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当前位置: 首页 > 文章 > 耐高温β-半乳糖苷酶的分离纯化与酶学性质分析 食品科学 2010,31 (23) 151-156
Position: Home > Articles > Purification and Enzymatic Characterization of Thermostable β-Galactosidase from Bacillus stearothermophilus XG24 FOOD SCIENCE 2010,31 (23) 151-156

耐高温β-半乳糖苷酶的分离纯化与酶学性质分析

作  者：
高兆建;侯进慧;孙会刚;刁进进
单  位：
徐州工程学院食品(生物)工程学院
关键词：
嗜热脂肪芽孢杆菌;β-半乳糖苷酶;分离纯化;性质
摘  要：
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-SepharoseFastFlow阴离子交换层析和SephadexG-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Cu2+、Ag+、Hg2+几乎完全抑制酶活性。以ONPG为底物酶的Km值为4.32mmol/L。SDS-PAGE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XG24β-半乳糖苷酶在乳制品工业中具有潜在的应用价值。
译  名：
Purification and Enzymatic Characterization of Thermostable β-Galactosidase from Bacillus stearothermophilus XG24
作  者：
GAO Zhao-jian,HOU Jin-hui,SUN Hui-gang,DIAO Jin-jin (College of Food (Biological) Engineering, Xuzhou Institute of Technology, Xuzhou 221008, China)
关键词：
Bacillus stearothermophilus;β-galactosidase;purification;characterization
摘  要：
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 temperature for the reaction of this enzyme were 6.5 and 65 ℃, respectively. It was stable at temperatures below 70 ℃ or in a pH range between 4.0 and 8.0. Its activity was notably promoted by Mg2+, Mn2+, Fe2+ and Co2+, whereas Cu2+, Ag+ and Hg2+ were almost able to entirely inhibit its activity. The Km towards ONPG was determined to be 4.32 mmol/L. The SDS-PAGE analysis revealed that this enzyme was a single-chain protein. Based on the results of Sephadex G-75 gel filtration chromatographic measurement, it was deduced that the apparent molecular weight of this enzyme was 64 kD. Therefore, Bacillus stearothermophilus XG24-derived thermostable β-galactosidase has high application potential in the dairy industry.

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