摘要`目的研究短双歧杆菌(Bifidobacterium breve) 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native gradient PAGE检查蛋白纯度。结果 native gradient PAGE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156×103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(0.01mmol/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0.168mmol/L、1.07mmol/L和0.456mmol/L，Vmax分别为4.55μmol/min、0.378μmol/min和0.614μmol/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60℃之内的热稳定性明显较好。

abstractsObjective　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+, Cu2+, Ag+ (1mmol/L) and PCMB (0.01mmol/L) had a strong inhibitory effect while Co2+, Mg2+, Ca2+, Mn2+, Zn2+ (1mmol/L), EDTA and DTT (10mmol/L) had no effect on the enzyme. The enzyme targeted specifically to α-galactoside linkages. Km-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.