Purification of coffee bean α-galactosidase by affinity chromatography

The preparation and properties of a specific adsorbent for the rapid and complete purification of α-d-galactosidase (α-d-galactoside galactohydrolase, EC 3.2.1.22) from green coffee beans by affinity chromatography is described. The adsorbent consists of N-ε-aminocaproyl-N-ε-aminocaproyl-α-d-galactopyranosylamine coupled to Sepharose. α-Galactosidase was adsorbed from a partially purified preparation obtained from a coffee bean extract, and eluted with either d-galactose or p-nitrophenyl α-d-galactopyranoside. The specific activity of the purified enzyme, tested with p-nitrophenyl α-d-galactopyranoside as substrate, was 94.5 units/mg protein, representing an 8600-fold purification of the original crude extract. Polyacrylamide gel electrophoresis at pH 8.3 revealed three protein bands, all of which possessed enzymic activity.