耐高温β-半乳糖苷酶的分离纯化与酶学性质分析.pdf

※生物工程 食品科学 2010, Vol. 31, No. 23 151 β- 高兆建，侯进慧，孙会刚，刁进进 (徐州工程学院食品(生物)工程学院，江苏 徐州 221008) ：为从嗜热脂肪芽孢杆菌(Bacillus stearothermophilus)XG24 发酵液中纯化到 β- 半乳糖苷酶，并对酶学性质 进行研究，利用硫酸铵分级盐析、DEAE-Sepharose Fast Flow 阴离子交换层析和Sephadex G-75 分子筛凝胶过滤层 析等方法进行分离纯化。结果表明：经过系列步骤纯化后，酶纯度提高了 54.5 倍，回收率 20.4% ，酶比活力达 32.7U/mg 。以邻- 硝基酚 -D - 半乳糖吡喃糖苷(ONPG)为底物，研究 β- 半乳糖苷酶的酶学性质。最适pH6.5 ，最 适作用温度 65 ℃。此菌株产β- 半乳糖苷酶在 70 ℃以下和pH4.0 ～8.0 范围内具有较好的稳定性；Mg2+ 、Mn2+ 、Fe2+ 和 Co2+ 对此酶有明显激活作用，而 Cu 2 + 、Ag + 、Hg 2+ 几乎完全抑制酶活性。以 ONPG 为底物酶的 K m 值为 4.32mmol/L 。SDS和凝胶过滤层析测得酶蛋白为单肽链蛋白，表观分子质量 64kD 。因此，嗜热脂肪芽孢 杆菌 XG24 β- 半乳糖苷酶在乳制品工业中具有潜在的应用价值。 ：嗜热脂肪芽孢杆菌； β- 半乳糖苷酶；分离纯化；性质 Purification and Enzymatic Characterization of Thermostable β-Galactosidase from Bacillus stearothermophilus XG24 GAO Zhao-jian ，HOU Jin-hui ，SUN Hui-gang，DIAO Jin-jin (College of Food (Biological) Engineering, Xuzhou Institute of Technology, Xuzhou 221008, China) Abstract ：The fermentation supernatant of Bacillus stearothermophilus XG24 received salting out with ammonium sulfate, separation on DEAE-Sepharose Fast Flow anion exchange column and purification on Sephadex G-75 gel filtration column to obtain high-purity thermostable β-galactosidase, which was subsequently subjected to enzymatic characterization with o-nitrophenyl- β-D-galactopyranoside (ONPG) as a substrate. After the above separation and purification procedures, the purity of this enzyme showed a 54.5-fold increase, with an activity recovery of 20.4%, and the specific activity of the purified enzyme was 32.7 U/mg protein. The optimal pH and temper