title: Trehalose phosphorylase from Pleurotus ostreatus: characterization and stabilization by covalent modification, and application for the synthesis of alpha,alpha-trehalose.
abstract: Trehalose phosphorylase from the basidiomycete Pleurotus ostreatus (PoTPase) was isolated from fungal fruit bodies through approximately 500-fold purification with a yield of 44%. Combined analyses by SDS-PAGE and gelfiltration show that PoTPase is a functional monomer of approximately 55 kDa molecular mass. PoTPase catalyzes the phosphorolysis of alpha,alpha-trehalose, yielding alpha-d-glucose 1-phosphate (alphaGlc 1-P) and alpha-d-glucose as the products. The optimum pH of PoTPase for alpha,alpha-trehalose phosphorolysis and synthesis is 6.8 and 6.2, respectively. Apparent substrate binding affinities (K(m)) were determined at pH 6.8 and 30 degrees C: alpha,alpha-trehalose (79 mM); phosphate (3.5 mM); d-glucose (40 mM); alphaGlc 1-P (4.1mM). A series of structural analogues of d-glucose were tested as glucosyl acceptors for the enzymatic reaction with alphaGlc 1-P, and robust activity with d-mannose (3%), 2-deoxy d-glucose (8%), 2-fluoro d-glucose (15%) and 2-keto-d-glucose (50%) was detected. Arsenate replaces, with 30% relative activity, phosphate in the conversion of alpha,alpha-trehalose, and vanadate strongly inhibits the enzyme activity (K(i) approximately 4 microM). PoTPase has a half-life (t(0.5)) of approximately 1 h at 30 degrees C in the absence of stabilizing compounds such as alpha,alpha-trehalose (300 mM; t(0.5)=11.5 h), glycerol (20%, w/v; t(0.5)=6.5h) or polyethylenglycol (PEG) 4000 (26%, w/v; t(0.5)=70 h). Covalent modification of PoTPase with activated derivatives of PEG 5000 increases the stability by up to 600-fold. Sucrose was converted to alpha,alpha-trehalose in approximately 60% yield using a coupled enzyme system composed of sucrose phosphorylase from Leuconostoc mesenteroides, glucose isomerase from Streptomyces murinus and the appropriately stabilized PoTPase.
authors.affiliation: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12, A-8010 Graz, Austria.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Schwarz
authors.forename: Christiane
authors.initials: C
authors.lastname: Goedl
authors.forename: Alphonse
authors.initials: A
authors.lastname: Minani
authors.forename: Bernd
authors.initials: B
authors.lastname: Nidetzky
mesh_terms: D002384:Catalysis; D004795:Enzyme Stability; D005964:Glucosyltransferases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006207:Half-Life; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D020076:Pleurotus / Q000201:enzymology*; D011487:Protein Conformation; D013379:Substrate Specificity; D013395:Sucrose / Q000378:metabolism; D013997:Time Factors; D014199:Trehalose / Q000096:biosynthesis*
chemical_list: D013395:Sucrose; D014199:Trehalose; D005964:Glucosyltransferases; C023181:alpha,alpha-trehalose phosphorylase