Exo- and Endo-1,5-α-L-Arabinanases and Prebiotic Arabino-Oligosaccharides Production

Abstract

There is growing interest in pentose-based prebiotic oligosaccharides as alternatives to traditional hexose-based prebiotics. Among these, arabino-oligosaccharides (AOS), derived from the enzymatic hydrolysis of arabinan polymers, have gained significant attention. AOS can selectively stimulate the growth of beneficial gut bacteria, including Bifidobacterium and Bacteroides species, and contribute to health-benefit functions such as blood sugar control, positioning AOS as a promising synbiotic candidate. For the industrial production of AOS, the development of efficient enzymatic processes is essential, with exo- and endo-1,5-α-L-arabinanases (exo- and endo-ABNs) playing a crucial catalytic role. Most ABNs belong to the glycoside hydrolase (GH) family 43, characterized by a five-bladed β-propeller fold structure. These enzymes hydrolyze internal α-1,5-L-arabinofuranosidic linkages, producing AOS with varying degrees of polymerization. Some ABNs GH43 were known to exhibit exo-type hydrolytic modes of action, producing specific AOS products such as arabinotriose. Additionally, exo-ABNs from GH93, which feature a six-bladed β-propeller fold, exclusively release arabinobiose through their exo-type catalytic mechanism. This review represents the first comprehensive analysis of exo- and endo-ABNs, offering scientific insights into their biotechnological potential for AOS production. It systematically compares enzyme classification, structural differences, catalytic mechanisms, paving the way for innovative applications in health, food, and pharmaceutical industries.

Keywords: 5-α-L-arabinanase (endo-ABN); Endo-1; arabino-oligosaccharides (AOS); enzymatic production; exo-1,5-α-L-arabinanase (exo-ABN); structure-function relationship.

Fig. 1. (A) Phylogenetic relationship of endo- and exo-arabinanases GH43 and GH93 and (B) Structures of multi-domain endo-arabinanases GH43.

The phylogenetic tree was drawn using MEGA11 software and iTOL web server. Multi-domain structures were analyzed using InterPro and SignalP web servers. The numbering assigned to each endoand exo-ABNs corresponds to the identifiers presented in Tables 2 and 3. The black triangle indicates a signal peptide for protein secretion.

Fig. 2. Three-dimensional structures of microbial exo- and endo-1,5-α-L-arabinanases (ABNs) GH43 and GH93 in complex with substrates.

Surface, cartoon, and closed-up structures are represented for (A) endo-ABN GH43 from Geobacillus stearothermophilus (PDB: 3CU9, catalytic residues of D27, D147, and E201 in blue) in complex with arabinobiose (3D61 in cyan), arabinotriose (3D5Z in magenta), and arabinopentaose (6F1G in green), (B) exo-ABN GH43 from Cellvibrio japonicus with arabinohexaose (PDB: 1GYD and 1GYE, catalytic residues of D38, D158, and E221 in blue), and (C) exo-ABN GH93 from Penicillium chrysogenum with arabinobiose (PDB: 3A72, catalytic residues of E170, E242, and Y337 in blue). The substrate-binding site for each enzyme is illustrated with designated subsite numbers. The substrate cleavage sites and non-reducing ends of substrates are marked with arrowheads and asterisks, respectively. Each structure is visualized using PyMOL molecular graphics tool.

Fig. 3. Scheme for enzymatic production of prebiotic arabino-oligosaccharides using exo- and endo-1,5-α-Larabinanases (ABNs) GH43 and GH93.

CjArb43A, exo-ABN GH43 from Cellvibrio japonicus; exo-ABF, α-Larabinofuranosidase with debranching activity.