Purification and characterisation of an extracellular beta-glucosidase with transglycosylation and exo-glucosidase activities from Fusarium oxysporum.
An extracellular beta-glucosidase from Fusarium oxysporum was purified to homogeneity by gel-filtration and ion-exchange chromatographies. The enzyme, a monomeric protein of 110 kDa, was maximally active at pH 5.0-6.0 and at 60 degrees C. It hydrolysed 1-->4-linked aryl-beta-glucosides and 1-->4-linked, 1-->3-linked and 1-->6-linked beta-glucosides. The apparent Km and kcat values for p-nitrophenyl beta-D-glucopyranoside (4-NpGlcp) and cellobiose were 0.093 (Km), 1.07 mM (kcat) and 1802 (Km), 461.5 min-1 (kcat), respectively. Glucose and gluconolactone inhibited the enzyme competitively with Ki values of 2.05 mM and 3.03 microM, respectively. Alcohols activated the enzyme; butanol showed maximum effect (2.2-fold at 0.5 M) while methanol increased the activity by 1.4-fold at 1 M. The enzyme catalysed the synthesis of methylglucosides, ethylglucoside and propylglucosides, as well as trisaccharides in the presence of different alcohols and disaccharides, respectively. In addition, the enzyme hydrolysed the unsubstituted and methylumbelliferyl cello-oligosaccharides [MeUmb(Glc)n] but the rate of hydrolysis decreased with increasing chain length. Analysis of products released from MeUmb(Glc)n as a function of time revealed that the enzyme attacked these substrates in a stepwise manner and from both ends. Thus, beta-glucosidase from F. oxysporum, with the above interesting properties, could be of commercial interest.
Christakopoulos
P
P
Department of Protein Engineering, Institute of Food Research, Reading Laboratory, England.
Goodenough
P W
PW
Kekos
D
D
Macris
B J
BJ
Claeyssens
M
M
Bhat
M K
MK
D000438:Alcohols / Q000494:pharmacology; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D005670:Fusarium / Q000201:enzymology*; D006031:Glycosylation; D007525:Isoelectric Focusing; D007700:Kinetics; D008970:Molecular Weight; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000302:isolation & purification* / Q000378:metabolism*
D000438:Alcohols; D001617:beta-Glucosidase