Selective isolation of endo-D-galacturonanase of Aspergillus niger based on interaction with tri(D-galactosiduronic acid) covalently bound to poly(hydroxyalkyl methacrylate).
A selective affinity-adsorbent for the extracellular endo-D-galacturonanase (E.C. 3.2.1.15) of Aspergillus niger was prepared by covalent coupling of tri(D-galactosiduronic acid) to Separon, a poly(hydroxyalkyl methacrylate) gel. Complexing of the enzyme with the adsorbent is pH dependent; maximal interaction occurs at the optimum pH for enzyme activity. The enzyme was quantitatively displaced from the adsorbent either by changing the pH or by bioelution with soluble tri(D-galactosiduronic acid) or other substrate. Within the range of substitution of Separon examined [content of tri(D-galactosiduronic acid) 1.7-6.7%] the amount of endo-D-galacturonanase retained was proportional to the content of affinity ligand. Under the same conditions, unsubstituted carrier did not complex with endo-D-galacturonanase. The dissociation constant of the affinity complex, as determined by zonal analysis, kinetic measurements, and by means of the adsorption isotherm KL (0.54 mmol.L-1), is close to the value (KI 0.44 mmol.L-1) obtained by the two first methods with soluble tri(D-galactosiduronic acid). The results show that adsorption of endo-D-galacturonanase on tri(D-galactosiduronic acid)-Separon is due exclusively to active-site-directed interaction with bound affinity-ligand.
D001234:Aspergillus niger / Q000201:enzymology*; D002846:Chromatography, Affinity; D006026:Glycoside Hydrolases / Q000302:isolation & purification*; D007700:Kinetics; D011096:Polygalacturonase / Q000302:isolation & purification* / Q000378:metabolism; D011109:Polymethacrylic Acids; D014312:Trisaccharides
D011109:Polymethacrylic Acids; D014312:Trisaccharides; C027498:hydroxyalkylmethacrylate carrier Separon E; C040907:tri(galactosiduronic acid); D006026:Glycoside Hydrolases; D011096:Polygalacturonase