Functional exploration of the glycoside hydrolase family GH113

Abstract

β-Mannans are a heterogeneous group of polysaccharides with a common main chain of β-1,4-linked mannopyranoside residues. The cleavage of β-mannan chains is catalyzed by glycoside hydrolases called β-mannanases. In the CAZy database, β-mannanases are grouped by sequence similarity in families GH5, GH26, GH113 and GH134. Family GH113 has been under-explored so far with six enzymes characterized, all from the Firmicutes phylum. We undertook the functional characterization of 14 enzymes from a selection of 31 covering the diversity of the family GH113. Our observations suggest that GH113 is a family with specificity towards mannans, with variations in the product profiles and modes of action. We were able to assign mannanase and mannosidase activities to four out of the five clades of the family, increasing by 200% the number of characterized GH113 members, and expanding the toolbox for fine-tuning of mannooligosaccharides.

Fig 1. Phylogenetic tree of family GH113.

Enzymes studied here are shown with red symbols: filled dots indicate that an activity was assigned to the enzyme, empty ones indicate that no activity could be demonstrated. Stars indicate that the target protein was not obtained in a soluble form. Enzymes characterized in previous studies are labeled in grey. A high-resolution phylogenetic tree is available in Supplementary Material, S1 Fig.

Fig 2. Analysis of the endo mode of action of two GH113 representatives.

The degradation of debranched β 1,4 mannan by A: CCO05952 (Clade 1) and B: AJZ27962 (Clade 2) was monitored using size-exclusion chromatography. M2: mannobiose, M3: mannotriose, M4: mannotetraose.

Fig 3. Analysis of the exo mode of action of five GH113 Clade 1 representatives.

A: The degradation of mannohexaose (M6) by EDP22100 was monitored by HPAEC-PAD. B: Analysis of end-products released by AGY82530, AGF59019, AIQ46016 and AIQ63165 after overnight incubation with M6. M1: Mannose, M2: mannobiose, M3: mannotriose, M6: mannohexaose.