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| A Novel Thermostable β-Galactosidase from Geobaciilus kaustophilus HTA42 | |
| 基金项目： | Supported by the Scientific and Technological Poject of Jilin Province of China（Nos.20130101127JC, 20130303101YY）. |
| 摘 要： | A novel thermostable β-galactosidase gene, designated as GkGallA, from the thermophilic bacterium Geobacillus kaustophilus HTA426 was cloned and heterologously overexpressed in Escherichia coli（E, coli）. Based on the sequence analysis, GkGallA belongs to the glycosyl hydrolase family 1 that was the first β-galactosidase of bacterial origins expressed by us in this family. The apparent molecular weight of GkGallA determined by sodium deodecyl sulfate-polyacrylamide gel electrophoresis is 52000. It exhibited the highest activity toward p-nitrophenyl-β-D-galactopyranoside at pH 7.8 and 70℃ and displayed high thermal stability, Divalent cations are prerequisite for the activity of GKGallA, with the highest activity in the presence of Mn2＋. Moreover, the three-dimensional structure of GkGaI1A was modeled to speculate the structure of the catalytic residues and the reac- tion mechanism. The catalytic residues consisting of Glu166 and Glu355 were verified by site-directed mutagenesis. |
| 关 键 词： | 半乳糖苷酶基因 耐高温 聚丙烯酰胺凝胶电泳 大肠杆菌 三维结构 芽孢杆菌属 对硝基苯基 二价阳离子 |
| 收稿时间： | 2014-03-17 |
A Novel Thermostable β-Galactosidase from Geobacillus kaustophilus HTA42 |
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| YU Shanshan,;YIN Hongbing,;WANG Xinying,;FENG Li,;XU Chunchun,;LI Jing,;HAN Hongxiang,;LIU Shuying. A Novel Thermostable β-Galactosidase from Geobacillus kaustophilus HTA42[J]. Chemical Research in Chinese University, 2014, 30(5): 778-784. DOI: 10.1007/s40242-014-4090-2 | |
| Authors: | YU Shanshan, YIN Hongbing, WANG Xinying, FENG Li, XU Chunchun, LI Jing, HAN Hongxiang, LIU Shuying |
| Affiliation: | 1. Jilin Ginseng Academy, Changchun 130117, P. R. China; 2. Affiliated Hospital, Changchun University of Chinese Medicine, Changchun 130117, P. R. China; 3. Jilin Agricultural University, Changchun 130118, P. R. China; 4. Changchun Center of Mass Spectrometry, Changchun Institute of Applied Chemistry, Chinese Academic of Science, Changchun 130022, P. R. China |
| Abstract: | A novel thermostable β-galactosidase gene, designated as GkGal1A, from the thermophilic bacterium Geobacillus kaustophilus HTA426 was cloned and heterologously overexpressed in Escherichia coli(E. coli). Based on the sequence analysis, GkGal1A belongs to the glycosyl hydrolase family 1 that was the first β-galactosidase of bacterial origins expressed by us in this family. The apparent molecular weight of GkGal1A determined by sodium deodecyl sulfate-polyacrylamide gel electrophoresis is 52000. It exhibited the highest activity toward p-nitrophenyl-β-D-galactopyranoside at pH 7.8 and 70℃ and displayed high thermal stability. Divalent cations are prerequisite for the activity of GKGal1A, with the highest activity in the presence of Mn2+. Moreover, the three-dimensional structure of GkGal1A was modeled to speculate the structure of the catalytic residues and the reaction mechanism. The catalytic residues consisting of Glu166 and Glu355 were verified by site-directed mutagenesis. |
| Keywords: | Geobacillus kaustophilus HTA426 β-Galactosidase Thermostability Glycoside hydrolase |
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