Purification and characterization of a glucose-tolerant beta-glucosidase from Aspergillus niger CCRC 31494.

An extracellular glucose-tolerant beta-glucosidase was purified to homogeneity by alcohol fractionation and preparative isoelectric focusing from Aspergillus niger CCRC 31494. The enzyme was a dimeric protein with a subunit of 49,000, and had its optimum activity at pH 5.0 and 55 degrees C. The enzyme was completely inhibited by 5 mM Ag+. Thiol groups and serine residues were not essential for its activity. Low concentrations of alcohols (10%) except for methanol could activate the enzyme. It was very specific for para-nitrophenyl-beta-D-glucoside (pNPG) and cellobiose. However, the enzyme also had some beta-xylosidase activity, but showed no activity towards alpha-linked glycosidic substrates. The Vmax of 124.4 U/mg and 21.6 U/mg were found for pNPG (Km = 21.7 mM) and para-nitrophenyl-beta-D-xyloside (pNPX) (Km = 14.2 mM), respectively. The enzyme was tolerant to glucose inhibition with a Ki of 543 mM, while fructose, galactose, mannose, and xylose were not inhibitory.