Purification and characterization of two different xylanases from the thermophilic actinomycete Microtetraspora flexuosa SIIX.
Two endoxylanases were isolated from the xylanolytic enzyme system of the thermophilic actinomycete Microtetraspora flexuosa SIIX, and purified by ammonium sulfate fractionation, DEAE-Sepharose chromatography, gel filtration on Sephacryl S 200 and fast protein liquid chromatography on Q-Sepharose. The molecular masses of xylanase I and II were 26.3 and 16.8 kDa, and isoelectric points were 8.4 and 9.45, respectively. Optimal enzyme activities were obtained at 80 degrees C and pH 6.0. The thermostability of both xylanases was greatly diminished during purification but could be restored by preincubation of the purified enzymes in the presence of xylan. The half-lives at 80 degrees C were approximately 25 min. The kinetic constants of xylanases I and II determined with Remazol-brilliant-blue xylan were Vmax of 1537 and 353 mumol.min-1.mg protein-1 and K(m) values of 2.44 and 1.07 mg.ml-1, respectively. Purified xylanases utilized xylan as well as small oligosaccharides such as xylotriose as substrate. They did not exhibit xylobiase or debranching activities. The predominant products of arabinoxylan hydrolysis were xylobiose and xylotriose, the latter being hydrolysed to xylobiose and xylose upon further incubation. In addition, fragments containing arabinose side chains accumulated. The xylanases did not act on crystalline or amorphous cellulose indicating a possible application in biobleaching processes.
Berens
S
S
Institut für Mikrobiologie und Biotechnologie, Universität Bonn, Germany.
Kaspari
H
H
Klemme
J H
JH
D000192:Actinomycetales / Q000201:enzymology*; D043364:Endo-1,4-beta Xylanases; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007526:Isoelectric Point; D007700:Kinetics; D008970:Molecular Weight; D013379:Substrate Specificity; D014995:Xylosidases / Q000302:isolation & purification* / Q000378:metabolism
D014995:Xylosidases; D043364:Endo-1,4-beta Xylanases