Characterization of a Glucose-, Xylose-, Sucrose-, and d-Galactose-Stimulated β-Glucosidase from the Alkalophilic Bacterium Bacillus halodurans C-125
Characterization of a Glucose-, Xylose-, Sucrose-, and d-Galactose-Stimulated β-Glucosidase from the Alkalophilic Bacterium Bacillus halodurans C-125
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DOI：
10.1007/s00284-010-9766-3
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发表时间：
2011-03
影响因子：
2.6
通讯作者：
Hu Xu;A. Xiong;Wei Zhao;Yong-Sheng Tian;R. Peng;Jian-min Chen;Q. Yao
中科院分区：
文献类型：
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作者：
Hu Xu;A. Xiong;Wei Zhao;Yong-Sheng Tian;R. Peng;Jian-min Chen;Q. Yao
从嗜碱性细菌嗜盐芽孢杆菌sc -125中合成β-葡萄糖苷酶基因Bhbgl，并通过pcr - two-step DNA synthesis （PTDS）方法在大肠杆菌中表达。Bhbgl含有一个1359 bp的开放阅读框（ORF），编码一个453个氨基酸的糖苷水解酶家族1 （GHF1）蛋白，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）证实重组Bhbgl的分子量为52,488 Da。该酶对-硝基苯-β-d-葡萄糖吡喃苷（oNPGlu）具有较高的比活性，表观km值为0.32 mM。以oNPGlu为底物时，Bhbgl的最适pH为~7.0℃，最适温度为50℃。该酶在碱性条件下相对稳定，在pH 9.5、4℃条件下孵育24 h，酶活性仍保持50%。5 mM Zn2+、Fe3+和Cd2+抑制Bhbgl活性，1 mM Mg2+和其他金属离子增强Bhbgl活性。至少四种糖（蔗糖、d-半乳糖、木糖、葡萄糖）在50至800 mM浓度范围内也能刺激酶活性。
The gene (Bhbgl) encoding a β-glucosidase from the alkalophilic bacteriumBacillus haloduransC-125 was synthesized chemically via the PCR-based two-step DNA synthesis (PTDS) method and expressed inEscherichia coli.Bhbglcontained an open reading frame (ORF) of 1359 bp encoding a 453-amino acid protein belonging to glycoside hydrolase family 1 (GHF1), and the deduced molecular mass of recombinant Bhbgl (52,488 Da) was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme exhibited a high specific activity witho-nitrophenyl-β-d-glucopyranoside (oNPGlu) and an apparentKmvalue of 0.32 mM. WithoNPGlu as the substrate, Bhbgl displayed pH and temperature optima of ~7.0 and 50°C, respectively. The enzyme was relatively stable under alkaline conditions and >50% activity was retained after incubation at pH 9.5 for 24 h at 4°C. Recombinant Bhbgl activity was inhibited by 5 mM Zn2+, Fe3+, or Cd2+, but was enhanced by 1 mM Mg2+and other metal ions. Enzyme activity was also stimulated by at least four sugars (sucrose,d-galactose, xylose, glucose) at concentrations ranging from 50 to 800 mM.