Characterization and application of carbohydrate-binding modules of beta-1,3-xylanase XYL4.
beta-1,3-Xylanase from Vibrio sp. strain AX-4 (XYL4) is a modular enzyme composed of an N-terminal catalytic module belonging to glycoside hydrolase family 26 and two putative carbohydrate-binding modules (CBMs) belonging to family 31 in the C-terminal region. To investigate the functions of these three modules, five deletion mutants lacking individual modules were constructed. The binding assay of these mutants showed that a repeating unit of the CBM was a non-catalytic beta-1,3-xylan-binding module, while the catalytic module per se was not likely to contribute to the binding activity when insoluble beta-1,3-xylan was used for the assay. The repeating CBMs were found to specifically bind to insoluble beta-1,3-xylan, but not to beta-1,4-xylan, Avicel, beta-1,4-mannan, curdlan, chitin or soluble glycol-beta-1,3-xylan. Both the enzyme and the binding activities for insoluble beta-1,3-xylan but not soluble glycol-beta-1,3-xylan were enhanced by NaCl in a concentration-dependent manner, indicating that the CBMs of XYL4 bound to beta-1,3-xylan through hydrophobic interaction. This property of the CBMs was successfully applied to the purification of a recombinant XYL4 from the cell extracts of Escherichia coli transformed with the xyl4 gene and the detection of beta-1,3-xylan-binding proteins including beta-1,3-xylanase from the extract of a turban shell, Turbo cornutus.
Kiyohara
Masashi
M
Department of Bioscience, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka 812-8581, Japan.
Sakaguchi
Keishi
K
Yamaguchi
Kuniko
K
Araki
Toshiyoshi
T
Ito
Makoto
M
D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity; D004586:Electrophoresis; D049851:Gastropoda / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017384:Sequence Deletion; D012965:Sodium Chloride / Q000494:pharmacology; D012995:Solubility / Q000187:drug effects; D013379:Substrate Specificity / Q000187:drug effects; D014020:Tissue Extracts; D014733:Vibrio / Q000187:drug effects / Q000201:enzymology*; D043325:Xylan Endo-1,3-beta-Xylosidase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D014990:Xylans / Q000378:metabolism
D050505:Mutant Proteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D014020:Tissue Extracts; D014990:Xylans; C055213:saccharide-binding proteins; D012965:Sodium Chloride; C065917:1,3-xylan; D043325:Xylan Endo-1,3-beta-Xylosidase