Abstract
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 Anaerostipes butyraticus (AbFEH) was heterologously expressed in Escherichia coli. 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 K (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.