-D-mannanase from *Bacillus circulans* proved useful in cleaving the mannan backbone of LM and LAM, allowing the reducing end of these molecules to be identified as Man*p*-(1→6) [Man*p*-(1→2)]-Ino. Although first reported 45 years ago, no easily accessible form of this enzyme was available to the research community, a fact that may in part be explained by a lack of knowledge of its complete gene sequence. Here, we report on the successful cloning of the complete endo-α-(1→6)-D-mannanase gene from *Bacillus circulans* TN-31, herein referred to as *emn*. We further report on the successful production and purification of the glycosyl hydrolase domain of this enzyme and its use to gain further insight into its substrate specificity using synthetic mannoside acceptors as well as LM and phosphatidyl-*myo*-inositol mannoside precursors purified from mycobacteria.

**Keywords:** endo-α-(1→6)-d-mannase; mannoside; *Mycobacterium*; lipomannan; lipoarabinomannan; phosphatidylinositol mannosides
