Studies on the substrate specificity of alpha- and beta-amylase of Entamoeba histolytica.
From the supernatant fraction of cell homogenates of Entamoeba histolytica alpha-amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) and beta-amylase (1,4-alpha-D-glucan maltohydrolase, EC 3.2.1.2) were separated and purified by gel filtration and isoelectric focusing, followed by DEAE- and CM-chromatography, respectively. Both enzymes catalyzed the degradation of amylose, amylopectin and glycogen. Hydrolysis of polysaccharides by alpha-amylase yielded as reaction products maltose, maltotriose, maltopentaose and maltohexaose, but no free glucose. beta-Amylase produced as main degradation product of glucopolysaccharides maltose and to a minor extend maltotriose, but no glucose. alpha- and beta-amylase were able to hydrolyze 4-nitrophenyl-alpha-D-glucooligosaccharides (Gn-pNP) containing more than two glucose units per molecule. Under identical conditions G6-pNP was cleaved at highest velocity by alpha-amylase, while beta-amylase exhibited the highest activity with G4-pNP as substrate. alpha-Amylase hydrolyzed G4-pNP, G5-pNP and G6-pNP yielding as main reaction product G2-pNP, but also the formation of G1-pNP and G3-pNP from G4-pNP, of G1-pNP, G3-pNP and G4-pNP from G5-pNP, of G1-pNP, G3-pNP, G4-pNP and G5-pNP from G6-pNP was observed. alpha-Amylase as endo-glucohydrolase attacked all glycosidic bonds in G6-pNP, G5-pNP and G4-pNP, while beta-amylase successively removed maltose units from the non-reducing ends of the glycosides.
E
E
Werries
F
F
Müller
D000681:Amylases / Q000378:metabolism*; D000687:Amylopectin / Q000378:metabolism; D000688:Amylose / Q000378:metabolism; D000818:Animals; D002850:Chromatography, Gel; D004748:Entamoeba histolytica / Q000201:enzymology*; D006003:Glycogen / Q000378:metabolism; D006868:Hydrolysis; D007525:Isoelectric Focusing; D009844:Oligosaccharides / Q000032:analysis / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism; D013379:Substrate Specificity; D000516:alpha-Amylases / Q000302:isolation & purification / Q000378:metabolism*; D001614:beta-Amylase / Q000302:isolation & purification / Q000378:metabolism*
D009844:Oligosaccharides; D011134:Polysaccharides; D006003:Glycogen; D000688:Amylose; D000687:Amylopectin; D000681:Amylases; D000516:alpha-Amylases; D001614:beta-Amylase