Expression and Characterization of a Methylated Galactose-Accommodating GH86 β‑Agarase from a Marine Bacterium

Agarose is the major component of agar, in which galactose units could be naturally modified by methyl groups. Although numerous agarases have been characterized, the capacity of agarases for accommodating methylated galactoses has been rarely investigated. In this study, we cloned, expressed, and characterized a novel GH86 family agarase Aga86A_Wa from a marine bacterium Wenyingzhuangia aestuarii OF219. The enzyme exhibited maximum activity at 30 °C and pH 6.5. Aga86A_Wa was a random endo-acting β-agarase. The smallest products of Aga86A_Wa were disaccharides. Besides typical agarose oligosaccharides, methylated oligosaccharides were also identified in the products by using liquid chromatography coupled with high-resolution mass spectrometry. It was confirmed that Aga86A_Wa could accommodate methylated galactoses at its −1 and +2 subsites. This is the first report on the sequence of the methyl group-tolerating agarase. Aga86A_Wa could be utilized as a biotechnological tool for producing methylated oligosaccharides and for the structural investigation of agarose.