Methyl-tolerant enzymes coupled with glycomics-based workflow facilitates porphyran structure characterization.

Abstract

Porphyran is a promising bioactive ingredient mainly composed of alternating (1→4)-α-l-galactose 6-sulfate (L6S) and (1→3)-β-d-galactose (G), with L6S units partially substituted by 3,6-anhydro-l-galactose (LA), and the O-6 position of G residues could be occasionally modified by methyl group (Me). Comprehensive research on porphyran requires efficient and precise structure characterization. In this study, a novel enzyme-glycomics strategy for porphyran structure analysis was developed. The approach involved adequately degradation of porphyran by methyl-tolerant agarase Aga86A_Wa and porphyranase Por16C_Wf. The resulting oligosaccharides were detected via UPSEC-HRMS and automatically analyzed using glycoinformatics software. The identified oligomers included (L6S-G)Me_0-1, (LA-G)_1-2, (L6S-G)(LA-G)Me_0-1, (L6S-G)₂(LA-G)Me_0-2, (L6S-G)(LA-G)₂Me_0-1. This confirms the identification of the consecutive homogeneous blocks (consisting of L6S-G or LA-G), alternating heterogeneous blocks (consisting of L6S-G and LA-G), and their methylated derivatives of porphyran. Furthermore, This method successfully distinguished the structure variations of porphyran in Porphyra from different harvest periods, species and producing areas. It provides a feasible approach for efficient and precise analyses of major and heterogeneous structure fragments of porphyran, and supports future investigations into porphyran structure-activity relationships.