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Enzymatic properties and substrate specificity of the trehalose phosphorylase from Caldanaerobacter subterraneus

Jef Van der Borght UGent, Chao Chen UGent, Lieve Hoflack UGent, Lucas Van Renterghem UGent, Tom Desmet UGent and Wim Soetaert UGent (2011) APPLIED AND ENVIRONMENTAL MICROBIOLOGY. 77(19). p.6939-6944
abstract
A putative glycoside phosphorylase from Caldanaerobacter subterraneus subsp. pacificus was recombinantly expressed in E. coli, after codon optimization and chemical synthesis of the encoding gene. The enzyme was purified by His-tag chromatography and was found to be specifically active towards trehalose, with an optimal temperature of 80°C. In addition, no loss of activity could be detected after 1 h incubation at 65°C, which means that it is the most stable trehalose phosphorylase reported so far. The substrate specificity was investigated in detail by measuring the relative activity on a range of alternative acceptors, applied in the reverse synthetic reaction, and determination of the kinetic parameters for the best acceptors. These results were rationalized based on the enzyme-substrate interactions observed in a homology model with a docked ligand. The specificity for the orientation of the acceptor's hydroxyl groups was found to decrease in the order C3 > C2 > C4. This results in a particularly high activity on the monosaccharides D-fucose, D-xylose, L-arabinose and D-galactose, as well as on L-fucose. However, determination of the kinetic parameters revealed that these acceptors bind less tightly in the active site than the natural acceptor D-glucose, resulting in drastically increased Km values. Nevertheless, the enzyme's high thermostability and broad acceptor specificity makes it a valuable candidate for industrial disaccharide synthesis.
Please use this url to cite or link to this publication:
author
organization
year
type
journalArticle (original)
publication status
published
subject
keyword
THERMOANAEROBACTER-BROCKII, EUGLENA-GRACILIS, GEN.-NOV., PURIFICATION, ENVIRONMENTS, DISACCHARIDE, METABOLISM, PACIFICUM, SWISS-MODEL WORKSPACE
journal title
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
Appl. Environ. Microbiol.
volume
77
issue
19
pages
6939 - 6944
Web of Science type
Article
Web of Science id
000295123300026
JCR category
BIOTECHNOLOGY & APPLIED MICROBIOLOGY
JCR impact factor
3.829 (2011)
JCR rank
29/157 (2011)
JCR quartile
1 (2011)
ISSN
0099-2240
DOI
10.1128/AEM.05190-11
project
Biotechnology for a sustainable economy (Bio-Economy)
language
English
UGent publication?
yes
classification
A1
copyright statement
I have transferred the copyright for this publication to the publisher
id
1908756
handle
http://hdl.handle.net/1854/LU-1908756
date created
2011-09-26 11:43:15
date last changed
2013-02-27 12:59:20
@article{1908756,
  abstract     = {A putative glycoside phosphorylase from Caldanaerobacter subterraneus subsp. pacificus was recombinantly expressed in E. coli, after codon optimization and chemical synthesis of the encoding gene. The enzyme was purified by His-tag chromatography and was found to be specifically active towards trehalose, with an optimal temperature of 80{\textdegree}C. In addition, no loss of activity could be detected after 1 h incubation at 65{\textdegree}C, which means that it is the most stable trehalose phosphorylase reported so far. The substrate specificity was investigated in detail by measuring the relative activity on a range of alternative acceptors, applied in the reverse synthetic reaction, and determination of the kinetic parameters for the best acceptors. These results were rationalized based on the enzyme-substrate interactions observed in a homology model with a docked ligand. The specificity for the orientation of the acceptor's hydroxyl groups was found to decrease in the order C3 {\textrangle} C2 {\textrangle} C4. This results in a particularly high activity on the monosaccharides D-fucose, D-xylose, L-arabinose and D-galactose, as well as on L-fucose. However, determination of the kinetic parameters revealed that these acceptors bind less tightly in the active site than the natural acceptor D-glucose, resulting in drastically increased Km values. Nevertheless, the enzyme's high thermostability and broad acceptor specificity makes it a valuable candidate for industrial disaccharide synthesis.},
  author       = {Van der Borght, Jef and Chen, Chao and Hoflack, Lieve and Van Renterghem, Lucas and Desmet, Tom and Soetaert, Wim},
  issn         = {0099-2240},
  journal      = {APPLIED AND ENVIRONMENTAL MICROBIOLOGY},
  keyword      = {THERMOANAEROBACTER-BROCKII,EUGLENA-GRACILIS,GEN.-NOV.,PURIFICATION,ENVIRONMENTS,DISACCHARIDE,METABOLISM,PACIFICUM,SWISS-MODEL WORKSPACE},
  language     = {eng},
  number       = {19},
  pages        = {6939--6944},
  title        = {Enzymatic properties and substrate specificity of the trehalose phosphorylase from Caldanaerobacter subterraneus},
  url          = {http://dx.doi.org/10.1128/AEM.05190-11},
  volume       = {77},
  year         = {2011},
}

Chicago
Van der Borght, Jef, Chao Chen, Lieve Hoflack, Lucas Van Renterghem, Tom Desmet, and Wim Soetaert. 2011. “Enzymatic Properties and Substrate Specificity of the Trehalose Phosphorylase from Caldanaerobacter Subterraneus.” Applied and Environmental Microbiology 77 (19): 6939–6944.
APA
Van der Borght, J., Chen, C., Hoflack, L., Van Renterghem, L., Desmet, T., & Soetaert, W. (2011). Enzymatic properties and substrate specificity of the trehalose phosphorylase from Caldanaerobacter subterraneus. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 77(19), 6939–6944.
Vancouver
1.
Van der Borght J, Chen C, Hoflack L, Van Renterghem L, Desmet T, Soetaert W. Enzymatic properties and substrate specificity of the trehalose phosphorylase from Caldanaerobacter subterraneus. APPLIED AND ENVIRONMENTAL MICROBIOLOGY. 2011;77(19):6939–44.
MLA
Van der Borght, Jef, Chao Chen, Lieve Hoflack, et al. “Enzymatic Properties and Substrate Specificity of the Trehalose Phosphorylase from Caldanaerobacter Subterraneus.” APPLIED AND ENVIRONMENTAL MICROBIOLOGY 77.19 (2011): 6939–6944. Print.