分枝犁头霉α-半乳糖苷酶的纯化和性质

PURIFICATION AND PROPERTIES OF α-GALACTOSIDASE FROM ABSIDIA RAMOSA

分枝犁头霉菌丝体球经金刚砂磨碎用水提取的粗酶液,用两种方法纯化,一是用制备垂直平板凝胶电泳;另一是经硫酸铵分级沉淀、Sephadex G-200、DEAE-Sephadex A50及羟基磷灰石等柱层析。这两种方法纯化后的样品经PAGE鉴定皆为均一带,无其他糖苷酶活性。酶反应最适温度为55—60℃,在50℃保温7h,剩余活力为50%,70℃保温10min,则全部失活。最适pH为5.0,pH6.0—8.5稳定。紫外吸收峰最高在波长272nm处,最低在248nm。激发光谱高峰在波长280nm,荧光发射光谱在300—400nm之间,最高在336nm。用浓度梯度PAGE测定分子量为323000,SDs-PAGE测亚基分子量为81000。薄层凝胶等电聚焦测pⅠ值为4.2。 Pb^(2+)和Mn^(2+)对酶有激活作用,Hg^(2+)、Ag^(2+)及Cu^(2+)有强烈的抑制作用。4mol/L尿素使酶活剩余70%,1.5mol/L盐酸胍使酶活剩余50%。2% SDS使酶活剩余25%。

α-galactosidase extracted from mycelium of Absidia ramosa was purified with two methods, one by using preparative slab gel electrophoresis; the other using ammonium sulfate fractionation, followed with gel filtration on Sephadex G-200, chromatography on DEAE-Sephadex A50, re-chromatography on DEAE-Sephadex A50 and chromatography on hydroxyapatite. The purified enzyme was homogeneous on disc gel electrophoresis without other glycosidase activity.The optimum reaction temperature of the enzyme was 55—60℃. The medium inactivity dose for this α-galactosidase was 50℃ for 7 h and inactivated completely at 70℃ for 10 min. This enzyme exhibited the optimum pH at 5.0 and was stable between pH 6.0 and 8.5.The UV absorption and fluorescence spectra of α-galactosidase were determined. The maximum absorption wavelength was at 272 nm and minimum 248 nm. For fluorescent analysis the excitation spectra was at 280 nm. The emission spectra was at between 300—400 nm, maximum at 336 nm.The molecular weight of purified α-galactosidase was determined to be about 323000 by concentration gradient PAGE and about 81000 by discodtinuous SDS-PAGE. The isolectric point was evaluated to be about 4.2 by polyacrylamide gel electrofocusing.The α-galactosidase activity was activited by Mn^(2+) but was completely inhibited by Hg^(2+), Ag^+ or Cu^(2+) ions. The α-galactosidase activity was remained 70% by 4mol/L urea, 50% by 1.5mol/L guanidine hydrochloride, and only 25% by 2% SDS.