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The quest for a thermostable sucrose phosphorylase reveals sucrose 6'-phosphate phosphorylase as a novel specificity

Tom Verhaeghe, Dirk Aerts UGent, Margo Diricks, Wim Soetaert UGent and Tom Desmet UGent (2014) APPLIED MICROBIOLOGY AND BIOTECHNOLOGY. 98(16). p.7027-7037
abstract
Sucrose phosphorylase is a promising biocatalyst for the glycosylation of a wide range of compounds, but its industrial application has been hampered by the low thermostability of known representatives. Hence, in this study, the putative sucrose phosphorylase from the thermophile Thermoanaerobacterium thermosaccharolyticum was recombinantly expressed and fully characterised. The enzyme showed significant activity on sucrose (optimum at 55 A degrees C), and with a melting temperature of 79 A degrees C and a half-life of 60 h at the industrially relevant temperature of 60 A degrees C, it is far more stable than known sucrose phosphorylases. Substrate screening and detailed kinetic characterisation revealed however a preference for sucrose 6'-phosphate over sucrose. The enzyme can thus be considered as a sucrose 6'-phosphate phosphorylase, a specificity not yet reported to date. Homology modelling and mutagenesis pointed out particular residues (Arg134 and His344) accounting for the difference in specificity. Moreover, phylogenetic and sequence analysis suggest that glycoside hydrolase 13 subfamily 18 might harbour even more specificities. In addition, the second gene residing in the same operon as sucrose 6'-phosphate phosphorylase was identified as well, and found to be a phosphofructokinase. The concerted action of both these enzymes implies a new pathway for the breakdown of sucrose, in which the reaction products end up at different stages of the glycolysis.
Please use this url to cite or link to this publication:
author
organization
year
type
journalArticle (original)
publication status
published
subject
keyword
DIRECTED EVOLUTION, GLUCOSYL TRANSFER, THERMOPHILIC BACTERIUM, NEISSERIA-POLYSACCHAREA, GEN. NOV., LEUCONOSTOC-MESENTEROIDES, SP-NOV., COMPLETE GENOME SEQUENCE, SEA HYDROTHERMAL VENT, Sucrose metabolism, Glycoside hydrolase family GH13, Thermostability, Sucrose 6'-phosphate phosphorylase, Sucrose phosphorylase, ENZYME STABILITY
journal title
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
Appl. Microbiol. Biotechnol.
volume
98
issue
16
pages
7027 - 7037
Web of Science type
Article
Web of Science id
000340087600012
JCR category
BIOTECHNOLOGY & APPLIED MICROBIOLOGY
JCR impact factor
3.337 (2014)
JCR rank
39/163 (2014)
JCR quartile
1 (2014)
ISSN
0175-7598
DOI
10.1007/s00253-014-5621-y
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
5766229
handle
http://hdl.handle.net/1854/LU-5766229
date created
2014-11-28 15:30:21
date last changed
2016-12-19 15:39:11
@article{5766229,
  abstract     = {Sucrose phosphorylase is a promising biocatalyst for the glycosylation of a wide range of compounds, but its industrial application has been hampered by the low thermostability of known representatives. Hence, in this study, the putative sucrose phosphorylase from the thermophile Thermoanaerobacterium thermosaccharolyticum was recombinantly expressed and fully characterised. The enzyme showed significant activity on sucrose (optimum at 55 A degrees C), and with a melting temperature of 79 A degrees C and a half-life of 60 h at the industrially relevant temperature of 60 A degrees C, it is far more stable than known sucrose phosphorylases. Substrate screening and detailed kinetic characterisation revealed however a preference for sucrose 6'-phosphate over sucrose. The enzyme can thus be considered as a sucrose 6'-phosphate phosphorylase, a specificity not yet reported to date. Homology modelling and mutagenesis pointed out particular residues (Arg134 and His344) accounting for the difference in specificity. Moreover, phylogenetic and sequence analysis suggest that glycoside hydrolase 13 subfamily 18 might harbour even more specificities. In addition, the second gene residing in the same operon as sucrose 6'-phosphate phosphorylase was identified as well, and found to be a phosphofructokinase. The concerted action of both these enzymes implies a new pathway for the breakdown of sucrose, in which the reaction products end up at different stages of the glycolysis.},
  author       = {Verhaeghe, Tom and Aerts, Dirk and Diricks, Margo and Soetaert, Wim and Desmet, Tom},
  issn         = {0175-7598},
  journal      = {APPLIED MICROBIOLOGY AND BIOTECHNOLOGY},
  keyword      = {DIRECTED EVOLUTION,GLUCOSYL TRANSFER,THERMOPHILIC BACTERIUM,NEISSERIA-POLYSACCHAREA,GEN. NOV.,LEUCONOSTOC-MESENTEROIDES,SP-NOV.,COMPLETE GENOME SEQUENCE,SEA HYDROTHERMAL VENT,Sucrose metabolism,Glycoside hydrolase family GH13,Thermostability,Sucrose 6'-phosphate phosphorylase,Sucrose phosphorylase,ENZYME STABILITY},
  language     = {eng},
  number       = {16},
  pages        = {7027--7037},
  title        = {The quest for a thermostable sucrose phosphorylase reveals sucrose 6'-phosphate phosphorylase as a novel specificity},
  url          = {http://dx.doi.org/10.1007/s00253-014-5621-y},
  volume       = {98},
  year         = {2014},
}

Chicago
Verhaeghe, Tom, Dirk Aerts, Margo Diricks, Wim Soetaert, and Tom Desmet. 2014. “The Quest for a Thermostable Sucrose Phosphorylase Reveals Sucrose 6’-phosphate Phosphorylase as a Novel Specificity.” Applied Microbiology and Biotechnology 98 (16): 7027–7037.
APA
Verhaeghe, T., Aerts, D., Diricks, M., Soetaert, W., & Desmet, T. (2014). The quest for a thermostable sucrose phosphorylase reveals sucrose 6’-phosphate phosphorylase as a novel specificity. APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 98(16), 7027–7037.
Vancouver
1.
Verhaeghe T, Aerts D, Diricks M, Soetaert W, Desmet T. The quest for a thermostable sucrose phosphorylase reveals sucrose 6’-phosphate phosphorylase as a novel specificity. APPLIED MICROBIOLOGY AND BIOTECHNOLOGY. 2014;98(16):7027–37.
MLA
Verhaeghe, Tom, Dirk Aerts, Margo Diricks, et al. “The Quest for a Thermostable Sucrose Phosphorylase Reveals Sucrose 6’-phosphate Phosphorylase as a Novel Specificity.” APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 98.16 (2014): 7027–7037. Print.