根霉胞内α-半乳糖苷酶的分离及其酶学性质

Purification and Characterization of Intracellular α-galactosidase from Rhizopus sp. A01

根霉Rhizopus sp.A01的菌丝体破碎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54.8倍,总酶活回收率达到27.3%,在SDS-PAGE上显示相对分子质量为85.6 ku的单一条带,凝胶过滤表明该酶表观相对分子质量为302 ku。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55℃,表观Km值为(0.242±0.027)mmol/L,表观kcat/Km值为4.089×105L/(mol.s);对蜜二糖和棉子糖有弱的水解作用,水解速度依次为138.3μmol/(h.mg)、19.7μmol/(h.mg)。水解活性受Fe2+和Fe3+的显著激活,但受Mn2+、Cu2+、Hg+和Mg2+等离子的强烈抑制。该酶活性在pH4.0～8.2保持稳定,在50℃时保温90 min,残余酶活达到了48%。

Using three-phase partitioning followed by filtration chromatography with Sephadex G - 100, an intra- cellular α-galactosidase from Rhizopus sp. A01 grown on soya bean dregs broth was purified to homogeneity with a 54. 8 - fold increase in specific activity and 27.3% recovery. The relative molecular weight of the enzyme was about 302 ku through gel filtration and 85.6 ku through SDS-polyacrylamide gel electrophoresis, suggesting that the native enzyme was a tetramer. The α-galactosidase showed high activity against p-nitrophenyl-α-d-galactopyranoside （pNP-Gal） but had little activity for melibiose and raffinose, and the optimal activity was observed at pH 4.5 and 55℃. The kinetic parameters of Km and kcat/Km were 0. 242 ＋ 0. 027 mmol/L and 4. 089 × 105 L/（mol· s） with pNPGal, and the rates of hydrolysis for melibiose and raffinose were 138.3 μmol/（h · mg） and 19.7 μmol/（h · mg）, respec-tively. The enzyme activity was significantly activated by Fe2^2＋ and Fe^3＋ , but strongly inhibited by Mn^2＋ ,Cu^2＋ ,Hg^＋ and Mg^2＋ at 5.0 mmol/L. The α-galactosidase was highly stable over pH range from 4.0 to 8.2 at 25 ℃ , and it re-tained approximately 48% of the original activity after incubation for 90min at 50℃.