Skip to main content

Lund University Publications

LUND UNIVERSITY LIBRARIES

Expression and Characterization of a Bifidobacterium adolescentis Beta-Mannanase Carrying Mannan-Binding and Cell Association Motifs

Kulcinskaja, Evelina LU ; Rosengren, Anna LU ; Ibrahim, Romany ; Kolenová, Katarina LU and Stålbrand, Henrik LU (2013) In Applied and Environmental Microbiology 79(1). p.133-140
Abstract
The gene encoding beta-mannanase (EC 3.2.1.78) BaMan26A from the bacterium Bifidobacterium adolescentis (living in the human gut) was cloned and the gene product characterized. The enzyme was found to be modular and to contain a putative signal peptide. It possesses a catalytic module of the glycoside hydrolase family 26, a predicted immunoglobulin-like module, and two putative carbohydrate-binding modules (CBMs) of family 23. The enzyme is likely cell attached either by the sortase mechanism (LPXTG motif) or via a C-terminal transmembrane helix. The gene was expressed in Escherichia coli without the native signal peptide or the cell anchor. Two variants were made: one containing all four modules, designated BaMan26A-101K, and one... (More)
The gene encoding beta-mannanase (EC 3.2.1.78) BaMan26A from the bacterium Bifidobacterium adolescentis (living in the human gut) was cloned and the gene product characterized. The enzyme was found to be modular and to contain a putative signal peptide. It possesses a catalytic module of the glycoside hydrolase family 26, a predicted immunoglobulin-like module, and two putative carbohydrate-binding modules (CBMs) of family 23. The enzyme is likely cell attached either by the sortase mechanism (LPXTG motif) or via a C-terminal transmembrane helix. The gene was expressed in Escherichia coli without the native signal peptide or the cell anchor. Two variants were made: one containing all four modules, designated BaMan26A-101K, and one truncated before the CBMs, designated BaMan26A-53K. BaMan26A-101K, which contains the CBMs, showed an affinity to carob galactomannan having a dissociation constant of 0.34 mu M(8.8 mg/liter), whereas BaMan26A-53K did not bind, showing that at least one of the putative CBMs of family 23 is mannan binding. For BaMan26A-53K, kappa(cat) was determined to be 444 s(-1) and K-m 21.3 g/liter using carob galactomannan as the substrate at the optimal pH of 5.3. Both of the enzyme variants hydrolyzed konjac glucomannan, as well as carob and guar gum galactomannans to a mixture of oligosaccharides. The dominant product from ivory nut mannan was found to be mannotriose. Mannobiose and mannotetraose were produced to a lesser extent, as shown by high-performance anion-exchange chromatography. Mannobiose was not hydrolyzed, and mannotriose was hydrolyzed at a significantly lower rate than the longer oligosaccharides. (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
in
Applied and Environmental Microbiology
volume
79
issue
1
pages
133 - 140
publisher
American Society for Microbiology
external identifiers
  • wos:000312931600015
  • pmid:23064345
  • scopus:84871881296
  • pmid:23064345
ISSN
0099-2240
DOI
10.1128/AEM.02118-12
language
English
LU publication?
yes
id
1ebb5d35-a1b5-4afb-a211-f53027363dc8 (old id 3480995)
date added to LUP
2016-04-01 10:13:19
date last changed
2022-04-27 19:49:13
@article{1ebb5d35-a1b5-4afb-a211-f53027363dc8,
  abstract     = {{The gene encoding beta-mannanase (EC 3.2.1.78) BaMan26A from the bacterium Bifidobacterium adolescentis (living in the human gut) was cloned and the gene product characterized. The enzyme was found to be modular and to contain a putative signal peptide. It possesses a catalytic module of the glycoside hydrolase family 26, a predicted immunoglobulin-like module, and two putative carbohydrate-binding modules (CBMs) of family 23. The enzyme is likely cell attached either by the sortase mechanism (LPXTG motif) or via a C-terminal transmembrane helix. The gene was expressed in Escherichia coli without the native signal peptide or the cell anchor. Two variants were made: one containing all four modules, designated BaMan26A-101K, and one truncated before the CBMs, designated BaMan26A-53K. BaMan26A-101K, which contains the CBMs, showed an affinity to carob galactomannan having a dissociation constant of 0.34 mu M(8.8 mg/liter), whereas BaMan26A-53K did not bind, showing that at least one of the putative CBMs of family 23 is mannan binding. For BaMan26A-53K, kappa(cat) was determined to be 444 s(-1) and K-m 21.3 g/liter using carob galactomannan as the substrate at the optimal pH of 5.3. Both of the enzyme variants hydrolyzed konjac glucomannan, as well as carob and guar gum galactomannans to a mixture of oligosaccharides. The dominant product from ivory nut mannan was found to be mannotriose. Mannobiose and mannotetraose were produced to a lesser extent, as shown by high-performance anion-exchange chromatography. Mannobiose was not hydrolyzed, and mannotriose was hydrolyzed at a significantly lower rate than the longer oligosaccharides.}},
  author       = {{Kulcinskaja, Evelina and Rosengren, Anna and Ibrahim, Romany and Kolenová, Katarina and Stålbrand, Henrik}},
  issn         = {{0099-2240}},
  language     = {{eng}},
  number       = {{1}},
  pages        = {{133--140}},
  publisher    = {{American Society for Microbiology}},
  series       = {{Applied and Environmental Microbiology}},
  title        = {{Expression and Characterization of a Bifidobacterium adolescentis Beta-Mannanase Carrying Mannan-Binding and Cell Association Motifs}},
  url          = {{http://dx.doi.org/10.1128/AEM.02118-12}},
  doi          = {{10.1128/AEM.02118-12}},
  volume       = {{79}},
  year         = {{2013}},
}