Abstract
Recent explorations of tool-like alginate lyases have been focused on their oligosaccharide-yielding properties and corresponding mechanisms, whereas most were reported as endo-type with α-L-guluronate (G) preference. Less is known about the β-D-mannuronate (M) preference, whose commercial production and enzyme application is limited. In this study, we elucidated Aly6 of Flammeovirga sp. strain MY04 as a novel M-preferred exolytic bifunctional lyase and compared it with AlgLs of Pseudomonas aeruginosa (Pae-AlgL) and Azotobacter vinelandii (Avi-AlgL), two typical M-specific endolytic lyases. This study demonstrated that the AlgL and heparinase_II_III modules play indispensable roles in determining the characteristics of the recombinant exo-type enzyme rAly6, which is preferred to degrade M-enriched substrates by continuously cleaving various monosaccharide units from the nonreducing end, thus yielding various size-defined ΔG-terminated oligosaccharides as intermediate products. By contrast, the endolytic enzymes Pae-rAlgL and Avi-rAlgL varied their action modes specifically against M-enriched substrates and finally degraded associated substrate chains into various size-defined oligosaccharides with a succession rule, changing from ΔM to ΔG-terminus when the product size increased. Furthermore, site-directed mutations and further protein structure tests indicated that H195NHSTW is an active, half-conserved, and essential enzyme motif. This study provided new insights into M-preferring lyases for novel resource discoveries, oligosaccharide preparations, and sequence determinations.