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.
Highlights
In alginate degraded by the recombinant enzymes Avi-rAlgL and Pae-rAlgL, unsaturated oligosaccharides with high DPs were the main products in the initial steps and gradually converted into smaller products (Figure S5A,B), eventually producing various size-defined fractions (Figure S5A,B,D), indicating that Avi-AlgL and Pae-AlgL are endotype alginate lyases, which is in agreement with existing reports [39,40,42,43]
To elucidate and compare the core values of alginate lyases with Mpreference in alginate degradation, direct oligosaccharide preparation, and associated enzymatic mechanisms, and to benefit novel tool-like enzyme exploration, we report comparative studies of Aly6 (AlgL-like module and Hep_II_III-like module) as a novel
This study demonstrated that the AlgL-like module and the Hep_II_III-like module in
Summary
Relatively little is known about these enzymes’ oligosaccharide products and corresponding substrate action modes, except their M-specificities or Mpreferences This lack of knowledge urgently needs to be overcome in order to achieve direct oligosaccharide preparation, structure identification, and functional exploration by using these AlgLs. In this study, wild-type, M-preferred and endolytic genes of AlgL from Pseudomonas aeruginosa (Pae-AlgL) and Azotobacter vinelandii (Avi-AlgL) respectively, sharing the same key motif of NNHSYW at the C-terminus, were initially codon-optimized, artificially synthesized, cloned and expressed in Escherichia coli strain BL21(DE3) for soluble proteins and comparison to the protein Aly of the marine-derived polysaccharide-degrading bacterium.
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