Purification and biochemical characteristics of beta-D-glucosidase from a thermophilic fungus, Thermomyces lanuginosus-SSBP.
The beta-D-glucosidase produced by Thermomyces lanuginosus-SSBP was purified to apparent homogeneity. The purified enzyme consisted of two identical subunits with a native molecular mass of 200 kDa. The purified beta-D-glucosidase only hydrolysed the glucoside substrates containing a terminal, non-reducing beta-D-glucose residue and was active on both aryl-beta-glucoside and cellobiose. This enzyme also exhibited less, but significant alpha-D-glucosidase activity and was capable of hydrolysing beta-1,6-linked diglucosides and gentiobiose. The Kapp m, V(max) and k(cat) values for p-nitrophenyl-beta-D-glucopyranoside were calculated to be 0.075 mM, 12.12 units/mg of protein and 44.44 glucose molecules released/s respectively. The beta-D-glucosidase retained its full activity after a 30 min incubation at 50 degrees C but was inactive after the same treatment at 70 degrees C. The enzyme appeared to be stable when the pH of the storage buffer was above 5.0. Maximal beta-D-glucosidase activity occurred at 65 degrees C and pH 6.0. This enzyme was competitively inhibited by glucose, cellobiose and salicin with K(i) values of 0.55, 0.52 and 0.81 mM respectively. The presence of Hg(2+) and N-bromosuccinimide inhibited the enzyme activity completely at 2 mM, while cysteine enhanced beta-D-glucosidase activity.
Lin
J
J
Department of Biochemistry and Microbiology, University of Zululand, Private Bag X 1001, Kwa Dlangezwa 3886, Republic of South Africa.
Pillay
B
B
Singh
S
S
D000596:Amino Acids / Q000737:chemistry; D002384:Catalysis; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000737:chemistry; D003904:Mitosporic Fungi / Q000201:enzymology*; D008970:Molecular Weight; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D000596:Amino Acids; D008670:Metals; D001617:beta-Glucosidase