a, GH92 α-mannosidases at high concentrations (50 μM) were incubated with yeast mannan for 5 h in the absence (labelled GH92) or in the presence (GH92/GH76) of the endo-α-1,6-mannanase BT3782. The GH92 α-mannosidases in this example were BT2199 (1), BT2130 (2) and BT3773 (3). The GH76 endo-α-1,6-mannanase only releases mannooligosaccharides in the presence of BT2199; see also Supplementary Information section 4.1. b, B. thetaiotaomicron was grown on yeast mannan or glucose. Yeast mannan was incubated with no bacterium (1), B. thetaiotaomicron previously cultured on yeast mannan (2) and B. thetaiotaomicron grown on glucose (3). The cells were incubated for 5 h at 37 °C with the polysaccharide without a nitrogen source and thus were not growing. The products released by the B. thetaiotaomicron cells, analysed by TLC, were mediated by the activity of enzymes presented on the surface of B. thetaiotaomicron, and not through the action of periplasmic mannanases and mannosidases. The black box highlights very low levels of high molecular weight mannooligosaccharides generated by the cells incubated in yeast mannan. c, B. thetaiotaomicron was cultured for up to 48 h (stationary phase) on yeast mannan. The supernatant of the culture at the time points indicated were analysed by TLC. In all panels the samples were chromatographed with the following α-1,6-mannooligosaccharides: mannose, M1; mannobiose, M2; mannotriose, M3; mannotetraose, M4. d, The absorbance of the culture used in c. e, BT3792 (GH76) endo-α-1,6-mannosidase is a retaining glycoside hydrolase. Enzymatic hydrolysis of 4-nitrophenyl α-d-mannopyranosyl-1,6-α-d-mannopyranoside (S) was monitored by 1H-NMR spectroscopy (500 MHz). The stacked spectra show the reaction progress over time. S H1α is the anomeric proton of the reducing end mannopyranoside of the substrate, and S H1′α is the anomeric proton of the non-reducing end mannopyranoside. The reaction proceeds with the initial formation of the product, the α-anomer of α-1,6-mannobiose (P-α, peaks P H1α and P H1′α ), which slowly mutarotates to the β-anomer (P-β, peaks P H1β and P H1′β ). f, TLC analysis of S. cerevisiae mannan incubated without enzyme (lane 1), BT3774 (lane 2), BT3792 (lane 3), and BT3774 and BT3792 (lane 4). M1–M4 are α-mannnooligosaccharide standards numbered according to their d.p. GH76 mannanase BT3792 does not attack the backbone of S. cerevisiae mannan unless the side chains are first removed by the GH38 α-mannosidase BT3774, confirming that this enzyme cleaves the mannose α-1,2-linked to the mannan backbone. The data in a, b and c are representative of two biological replicates, while the data in f are representative of two technical replicates.