Cloning, purification and characterization of an alkali-stable endoxylanase from thermophilic Geobacillus sp. 71.
The gene encoding a xylanase from Geobacillus sp. 71 was isolated, cloned, and sequenced. Purification of the Geobacillus sp 7.1 xylanase, XyzGeo71, following overexpression in E. coli produced an enzyme of 47 kDa with an optimum temperature of 75°C. The optimum pH of the enzyme is 8.0, but it is active over a broad pH range. This protein showed the highest sequence identity (93%) with the xylanase from Geobacillus thermodenitrificans NG80-2. XyzGeo71 contains a catalytic domain that belongs to the glycoside hydrolase family 10 (GH10). XyzGeo71 exhibited good pH stability, remaining stable after treatment with buffers ranging from pH 7.0 to 11.0 for 6 h. Its activity was partially inhibited by Al(3+) and Cu(2+) but strongly inhibited by Hg(2+). The enzyme follows Michaelis-Menten kinetics, with K(m) and V(max) values of 0.425 mg xylan/ml and 500 μmol/min.mg, respectively. The enzyme was free from cellulase activity and degraded xylan in an endo fashion. The action of the enzyme on oat spelt xylan produced xylobiose and xylotetrose.
Department of Biology, Faculty of Sciences, Karadeniz Technical University, 61080 Trabzon, Turkey. sabriye@ktu.edu.tr
Sabriye
S
Canakcı
Zeliha
Z
Cevher
Kadriye
K
Inan
Muslum
M
Tokgoz
Fatmagul
F
Bahar
Murat
M
Kacagan
Fulya Ay
FA
Sal
Ali Osman
AO
Belduz
D000468:Alkalies; D018554:Avena / Q000378:metabolism; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004187:Disaccharides / Q000378:metabolism; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D056506:Geobacillus / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017385:Sequence Homology; D013696:Temperature; D014990:Xylans / Q000378:metabolism
D000468:Alkalies; D004269:DNA, Bacterial; D004187:Disaccharides; D004791:Enzyme Inhibitors; D008670:Metals; D011994:Recombinant Proteins; D014990:Xylans; D043364:Endo-1,4-beta Xylanases; C004173:xylobiose