title: The quest for a thermostable sucrose phosphorylase reveals sucrose 6'-phosphate phosphorylase as a novel specificity.
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 °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.
authors.affiliation: Centre for Industrial Biotechnology and Biocatalysis, Department of Biochemical and Microbial Technology, Ghent University, Coupure Links 653, 9000, Ghent, Belgium.
authors.forename: Tom
authors.initials: T
authors.lastname: Verhaeghe
authors.forename: Dirk
authors.initials: D
authors.lastname: Aerts
authors.forename: Margo
authors.initials: M
authors.lastname: Diricks
authors.forename: Wim
authors.initials: W
authors.lastname: Soetaert
authors.forename: Tom
authors.initials: T
authors.lastname: Desmet
mesh_terms: D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D015870:Gene Expression; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D013395:Sucrose / Q000031:analogs & derivatives* / Q000378:metabolism; D013403:Sugar Phosphates / Q000378:metabolism*; D013696:Temperature; D046950:Thermoanaerobacterium / Q000201:enzymology* / Q000235:genetics
chemical_list: D004269:DNA, Bacterial; D011994:Recombinant Proteins; D013403:Sugar Phosphates; C535046:sucrose-6-phosphate; D013395:Sucrose; D005964:Glucosyltransferases; C025901:sucrose phosphorylase