Advanced

Carbohydrate-binding modules from a thermostable Rhodothermus marinus xylanase : Cloning, expression and binding studies

Abou Hachem, Maher LU ; Nordberg Karlsson, Eva LU ; Bartonek-Roxå, Eva LU ; Raghothama, Srinivasrao ; Simpson, Peter J. ; Gilbert, Harry J. ; Williamson, Michael P. and Holst, Olle LU (2000) In Biochemical Journal 345(1). p.53-60
Abstract

The two N-terminally repeated carbohydrate-binding modules (CBM4-1 and CBM4-2) encoded by xyn10A from Rhodothermus marinus were produced in Escherichia coli and purified by affinity chromatography. Binding assays to insoluble polysaccharides showed binding to insoluble xylan and to phosphoric-acid-swollen cellulose but not to Avicel or crystalline cellulose. Binding to insoluble substrates was significantly enhanced by the presence of Na+ and Ca2+ ions. The binding affinities for soluble polysaccharides were tested by affinity electrophoresis; strong binding occurred with different xylans and β-glucan. CBM4-2 displayed a somewhat higher binding affinity than CBM4-1 for both soluble and insoluble substrates but both... (More)

The two N-terminally repeated carbohydrate-binding modules (CBM4-1 and CBM4-2) encoded by xyn10A from Rhodothermus marinus were produced in Escherichia coli and purified by affinity chromatography. Binding assays to insoluble polysaccharides showed binding to insoluble xylan and to phosphoric-acid-swollen cellulose but not to Avicel or crystalline cellulose. Binding to insoluble substrates was significantly enhanced by the presence of Na+ and Ca2+ ions. The binding affinities for soluble polysaccharides were tested by affinity electrophoresis; strong binding occurred with different xylans and β-glucan. CBM4-2 displayed a somewhat higher binding affinity than CBM4-1 for both soluble and insoluble substrates but both had similar specificities. Binding to short oligosaccharides was measured by NMR; both modules bound with similar affinities. The binding of the modules was shown to be dominated by enthalpic forces. The binding modules did not contribute with any significant synergistic effects on xylan hydrolysis when incubated with a Xyn10A catalytic module. This is the first report of family 4 CBMs with affinity for both insoluble xylan and amorphous cellulose.

(Less)
Please use this url to cite or link to this publication:
author
; ; ; ; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
CBD, Hemicellulose binding, Modular proteins, Xyn10A
in
Biochemical Journal
volume
345
issue
1
pages
8 pages
publisher
Portland Press
external identifiers
  • scopus:0033976867
  • pmid:10600638
ISSN
0264-6021
DOI
10.1042/0264-6021:3450053
language
English
LU publication?
yes
id
25e86bb3-1109-47ae-9d48-a87f56c20a5f
date added to LUP
2018-11-14 21:16:41
date last changed
2021-09-01 05:15:21
@article{25e86bb3-1109-47ae-9d48-a87f56c20a5f,
  abstract     = {<p>The two N-terminally repeated carbohydrate-binding modules (CBM4-1 and CBM4-2) encoded by xyn10A from Rhodothermus marinus were produced in Escherichia coli and purified by affinity chromatography. Binding assays to insoluble polysaccharides showed binding to insoluble xylan and to phosphoric-acid-swollen cellulose but not to Avicel or crystalline cellulose. Binding to insoluble substrates was significantly enhanced by the presence of Na<sup>+</sup> and Ca<sup>2+</sup> ions. The binding affinities for soluble polysaccharides were tested by affinity electrophoresis; strong binding occurred with different xylans and β-glucan. CBM4-2 displayed a somewhat higher binding affinity than CBM4-1 for both soluble and insoluble substrates but both had similar specificities. Binding to short oligosaccharides was measured by NMR; both modules bound with similar affinities. The binding of the modules was shown to be dominated by enthalpic forces. The binding modules did not contribute with any significant synergistic effects on xylan hydrolysis when incubated with a Xyn10A catalytic module. This is the first report of family 4 CBMs with affinity for both insoluble xylan and amorphous cellulose.</p>},
  author       = {Abou Hachem, Maher and Nordberg Karlsson, Eva and Bartonek-Roxå, Eva and Raghothama, Srinivasrao and Simpson, Peter J. and Gilbert, Harry J. and Williamson, Michael P. and Holst, Olle},
  issn         = {0264-6021},
  language     = {eng},
  month        = {01},
  number       = {1},
  pages        = {53--60},
  publisher    = {Portland Press},
  series       = {Biochemical Journal},
  title        = {Carbohydrate-binding modules from a thermostable Rhodothermus marinus xylanase : Cloning, expression and binding studies},
  url          = {http://dx.doi.org/10.1042/0264-6021:3450053},
  doi          = {10.1042/0264-6021:3450053},
  volume       = {345},
  year         = {2000},
}