A Novel Bifunctional Endolytic Alginate Lyase with Variable Alginate-Degrading Modes and Versatile Monosaccharide-Producing Properties

Endo-type alginate lyases usually degrade alginate completely into various sizedefined unsaturated oligosaccharide products (≥disaccharides), while exoenzymes primarily produce monosaccharide products including saturated mannuronate (M) and guluronate (G) units and particularly unsaturated units.Recently, two bifunctional alginate lyases have been identified as endolytic but M-and G-producing with variable action modes.However, endolytic -producing alginate lyases remain undiscovered.Herein, a new Flammeovirga protein, Aly2, was classified into the polysaccharide lyase 7 superfamily.The recombinant enzyme and its truncated protein showed similar stable biochemical characteristics.Using different sugar chains as testing substrates, we demonstrated that the two enzymes are bifunctional while G-preferring, endolytic whereas monosaccharide-producing.Furthermore, the catalytic module of Aly2 can vary the action modes depending on the terminus type, molecular size, and M/G content of the substrate, thereby yielding different levels of M, G, and units.Notably, the enzymes preferentially produce units when digesting small size-defined oligosaccharide substrates, particularly the smallest substrate (unsaturated tetrasaccharide fractions).Deletion of the non-catalytic region of Aly2 caused weak changes in the action modes and biochemical characteristics.This study provided extended insights into alginate lyase groups with variable action modes for accurate enzyme use.