# Catalytic Mode and Product Specificity of an α-Agarase Reveal Its Direct Catalysis for the Production of Agarooligosaccharides

## TL;DR

This study reveals how an α-agarase enzyme breaks down agarose into specific agarooligosaccharides, providing insights for enzyme engineering in food and medicine.

## Contribution

The study identifies the catalytic residues and mechanism of an α-agarase for producing specific agarooligosaccharides.

## Key findings

- Cm-AGA degrades agarose into agarotetraose and agarohexaose.
- Asp994 and Glu1129 are identified as catalytic residues through mutagenesis.
- Cm-AGA acts only on agarose oligosaccharides with a degree of polymerization greater than four.

## Abstract

Many α-agarases have been characterized and are utilized for producing agarooligosaccharides through the degradation of agar and agarose, which are considered valuable for applications in the food and medicine industries. However, the catalytic mechanism and product transformation process of α-agarase remain unclear, limiting further enzyme engineering for industrial applications. In this study, an α-agarase from Catenovulum maritimus STB14 (Cm-AGA) was employed to degrade agarose oligosaccharides (AGOs) with varying degrees of polymerization (DPs) to investigate the catalytic mechanism of α-agarases. The results demonstrated that Cm-AGA could degrade agarose into agarotetraose and agarohexaose. The reducing ends of agarotetraose and agarohexaose spontaneously release unstable 3,6-anhydro-α-l-galactose molecules, which were further degraded into agarotriose and agaropentose. Cm-AGA cannot act on α-1,3-glucoside bonds in agarotriose, agarotetraose, neoagarobiose, and neoagarotetraose but can act on AGOs with a DP greater than four. The product analysis was further verified by β-galactosidase hydrolysis, which specifically cleaves the non-reducing glycosidic bond of agarooligosaccharides. Multiple sequence alignment results showed that two conserved residues, Asp994 and Glu1129, were proposed as catalytic residues and were further identified by site-directed mutagenesis. Molecular docking of Cm-AGA with agaroheptose revealed the potential substrate binding mode of the α-agarase. These findings enhance the understanding of Cm-AGA’s catalytic mode and could guide enzyme engineering for modulating the production of agarooligosaccharides.