Expression and characterization of a GH39 β-xylosidase II from Caulobacter crescentus.
In the present work, the gene xynB2, encoding a β-xylosidase II of the Glycoside Hydrolase 39 (GH39) family, of Caulobacter crescentus was cloned and successfully overexpressed in Escherichia coli DH10B. The recombinant protein (CcXynB2) was purified using nickel-Sepharose affinity chromatography, with a recovery yield of 75.5 %. CcXynB2 appeared as a single band of 60 kDa on a sodium dodecyl sulfate polyacrylamide gel and was recognized by a specific polyclonal antiserum. The predicted CcXynB2 protein showed a high homology with GH39 β-xylosidases of the genus Xanthomonas. CcXynB2 exhibited an optimal activity at 55 °C and a pH of 6. CcXynB2 displayed stability at pH values of 4.5-7.5 for 24 h and thermotolerance up to 50 °C. The K                 (M) and V                 (Max) values were 9.3 ± 0.45 mM and 402 ± 19 μmol min(-1) for ρ-nitrophenyl-β-D-xylopyranoside, respectively. The purified recombinant enzyme efficiently produced reducing sugars from birchwood xylan and sugarcane bagasse fibers pre-treated with a purified xylanase. As few bacterial GH39 family β-xylosidases have been characterized, this work provides a good contribution to this group of enzymes.
Centro de Ciências Exatas e Tecnológicas, Universidade Estadual do Oeste do Paraná, Rua Universitária, 2069, Cascavel, Paraná, 85814-110, Brazil.
Juliana Moço
JM
Corrêa
Luciana
L
Graciano
Josielle
J
Abrahão
Eduardo Alexandre
EA
Loth
Rinaldo Ferreira
RF
Gandra
Marina Kimiko
MK
Kadowaki
Caroline
C
Henn
Rita de Cássia Garcia
Rde C
Simão
D018533:Biomass; D016935:Caulobacter crescentus / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006868:Hydrolysis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D031786:Saccharum / Q000737:chemistry; D014990:Xylans / Q000378:metabolism; D014995:Xylosidases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D011994:Recombinant Proteins; D014990:Xylans; D014995:Xylosidases