关键词: 双水相萃取, 蜜二糖, 对硝基苯-α-D-吡喃半乳糖苷, 同二聚体, α-半乳糖苷酶

Abstract: After the end of the fermentation of soybean dregs by Aspergillus niger L. for α-galactosidase production, the fermentation broth supernatant was harvested as crude enzyme solution. Electrophoretically pure α-galactosidase was obtained from the crude enzyme solution by aqueous two-phase extraction in polyethylene glycol-6000-KH2PO4 system and Sephadex G-100 gel filtration, resulting in a purification factor of 36.4 and a total activity recovery of 70%. The molecular weight of the purified enzyme was approximately 125 kD as determined by Sephadex G-100 gel filtration chromatography and 58.5 kD as determined by SDS-PAGE, suggesting that the native enzyme was a homodimer. The optimum pH and temperature for the hydrolysis of p-nitrophenyl-α-D-galactopyranoside (pNPGal) by the enzyme were 4.0 and 65 ℃, respectively, and the apparent Km and kcat/Km were 0.915 mmol/L and 1.07 × 105 L/(mol•s), respectively, whereas those for melibiose were 21.0 mmol/L and 9.96 × 103 L/(mol•s), respectively. The enzyme activity was inhibited by some metal ions, especially Fe2+, Mn2+ and Hg+, but was stable in the pH range of 1.5 to 8.2. After 60 min of exposure to 60 ℃, 60% of the original activity was retained. Therefore, the enzyme has good acid and thermal stability.

Key words: aqueous two-phase extraction, melibiose, p-nitrophenyl-α-D-galactopyranoside, homodimer, α-galactosidase