Purification and characterization of a less randomly acting endo-1,4-beta-D-glucanase from the culture filtrates of Fusarium oxysporum.
An extracellular endo-1,4-beta-D-glucanase from Fusarium oxysporum was purified by affinity chromatography and gel filtration. The enzyme purified in this way was homogeneous when judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing-polyacrylamide gel electrophoresis. The protein corresponded to a molecular mass and pI value of 41.7 kDa and 6.4, respectively. It was optimally active at pH 4.5 and at 55 degrees C. The enzyme hydrolyzed carboxymethylcellulose (CMC) and unsubstituted and substituted cello-oligosaccharides but was inactive on Avicel, filter paper, xylan, cellobiose, p-nitrophenyl-beta-D-glucoside, and p-nitrophenyl-beta-D-xyloside. However, the enzyme effected only a small change in viscosity of CMC per unit increase of reducing sugar. When cellotriose, cellotetraose, and cellopentaose were used as substrates, the enzyme released mainly cellobiose. Use of 4-methylumbelliferyl cello-oligosaccharides and the determination of bond cleavage frequency revealed that the enzyme preferentially hydrolyzed the glycosidic bond adjacent to 4-methylumbelliferone. Thus, the purified enzyme appeared to be a less randomly acting endoglucanase.
Department of Protein Engineering, Reading Laboratory, Earley Gate, Berks, United Kingdom.
P
P
Christakopoulos
D
D
Kekos
B J
BJ
Macris
M
M
Claeyssens
M K
MK
Bhat
D002266:Carboxymethylcellulose Sodium / Q000378:metabolism; D002480:Cellulase / Q000302:isolation & purification / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism; D004795:Enzyme Stability; D005670:Fusarium / Q000201:enzymology*; D016695:Glycosyltransferases / Q000378:metabolism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D006923:Hymecromone / Q000031:analogs & derivatives; D007526:Isoelectric Point; D008970:Molecular Weight; D009844:Oligosaccharides / Q000378:metabolism; D013379:Substrate Specificity
D009844:Oligosaccharides; D006923:Hymecromone; D002482:Cellulose; D016695:Glycosyltransferases; D002480:Cellulase; D002266:Carboxymethylcellulose Sodium