Selective isolation of endo-d-galacturonanase of Aspergillus niger based on interaction with tri(d-galactosiduronic acid) covalently bound to poly(hydroxyalkyl methacrylate)
✍ Scribed by Ľubomíra Rexová-Benková; Jiřina Omelková; Karel Filka; Jan Kocourek
- Book ID
- 102992235
- Publisher
- Elsevier Science
- Year
- 1983
- Tongue
- English
- Weight
- 833 KB
- Volume
- 122
- Category
- Article
- ISSN
- 0008-6215
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✦ Synopsis
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(Dgalactosiduronic acid) to Separon, a poly(hydroxyalky1 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 KI, (0.54 mmol.L-'), is close to the value (K, 0.44 mmol.L-') 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.