Synthesis of Raffinose by Fungal α-Galacotosidase from Absidia corymbifera

In order to investigate the optimal conditions for raffinose synthesis, alpha-galactosidase was purified from Absidia corymbifera IFO8084 with a recovery yield of approximately 8.1% (8.36 mg). The molecular weight of the wild-type alpha-galactosidase was about 83 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The native molecular mass of the enzyme was approximately 330 kDa by gel filtration chromatography, indicating that alpha-galactosidase from A. corymbifera IFO8084 is a homotetrameric enzyme. The purified enzyme displayed optimal enzyme activity at pH 4.5 and 60 degrees C. When the purified alpha-galactosidase was incubated in a substrate solution of sucrose and D-galactose for 48 hr at 37 degrees C, raffinose was synthesized and was confirmed by thin layer chromatography (TLC), high performance liquid chromatography (HPLC), and C-13-nuclear magnetic resonance (C-13-NMR) spectrometer analysis. Maximum rates of conversion were observed with 1.67 M galactose, 2.04 M sucrose, and 100 U alpha-galactosidase at pH 6.0 and 70 degrees C. Under the optimized conditions, the overall conversion ratio was 10%(w/v), representing 2.5 times the synthesis yield that would be possible without the optimized conditions.