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

Appl Microbiol Biotechnol. 2014 Aug;98(16):7027-37. doi: 10.1007/s00253-014-5621-y. Epub 2014 Mar 6.


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 °C), and with a melting temperature of 79 °C and a half-life of 60 h at the industrially relevant temperature of 60 °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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cloning, Molecular
  • DNA Mutational Analysis
  • DNA, Bacterial / chemistry
  • DNA, Bacterial / genetics
  • Enzyme Stability
  • Gene Expression
  • Glucosyltransferases / chemistry
  • Glucosyltransferases / genetics
  • Glucosyltransferases / metabolism*
  • Molecular Sequence Data
  • Phosphorylation
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Sequence Analysis, DNA
  • Substrate Specificity
  • Sucrose / analogs & derivatives*
  • Sucrose / metabolism
  • Sugar Phosphates / metabolism*
  • Temperature
  • Thermoanaerobacterium / enzymology*
  • Thermoanaerobacterium / genetics


  • DNA, Bacterial
  • Recombinant Proteins
  • Sugar Phosphates
  • sucrose-6-phosphate
  • Sucrose
  • Glucosyltransferases
  • sucrose phosphorylase

Associated data

  • GENBANK/KF914648
  • GENBANK/KF914649