Characterization of a Novel Porphyranase Accommodating Methyl-galactoses at Its Subsites.

Porphyran is the major polysaccharide of laver and mainly composed of 3-linked β-d-galactopyranose (G) and 4-linked α-l-galactopyranose-6-sulfate (L6S) units. Structural heterogeneity of porphyran highly originates from the natural methylation on the O-6 position of G units (GMe). Here, a GH16 porphyranase Por16C_Wf was cloned from a porphyran-related polysaccharide utilization locus of Wenyingzhuangia fucanilytica and expressed in Escherichia coli. It hydrolyzed porphyran in a random endo-acting manner. Using a glycomics strategy combining liquid chromatography-mass spectrometry and glycoinformatics, the subsite specificity was clarified. Por16C_Wf accommodated both G and GMe at subsites -1 and +2. This is the first report on the sequence of porphyranases hydrolyzing consecutive methyl-porphyranobiose moieties, which shed light on the diversity in subsite specificity of porphyranases. Por16C_Wf was the first characterized enzyme in subfamily 14 of the GH16 family. The defined and novel activity of Por16C_Wf implied that it could serve as a favorable tool in the full degradation and structural investigation of porphyran.