title: Enzymatic properties and substrate specificity of the trehalose phosphorylase from Caldanaerobacter subterraneus.
abstract: A putative glycoside phosphorylase from Caldanaerobacter subterraneus subsp. pacificus was recombinantly expressed in Escherichia 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 toward trehalose, with an optimal temperature of 80°C. In addition, no loss of activity could be detected after 1 h of 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 determining 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 following order: C-3 > C-2 > C-4. 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 K(m) values. Nevertheless, the enzyme's high thermostability and broad acceptor specificity make it a valuable candidate for industrial disaccharide synthesis.
authors.affiliation: Department of Biochemical and Microbial Technology, Center of Expertise-Industrial Biotechnology and Biocatalysis, Ghent University, Coupure Links 653, 9000 Ghent, Belgium.
authors.forename: Jef
authors.initials: J
authors.lastname: Van der Borght
authors.forename: Chao
authors.initials: C
authors.lastname: Chen
authors.forename: Lieve
authors.initials: L
authors.lastname: Hoflack
authors.forename: Lucas
authors.initials: L
authors.lastname: Van Renterghem
authors.forename: Tom
authors.initials: T
authors.lastname: Desmet
authors.forename: Wim
authors.initials: W
authors.lastname: Soetaert
mesh_terms: D020134:Catalytic Domain; D002846:Chromatography, Affinity; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006094:Gram-Positive Bacteria / Q000201:enzymology*; D006358:Hot Temperature; D007700:Kinetics; D008958:Models, Molecular; D009005:Monosaccharides / Q000378:metabolism*; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013997:Time Factors
chemical_list: D009005:Monosaccharides; D011994:Recombinant Proteins; D005964:Glucosyltransferases; C023181:alpha,alpha-trehalose phosphorylase