Purification and properties of beta-mannanases I and II from the germinated seeds of Trifolium repens. Mode of galactomannan degradation in vitro.
Two beta-mannanases (beta-mannosidases, EC 3.2.1.25) purified from the germinated seeds of Trifolium repens by a procedure that included chromatography on hydroxyapatite, gel filtration on acrylamide/agarose (Ultragel 5/4) and preparative polyacrylamide-gel-electrophoresis. The final purification step completely resolved two beta-mannanases with distinct specificities, which were termed beta-mannanase I and beta-mannanase II. beta-Mannanase I was purified 1400-fold and beta-mannanase II 1000-fold. The purified enzymes showed a single protein band when examined by polyacrylamide-gel disc electrophoresis. beta-Mannanase I, apparent mol.wt. 43 000, accounted for 49% of the total activity recovered from the final step of purification. beta-Mannanase II, apparent mol.wt. 38 000, accounted for the remaining 51% of activity. Molecular-weight determinations were by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and by the electrophoretic method of Hendrick & Smith [(1968) Arch. Biochem. Biophys. 126, 155-164]. The substrate specificities of both enzymes were examined with the galactomannans of T. repens and of Medicago sativa, as well as with manno-oligosaccharides. The pH optimum was between pH 5.1 and 5.6 for both enzymes.
D004789:Enzyme Activation; D005685:Galactans / Q000378:metabolism; D005690:Galactose / Q000031:analogs & derivatives; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008351:Mannans / Q000378:metabolism; D008361:Mannosidases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D012639:Seeds / Q000201:enzymology*; D013379:Substrate Specificity
D005685:Galactans; D008351:Mannans; C012990:galactomannan; D008361:Mannosidases; D005690:Galactose