The cloning, expression, purification, characterization and modeled structure of Caulobacter crescentus β-Xylosidase I.
The xynB1 gene (CCNA 01040) of Caulobacter crescentus that encodes a bifunctional enzyme containing the conserved β-Xylosidase and α-L-Arabinofuranosidase (β-Xyl I-α-L-Ara) domains was amplified by PCR and cloned into the vector pJet1.2Blunt. The xynB1 gene was subcloned into the vector pPROEX-hta that produces a histidine-fused translation product. The overexpression of recombinant β-Xyl I-α-L-Ara was induced with IPTG in BL21 (DE3) and the resulting intracellular protein was purified with pre-packaged nickel-Sepharose columns. The recombinant β-Xyl I-α-L-Ara exhibited a specific β-Xylosidase I activity of 1.25 U mg(-1) to oNPX and a specific α-L-Arabinofuranosidase activity of 0.47 U mg(-1) to pNPA. The predominant activity of the recombinant enzyme was its β-Xylosidase I activity, and the enzymatic characterization was focused on it. The β-Xylosidase I activity was high over the pH range 3-10, with maximal activity at pH 6. The enzyme activity was optimal at 45 °C, and a high degree of stability was verified over 240 min at this temperature. Moreover, β-Xylosidase activity was inhibited in the presence of the metals Zn(2+) and Cu(2+), and the enzyme exhibited K(M) and V(Max) values of 2.89 ± 0.13 mM and 1.4 ± 0.04 μM min(-1) to oNPX, respectively. The modeled structure of β-xylosidase I showed that its active site is highly conserved compared with other structures of the GH43 family. The increase in the number of contact residues responsible for maintaining the dimeric structure indicates that this dimer is more stable than the tetramer form.
Centro de Ciências Exatas e Tecnológicas, Universidade Estadual do Oeste do Paraná, Cascavel, PR, Brazil.
Luciana
L
Graciano
Juliana Moço
JM
Corrêa
Rinaldo Ferreira
RF
Gandra
Flavio Augusto Vicente
FA
Seixas
Marina Kimiko
MK
Kadowaki
Silvio César
SC
Sampaio
José Luis da Conceição
JL
Silva
Clarice Aoki
CA
Osaku
Rita de Cássia Garcia
Rde C
Simão
D000595:Amino Acid Sequence; D016935:Caulobacter crescentus / Q000201:enzymology* / Q000235:genetics*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D006026:Glycoside Hydrolases / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D014995:Xylosidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D004269:DNA, Bacterial; D006026:Glycoside Hydrolases; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase; C019713:alpha-N-arabinofuranosidase