Rational affinity purification of native Streptomyces family 10 xylanase.
Xylanase SoXyn10A from Streptomyces olivaceoviridis E-86 comprises a family 10 catalytic module linked to a family 13 carbohydrate-binding module (SoCBM13). The SoCBM13 has a beta-trefoil structure, with binding sites in each subdomain (alpha, beta and gamma). Subdomain alpha, but not subdomains beta and gamma, binds tightly to lactose. It was, therefore, thought that immobilized lactose could be used for the affinity purification of SoXyn10A. Lactosyl-Sepharose was prepared and tested as an affinity matrix. SoXyn10A produced from the cloned xyn10A gene by Escherichia coli, and native SoXyn10A in culture supernatants from S. olivaceoviridis, were purified to homogeneity in a single step by affinity chromatography using this matrix. This simple purification of SoXyn10A makes the enzyme an attractive candidate for applications requiring xylanase. The CBM also has the potential for use as an affinity tag for the purification of other proteins.
Ito
Shigeyasu
S
Department of Material and Biological Chemistry, Faculty of Science, Yamagata University, Yamagata 990-8560, Japan.
Kuno
Atsushi
A
Suzuki
Ryuichiro
R
Kaneko
Satoshi
S
Kawabata
Yasuyuki
Y
Kusakabe
Isao
I
Hasegawa
Tsunemi
T
D015153:Blotting, Western; D002846:Chromatography, Affinity / Q000191:economics / Q000379:methods*; D004187:Disaccharides; D004591:Electrophoresis, Polyacrylamide Gel; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D012685:Sepharose; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D014990:Xylans / Q000378:metabolism; D014995:Xylosidases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D004187:Disaccharides; D011994:Recombinant Proteins; D014990:Xylans; D012685:Sepharose; D014995:Xylosidases