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.
Yan
T R
TR
Department of Bioengineering, Tatung Institute of Technology, Taipei, Taiwan, Republic of China. tryan@140.129.35.12
Lin
C L
CL
D001234:Aspergillus niger / Q000201:enzymology*; D005591:Chemical Fractionation; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D000431:Ethanol / Q000737:chemistry / Q000494:pharmacology; D005632:Fructose / Q000494:pharmacology; D005690:Galactose / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism / Q000494:pharmacology*; D005960:Glucosides / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D008358:Mannose / Q000494:pharmacology; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D012694:Serine / Q000737:chemistry; D012834:Silver / Q000494:pharmacology; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013696:Temperature; D014994:Xylose / Q000494:pharmacology; D001617:beta-Glucosidase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D004791:Enzyme Inhibitors; D005960:Glucosides; D008670:Metals; D013438:Sulfhydryl Compounds; C025193:4-nitrophenyl beta-D-glucoside; D005632:Fructose; D000431:Ethanol; D012834:Silver; D012694:Serine; D014994:Xylose; D001617:beta-Glucosidase; D005947:Glucose; D008358:Mannose; D005690:Galactose