[#1998-05] Purification and Characterization of a Trehalose Synthase from the Basidiomycete Grifola frondosa (Maitake), Koki Saito et al.

Purification and Characterization of a Trehalose Synthase from the Basidiomycete Grifola frondosa ( Maitake )

KOKI SAITO,¹ TOSHIYA KASE,¹ EIICHI TAKAHASHI,¹ EISAKU TAKAHASHI,¹ AND SUEHARU HORINOUCHI²*

Nishiki Research Laboratories, Kureha Chemical Industry Co., Ltd.,Fukushima, Japan,¹ and Department of Biotechnology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Tokyo, Japan²

A trehalose synthase (TSase) that catalyzes the synthesis of trehalose from D-glucose and α-D-glucose 1-phosphate (α-D-glucose 1-P) was detected in a basidiomycete, Grifola frondosa ( Maitake ). 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 sulfate­polyacrylamide gel electrophoresis. Although TSase catalyzed the phosphorolysis of trehalose to D-glucose and α-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°C and 35 to 37.5°C, 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 a-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 α-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°C, 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.