短双歧杆菌α-D-半乳糖苷酶的纯化及性质

Purification and characterization of α-D-galactosidase from Bifidobacterium breve

目的 研究短双歧杆菌 (Bifidobacteriumbreve)α D 半乳糖苷酶的纯化及性质。方法 采用硫酸铵分级沉淀及柱层析法纯化酶蛋白 ,垂直板状SDS PAGE和nativegradientPAGE检查蛋白纯度。结果 nativegradientPAGE电泳后活性染色确定无细胞粗酶液中含有一种α D 半乳糖苷酶 ,纯化后的酶蛋白是一个相对分子质量 (Mr)约为 15 6× 10 3 的双亚基蛋白质 ,等电点pI为 5 .3,最适反应温度为 37℃ ,在 40℃以下稳定 ,70℃时失去所有的酶活性。最适反应pH为 5 .5～ 6 .5 ,酶在pH5 .5～ 9.5范围内稳定。Hg+ 、Cu2 + 、Ag+ (各 1mmol/L)和PCMB(0 .0 1mmol/L)强烈抑制酶活性 ,Co2 + 、Mg2 + 、Ca2 + 、Mn2 + 、Zn2 + (各 1mmol/L)、EDTA和DTT(各 10mmol/L)对酶活性没有抑制。酶专一性水解α D 半乳糖苷键。酶对p 硝基苯酚 α D 半乳糖苷、m 硝基苯酚 α D 半乳糖苷和o 硝基苯酚 α D 半乳糖苷的Km分别为 0 .16 8mmol/L、1.0 7mmol/L和 0 .45 6mmol/L ,Vmax分别为 4.5 5 μmol/min、0 .378μmol/min和 0 .6 14μmol/min。结论 B .breve的α D 半乳糖苷酶与少数国外报道的其他双歧杆菌的α D 半乳糖苷酶有相似之处 ,也有不同点。其Mr 不同 ,在 6 0℃之内的热稳定性明显较好。

Objective Purification and characterization of α-D-galactosidease from Bifidobacterium breve. Methods The enzyme was purified by ammonium sulfate-fractionation, DEAE Sepharose Fast Flow, Gigapite K-100S and Sephadex G-150 column chromatography. SDS-PAGE and native gradient PAGE were used for the identification of the protein. Results B. breve produced one kind of α-D-galactosidease. The enzyme was purified from the cell extract of B.breve. It was a homodimer with a molecular weight of about 156000 and pI 5.3. The enzyme showed optimal activity at 37℃ and pH5.5-6.5. Hg+, Cu 2+, Ag+ (1mmol/L) and PCMB ( 0.01mmol/L) had a strong inhibitory effect while Co 2+, Mg 2+, Ca 2+, Mn 2+, Zn 2+ (1mmol/L), EDTA and DTT (10mmol/L) had no effect on the enzyme. The enzyme targeted specifically to α-galactoside linkages. K m-values for p-nitrophenyl-α-D-galactopyranoside (pNP-α-D-Gal), m-NP-α-D-Gal and o-NP-α-D-Gal were 0.168mmol/L, 1.07mml/L and 0.456mmol/L, respectively. Conclusion In spite of some similarities, α-D-galactosidease from B.breve showed the different molecular mass and better thermal stability at 60℃ compared with that form other bifidobacteria.