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 K L (0.54 mmol.L −1), is close to the value ( K 1 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.