**1. Introduction**

Two complex α-(1→6)-linked D-mannose-containing lipoglycans, lipomannan (LM) and lipoarabinomannan (LAM) populate the cell envelope of mycobacteria [1]. They are essential to the integrity of the cell envelope [2] and play important roles in the immunopathogenesis of mycobacterial infections [1,3,4]. LM and LAM are believed to share a mannosylated phosphatidyl-*myo*-inositol lipid anchor and a mannan backbone composed of α-(1→6)-linked-D-mannopyranosyl residues occasionally branched with α-(1→2)-D-mannopyranosyl residues [1]. Despite recent advances in the biosynthetic pathway of these lipoglycans, the fine details of the structure of their mannan backbone, specifically, the precise distribution of the α-(1→2)-D-mannopyranosyl residues along the mannan backbone, is not known with certainty. To help in these analyses, two types of glycosyl hydrolases capable of hydrolyzing the mannan backbone of LM and LAM have traditionally been used: An exo-type

α-(1→2,3,6)-mannosidase (EC # 3.2.1.24), which cleaves the terminal mannopyranosyl residues from the non-reducing end [5] and an endo-α-(1→6)-D-mannanase (EC # 3.2.1.101), which cleaves the glycosidic bond between two internal mannopyranosyl residues [6]. While the first enzyme can be purified from Jack bean and is commercially available, to the best of our knowledge, no commercial source of the second enzyme is available. The endomannanase hydrolyzing α-(1→6)-D-mannopyranosyl residues was discovered in 1974 by Ballou and co-workers [7] who then went on to purify this enzyme from the soil bacterium, *Bacillus circulans* [8], and subsequently shared this enzyme with other laboratories [6,9]. Some 24 years later, the gene encoding this enzyme, *aman6*, was identified by reverse genetics [10] but found to encode a protein only about half the size of that purified by Ballou et al., even though the recombinant product of the gene apparently displayed the expected catalytic activity on an (undefined) α-(1→6)-D-mannan substrate releasing 6α-mannotriose and 6α-mannobiose [10] [Figure S1]. Efforts by our laboratory to re-clone this gene by PCR based on its published sequence consistently failed, suggestive of potential rearrangements in the *Sau*3AI-digested chromosomal DNA libraries used in the isolation of *aman6* [10]. As a part of our continued effort to decipher the biosynthetic pathway of LM and LAM and establish the fine details of their structure, here, we report on the cloning of the full-size endo-α-(1→6)-D-mannanase gene (which we named *emn*) and its use to recombinantly express and purify the glycosyl hydrolase domain of this enzyme. The cleavage properties of the glycosyl hydrolase domain of Emn on synthetic mannoside acceptors, purified LM from *Mycobacterium smegmatis* and biosynthetic precursors, phosphatidyl-*myo*-inositol di- and hexa-mannosides, were investigated using mass spectrometry.

#### **2. Results and Discussion**
