Purification and characterization of a trehalose synthase from the basidiomycete grifola frondosa
A trehalose synthase (TSase) that catalyzes the synthesis of trehalose from D-glucose and alpha-D-glucose 1-phosphate (alpha-D-glucose 1-P) was detected in a basidiomycete, Grifola frondosa. TSase was purified 106-fold to homogeneity with 36% recovery by ammonium sulfate precipitation and several steps of column chromatography. The native enzyme appears to be a dimer since it has apparent molecular masses of 120 kDa, as determined by gel filtration column chromatography, and 60 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Although TSase catalyzed the phosphorolysis of trehalose to D-glucose and alpha-D-glucose 1-P, in addition to the synthesis of trehalose from the two substrates, the TSase equilibrium strongly favors trehalose synthesis. The optimum temperatures for phosphorolysis and synthesis of trehalose were 32.5 to 35 degreesC and 35 to 37.5 degreesC, respectively. The optimum pHs for these reactions were 6.5 and 6.5 to 6.8, respectively. The substrate specificity of TSase was very strict: among eight disaccharides examined, only trehalose was phosphorolyzed, and only alpha-D-glucose 1-P served as a donor substrate with D-glucose as the acceptor in trehalose synthesis. Two efficient enzymatic systems for the synthesis of trehalose from sucrose were identified. In system I, the alpha-D-glucose 1-P liberated by 1.05 U of sucrose phosphorylase was linked with D-glucose by 1.05 U of TSase, generating trehalose at the initial synthesis rate of 18 mmol/h in a final yield of 90 mol% under optimum conditions (300 mM each sucrose and glucose, 20 mM inorganic phosphate, 37.5 degreesC, and pH 6.5). In system II, we added 1.05 U of glucose isomerase and 20 mM MgSO4 to the reaction mixture of system I to convert fructose, a by-product of the sucrose phosphorylase reaction, into glucose. This system generated trehalose at the synthesis rate of 4.5 mmol/h in the same final yield.
Saito
K
Nishiki Research Laboratories, Kureha Chemical Industry Co., Ltd., Nishiki-machi, Iwaki-shi, Fukushima 974-8686, Japan.
Kase
T
Takahashi
E
Horinouchi
S