Expression and Characterization of Two α-l-Arabinofuranosidases from Talaromyces amestolkiae: Role of These Enzymes in Biomass Valorization.

ABSTRACT

α-l-arabinofuranosidases are glycosyl hydrolases that catalyze the break between α-l-arabinofuranosyl substituents or between α-l-arabinofuranosides and xylose from xylan or xylooligosaccharide backbones. While they belong to several glycosyl hydrolase (GH) families, there are only 24 characterized GH62 arabinofuranosidases, making them a small and underrepresented group, with many of their features remaining unknown. Aside from their applications in the food industry, arabinofuranosidases can also aid in the processing of complex lignocellulosic materials, where cellulose, hemicelluloses, and lignin are closely linked. These materials can be fully converted into sugar monomers to produce secondary products like second-generation bioethanol. Alternatively, they can be partially hydrolyzed to release xylooligosaccharides, which have prebiotic properties. While endoxylanases and ß-xylosidases are also necessary to fully break down the xylose backbone from xylan, these enzymes are limited when it comes to branched polysaccharides. In this article, two new GH62 α-l-arabinofuranosidases from Talaromyces amestolkiae (named ARA1 and ARA-2) have been heterologously expressed and characterized. ARA-1 is more sensitive to changes in pH and temperature, whereas ARA-2 is a robust enzyme with wide pH and temperature tolerance. Both enzymes preferentially act on arabinoxylan over arabinan, although ARA-1 has twice the catalytic efficiency of ARA-2 on this substrate. The production of xylooligosaccharides from arabinoxylan catalyzed by a T. amestolkiae endoxylanase was significantly increased upon pretreatment of the polysaccharide with ARA-1 or ARA-2, with the highest synergism values reported to date. Finally, both enzymes (ARA-1 or ARA-2 and endoxylanase) were successfully applied to enhance saccharification by combining them with a ß-xylosidase already characterized from the same fungus.