Figure 5

Thin layer chromatography analysis of barley β-glucan and oligosaccharide degradation products by purified EngU and Clostridium thermocellum LicB. The samples were incubated in 50 mM McIlvaine buffer pH 6 at 80 °C for EngU and 65 °C for LicB. The reactions contained 15 µg/ml EngU or 7 µg/ml LicB and 0.5% barley β-glucan or 0.1% of the defined oligosaccharides cellopentaose (G₅), cellotriose (G₃), or the mixed-linkage glucotetraoses G_{4 b} (G4G4G3G) and G_{4 c} (G4G3G4G). The cellooligosaccharide standard used (G_1–6) is a mixture of 0.1% each of glucose, cellobiose, -triose, -pentaose and -hexaose. The mobile phase was butanol:ethanol:water (in a volume ratio of 5:5:4) in (a) and acetonitrile:water (in a volume ratio of 8:2) in (b) and (c). While LicB preferentially cleaves β-1,4 linkages of glucose moieties that are substituted at C3, EngU preferentially cleaves β-1,3 linkages of glucose moieties that are substituted at C4 (see Fig. 6), resulting in mainly β-glucooligosaccharides with a β-1,3 linkage (LicB) or mainly cellotriose (EngU) from mixed-linkage β-glucan (TLC sheets A and B).