Characterization of novel glycosyl hydrolases discovered by cell wall glycan directed monoclonal antibody screening and metagenome analysis of maize aerial root mucilage.

Abstract

An indigenous maize landrace from the Sierra Mixe region of Oaxaca, Mexico exhibits extensive formation of aerial roots which exude large volumes of a polysaccharide-rich gel matrix or "mucilage" that harbors diazotrophic microbiota. We hypothesize that the mucilage associated microbial community carries out multiple functions, including disassembly of the mucilage polysaccharide. In situ, hydrolytic assay of the mucilage revealed endogenous arabinofuranosidase, galactosidase, fucosidase, mannosidase and xylanase activities. Screening the mucilage against plant cell wall glycan-specific monoclonal antibodies recognized the presence of carbohydrate epitopes of hemicellulosic polysaccharides like xyloglucan (both non-fucosylated and fucosylated), xylan (both substituted and unsubstituted xylan domains) and pectic-arabinogalactans, all of which are potential carbon sources for mucilage microbial residents. Mucilage metagenome annotation using MG-RAST identified the members forming the microbial community, and gene fragments with predicted functions associated with carbohydrate disassembly. Data from the in situ hydrolytic activity and monoclonal antibody screening assays were used to guide the selection of five full length genes with predicted glycosyl hydrolase function from the GenBank database that were similar to gene fragments of high relative abundance in the mucilage metagenomes. These five genes were then synthesized for recombinant production in Escherichia coli. Here we report the characterization of an α-N-arabinofuranosidase (GH51) and an oligosaccharide reducing-end xylanase (GH8) from Flavobacterium johnsoniae; an α-L-fucosidase (GH29) and a xylan β-1,4 xylosidase (GH39) from Spirosoma linguale, and a β-mannosidase (GH2) from Agrobacterium fabrum. Biochemical characterization of these enzymes revealed a β-Mannosidase that also exhibits a secondary activity towards the cleavage of galactosyl residues. We also describe two xylanases (GH8 and GH39) from underexplored glycosyl hydrolase families, one thermostable α-L-Fucosidase (GH29) and a thermostable α-N-Arabinofuranosidase (GH51).