[alpha-L-Rhamnosidase from Fagopyrum esculentum: purification and some properties (author's transl)].
An alpha-L-rhamnosidase from the seeds of Fagopyrum esculentum (saracen corn) has previously been identified, and the effect of the enzyme on rhamnoisic bonds has been studied with various flavonoid glycosides. This alpha-L-rhamnosidase can be useful in structural studies, and a preliminary report of this study has appeared. The present paper describes the extensive purification of the enzyme and the determination of its properties. The purification involved extraction, ammonium sulfate fractionation and chromatography on Sephadex G 75, DEAE-Sephadex and Ultrogel AcA-44. The alpha-L-rhamnosidase was purified about 9600 fold and the final enzyme preparation was practically pure according to the criteria of disc electrophoresis. The molecular weight of this alpha-L-rhamnosidase, calculated from data obtained by disc gel electrophoresis and gel filtration, was about 70 000. Isoelectric focusing established the isoelectric point to be 3.7. The behaviour of the enzyme on a concanavalin-A-Sepharose column suggests the presence of residues resembling alpha-D-mannose or alpha-D-glucose in the protein. The various kinetic parameters, Kcat, Km and the Kcat/Km ratio have been determined at pH 5 on the following substrates: p-nitrophenyl-alpha-L-rhamnoside and rutinose (6-O-alpha-L-rhamnosyl-D-glucopyranose). All kinetics exhibit a Michaelian behaviour and the Km for the former substrate was 0.33 mM and for the latter, 2.2 mM. The Kcat/Km ratio corroborates the greater specificity of the enzyme for p-nitrophenyl-alpha-L-rhamnoside. L-Rhamnose, L-lyxose, 6-deoxy-D-glucose and methyl-alpha-D-mannoside were shown to behave strictly as competitive inhibitors of alpha-L-rhamnosidase activity; it seems that the methyl group of L-rhamnose is important for substrate binding to the enzyme.
D001667:Binding, Competitive; D002846:Chromatography, Affinity; D006026:Glycoside Hydrolases / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D011485:Protein Binding; D012210:Rhamnose / Q000378:metabolism; D012639:Seeds / Q000201:enzymology*
D006026:Glycoside Hydrolases; D012210:Rhamnose
alpha-L-Rhamnosidase de Fagopyrum esculentum. Purification et quelques propriétés