Production, purification, and characterization of a potential thermostable galactosidase for milk lactose hydrolysis from Bacillus stearothermophilus
Production, purification, and characterization of a potential thermostable galactosidase for milk lactose hydrolysis from Bacillus stearothermophilus
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嗜热脂肪芽孢杆菌用于牛奶乳糖水解的潜在热稳定半乳糖苷酶的生产、纯化和表征
DOI：
10.3168/jds.2007-617
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发表时间：
2008-05-01
影响因子：
3.5
通讯作者：
Zhang, H.
中科院分区：
文献类型：
--
作者：
Chen, W.;Chen, H.;Zhang, H.
-半乳糖苷酶，通常被称为乳糖酶，是乳制品加工中最重要的酶之一；它催化乳糖水解成其组成单糖葡萄糖和半乳糖。本研究从嗜热脂肪芽孢杆菌中克隆了耐热性β -半乳糖苷酶基因bgaB，并在枯草芽孢杆菌WB600中表达。重组酶通过热处理、硫酸铵分馏、离子交换和凝胶过滤层析技术进行纯化。纯化后的β -半乳糖苷酶在十二烷基硫酸钠- page凝胶中表现为单个蛋白带，分子量约为70 kDa。聚丙烯酰胺凝胶等电聚焦测定其等电点接近5.1。该β -半乳糖苷酶活性的最佳温度为70℃，pH为7.0。该酶在65℃和70℃下的热失活动力学和半衰期分别为50和9 h， K-m和V-max分别为2.96 mM和6.62 mu mol/min / mg。金属阳离子和EDTA不能激活该酶，一些二价金属离子Fe2+、Zn2+、Cu2+、Pb2+和Sn2+抑制了该酶的活性。巯基阻断剂对酶活性无影响，巯基阻断剂使酶失活。这种酶在水解牛奶中的乳糖时具有高水平的半乳糖基化活性。结果表明，该重组热稳定酶既适用于乳糖的水解，也适用于牛奶加工中半乳糖低聚糖的生产。
beta-Galactosidase, commonly named lactase, is one of the most important enzymes used in dairy processing; it catalyzes the hydrolysis of lactose to its constituent monosaccharides glucose and galactose. Here, a thermostable beta-galactosidase gene bgaB from Bacillus stearothermophilus was cloned and expressed in B. subtilis WB600. The recombinant enzyme was purified by a combination of heat treatment, ammonium sulfate fractionation, ion exchange, and gel filtration chromatography techniques. The purified beta-galactosidase appeared as a single protein band in sodium dodecyl sulfate-PAGE gel with a molecular mass of approximately 70 kDa. Its isoelectric point, determined by polyacrylamide gel isoelectric focusing, was close to 5.1. The optimum temperature and pH for this beta-galactosidase activity were 70 degrees C and pH 7.0, respectively. Kinetics of thermal inactivation and half-life times for this thermostable enzyme at 65 and 70 degrees C were 50 and 9 h, respectively, and the K-m and V-max values were 2.96 mM and 6.62 mu mol/min per mg. Metal cations and EDTA could not activate this thermostable enzyme, and some divalent metal ions, namely, Fe2+, Zn2+, Cu2+, Pb2+, and Sn2+, inhibited its activity. Thiol reagents had no effect on the enzyme activity, and sulfhydryl group blocking reagents inactivated the enzyme. This enzyme possessed a high level of transgalactosylation activity in hydrolysis of lactose in milk. The results suggest that this recombinant thermostable enzyme may be suitable for both the hydrolysis of lactose and the production of galacto-oligosaccharides in milk processing.