Characterization of a GH3 Family β-Glucosidase from Dictyoglomus turgidum and Its Application to the Hydrolysis of Isoflavone Glycosides in Spent Coffee Grounds

A recombinant beta-glucosidase from Dictyoglomus turgidum was purified with a specific activity of 31 U/mg by His-Trap affinity chromatography. D. turgidum beta-glucosidase was identified as a memmber of the glycoside hydrolase (GH) 3 family on the basis of its amino acid sequence. The native enzyme existed as an 86 kDa monomer with an activity maximum at pH 5 and 85 degrees C with a half-life of 334 min. The hydrolytic activity of the enzyme with aryl-glycoside substrates was the highest for p-nitrophenyl (pNP)-beta-D-glucopyranoside (with a K-m of 1.3 mM and a k(cat) of 13900 1/s), followed by oNP-beta-D-glucopyranoside, pNP-beta-D-xylopyranoside, pNP-beta-D-fucopyranoside, and pNP-beta-D-galactopyranoside. However, no activity was observed for oNP-beta-D-galactopyranoside, pNP-alpha-D-glucopyranoside, pNP-alpha-D-glucopyranoside, pNP-beta-D-mannopyranoside, pNP-beta-L-arabinopyranoside, and pNP-alpha-L-rhamnopyranoside. The hydrolytic activity of the beta-glucosidase for coffee isoflavones followed the order genistin (with a K-m of 0.67 mM and a k(cat) of 5750 1/s) > daidzin > ononin > glycitin. The concentrations of daidzin in ground coffee and spent coffee grounds were 160 and 107 mu g/g, respectively, but other isoflavones were present at low concentrations or absent. The enzyme completely hydrolyzed 1.2 mM daidzin in spent coffee grounds after 2 h, with a productivity of 0 6 mM/h. This is the first report concerning the enzymatic hydrolysis of isoflavone glycosides in spent coffee grounds.