An endo-1,4-beta-mannanase (MA62) isolated from Sclerotium rolfsii was found to form mixed oligosaccharides from C-14 labeled mannose, glucose, and galactose in the presence of mannotetraose. When this enzyme was incubated with galactose and mannotetraose the main reaction product was 6(1)-alpha-D-galactosyl-beta-D-mannotriose (GM3), which was purified (95%) using charcoal/celite column chromatography and a centrifugal accelerated thin-layer chromatography system. GM3 was also isolated from the reaction mixture resulting from the hydrolysis of Ceratonia siliqua galactomannan using mannanases from Schizophyllum commune. The structure of GM3 was confirmed using C-13 and H-1-NMR. (C) 2000 Elsevier Science Inc. All rights reserved.