Characterization of a Fructan Exohydrolase from the Gut Bacterium <i>Anaerostipes butyraticus</i>

Glycoside hydrolase family 32 (GH32) enzymes play key roles in fructooligosaccharide metabolism in gut bacteria. In this study, a GH32 enzyme (GenBank code, GFO85652) containing carbohydrate binding module 66 (CBM66) from the gut bacterium <i>Anaerostipes butyraticus</i> (AbFEH) was heterologously expressed in <i>Escherichia coli</i>. We constructed an expression plasmid that does not contain sequences for the N-terminal signal peptide and the C-terminal region potentially involving cell-wall binding. The enzyme obtained (AbFEH∆C) was purified and characterized. Thin-layer chromatography and high-performance liquid chromatography analyses revealed that AbFEH∆C produced fructose from all the substrates, sucrose, 1-kestose, inulin, and levan, and intermediate oligosaccharide products were not observed. The ratio of activities towards sucrose, 1-kestose, nystose, inulin, and levan was 6:100:83:8:95 under the conditions of this study. A region containing M and CBM66 domains was further removed from AbFEH∆C, and the activities for both 1-kestose and levan of this mutant enzyme were about 400-fold lower than those of AbFEH∆C. Kinetic analysis indicated a low <i>K</i> _m value for levan, while requiring higher substrate concentrations for 1-kestose and sucrose. Comparison of the predicted structure of AbFEH with crystal structures of some GH32 enzymes indicated that residues at subsite -1 were almost completely conserved, while some key residues found in GH32 enzymes were not present at subsites +1 and +2 in AbFEH. These observations suggest that AbFEH functions as fructan exohydrolase that exhibits low sucrose-hydrolyzing activity.