【摘要】 根霉（Rhizopus sp.A03）发酵豆渣产α-半乳糖苷酶,粗酶液依次经过三相分离、Sephadex G-100凝胶过滤获得了电泳纯的α-半乳糖苷酶,纯化了2.3倍,总酶活回收率达到25.9%,SDS-PAGE显示其相对分子质量为168.8 kDa。该酶水解对硝基苯-α-D-吡喃半乳糖苷的最适pH值为4.5,最适温度为45℃,表观Km值为0.340±0.026 mmol/L,表观kcat/K_m值为2.866×10⁴ mol-1/（L·s）;水解蜜二糖和棉子糖的表观速率均为10.0μmol/（h·mg）;水解活性受Fe³⁺、Cu²⁺、Mn²⁺和Hg⁺等离子的强烈抑制,但Fe²⁺对酶活性具有显著的激活作用。该酶活性在pH4.0～8.9保持稳定,在45℃时保温60min,残余酶活达到了77.4%。更多还原

【Abstract】 Using three-phase partitioning flowed by filtration chromatography with Sephadex G-100,a form ofα-galactosidase from Rhizopus sp.A03 grown on soya bean dreg broth was purified to homogeneity with a 2.3-fold increase in specific activity and 25.9%of recovery.The enzyme showed a monomer with apparent molecular mass of 168.8 kDa by SDS-polyacrylamide gel electrophoresis and gel filtration.The a-galactosidase showed high activity against p-nitrophenyl-α-d-galactopyranoside（pNPGal） but had slight activity for melibiose and raffinose,and the optimal activity was observed at pH 4.5 and 45℃.The kinetic parameters of K_m and k_cat/K_m were 0.340±0.026 mmol/L and 2.866×10⁴ mol/（L·s） with pNPGal,and the rate of hydrolysise for melibiose was 10.0μmol/（h·mg） as much as that for raffinose.The enzyme activity was activated by Fe²⁺,but strongly inhibited by Fe³⁺,Cu²⁺,Mn²⁺ and Hg⁺ at 5.0 mmol/L.Theα-galactosidase was highly stable over pH range of 4.0-8.9 at 25℃,and its activity retained approximately 77.4%of the original activity after incubation for 60min at 45℃.更多还原