Trehalose phosphorylase from Pleurotus ostreatus: Characterization and stabilization by covalent modification, and application for the synthesis of α,α-trehalose

Trehalose phosphorylase from the basidiomycete Pleurotus ostreatus (PoTPase) was isolated from fungal fruit bodies through ∼500-fold purification with a yield of 44%. Combined analyses by SDS-PAGE and gelfiltration show that PoTPase is a functional monomer of ∼55 kDa molecular mass. PoTPase catalyzes the phosphorolysis of α,α-trehalose, yielding α-d-glucose 1-phosphate (αGlc 1-P) and α-d-glucose as the products. The optimum pH of PoTPase for α,α-trehalose phosphorolysis and synthesis is 6.8 and 6.2, respectively. Apparent substrate binding affinities (Km) were determined at pH 6.8 and 30 °C: α,α-trehalose (79 mM); phosphate (3.5 mM); d-glucose (40 mM); αGlc 1-P (4.1 mM). A series of structural analogues of d-glucose were tested as glucosyl acceptors for the enzymatic reaction with αGlc 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 α,α-trehalose, and vanadate strongly inhibits the enzyme activity (Ki ∼4 μM). PoTPase has a half-life (t0.5) of approximately 1 h at 30 °C in the absence of stabilizing compounds such as α,α-trehalose (300 mM; t0.5 = 11.5 h), glycerol (20%, w/v; t0.5 = 6.5 h) or polyethylenglycol (PEG) 4000 (26%, w/v; t0.5 = 70 h). Covalent modification of PoTPase with activated derivatives of PEG 5000 increases the stability by up to 600-fold. Sucrose was converted to α,α-trehalose in ∼60% yield using a coupled enzyme system composed of sucrose phosphorylase from Leuconostoc mesenteroides, glucose isomerase from Streptomyces murinus and the appropriately stabilized PoTPase.