Novel xylan degrading enzymes from polysaccharide utilizing loci of Prevotella copri DSM18205

Abstract

Prevotella copri DSM18205 is a bacterium, classified under Bacteroidetes that can be found in the human gastrointestinal tract (GIT). The role of P. copri in the GIT is unclear, and elevated numbers of the microbe have been reported both in dietary fiber-induced improvement in glucose metabolism but also in conjunction with certain inflammatory conditions. These findings raised our interest in investigating the possibility of P. copri to grow on xylan, and identify the enzyme systems playing a role in digestion of xylan-based dietary fibers in P. copri, which currently are unexplored. Two xylan degrading polysaccharide utilizing loci (PUL10 and 15) were found in the genome, with three and eight GH-encoding genes, respectively. Three of the eight gene products were successfully produced in Escherichia coli: One monomeric two-domain extracellular enzyme from GH43 (subfamily 12, in PUL10, 60 kDa) and two dimeric single module enzymes from PUL15, one extracellular GH10 (41 kDa), and one intracellular GH43 subfamily 1 enzyme (37 kDa). The GH43_12 enzyme was hydrolysing arabinofuranose residues from different substrates, and a model of the 3D-structure revealed a single arabinose binding pocket. The GH10 (1) and GH43_1 are cleaving the xylan backbone. Hydrolysis products of GH10 (1) were DP2-4, and seven subsites (-3 to +4) were predicted in the 3D-model of the GH10 active site. GH43_1 mainly produced xylose (in line with its intracellular location). Based on our results we propose that in PUL15, GH10 (1) is an extracellular endo-1,4-{beta}-xylanase, that hydrolyses mainly glucuronosylated xylan polymers to xylooligosaccharides (XOS); while, GH43_1 in the same PUL, is an intracellular {beta}-xylosidase, catalysing complete hydrolysis of the XOS to xylose. In PUL10, the characterized GH43_12 is an arabinofuranosidase, with a role in degradation of arabinoxylan, catalysing removal of arabinose-residues on xylan polymers.