Current commercial bioethanol processes often fail to saccharify hemicelluloses like the complex arabinoxylans found in corn-based feedstocks, lowering product yields. Several thermophilic anaerobic bacteria species can natively degrade these arabinoxylans, providing a source for identifying key enzymes to improve these processes. One such species is Acetivibrio thermocellus (basionym: Clostridium thermocellum), which possesses a large inventory of lignocellulolytic Carbohydrate-Active enZymes (CAZymes). It efficiently degrades glucuronoarabinoxylan (GAX) from corn. Yet, it is unclear what enzymes are most responsible for this ability. To explore this, we have cloned and expressed a number of CAZymes from A. thermocellus DSM1313 in Escherichia coli for characterization alone and in mixtures. In this process, we identified a previously uncharacterized Glycoside Hydrolase (GH) family 43 enzyme, encoded by Clo1313_2794, with strong ⍺-l-arabinofuranosidase activity (EC 3.2.1.55). Through further characterization we describe the function and structure of this enzyme, now named AtAbf43C. The optimal temperature and pH of AtAbf43C are 65 ℃ and 5.5, respectively. With liquid chromatography-mass spectrometry (LC-MS) analysis of polysaccharides and oligosaccharides hydrolyzed by AtAbf43C, we show this enzyme acts in an exo manner primarily towards ⍺-1,5 linked arabino-oligosaccharides. By obtaining multiple crystal structures of the full AtAbf43C protein and its domains, we elucidate the structural basis for this activity. While this enzyme shows no activity on arabinoxylans directly, it is a promising candidate for inclusion in mixtures with other GH enzymes for improving arabinoxylan degradation. Through these efforts we hope to improve the saccharification of feedstocks above the current industrial standard, enabling increased industrial bioethanol yields while providing insights into the function of A. thermocellus’s lignocellulolytic enzymes.
