A Novel Thermostable β-Galactosidase from Geobacillus kaustophilus HTA42  

作　　者：YU Shanshan YIN Hongbing WANG Xinying FENG Li XU Chunchun LI Jing HAN Hongxiang LIU Shuying 

机构地区：[1]Jilin Ginseng Academy, Changchun University of Chinese Medicine Changehun 130117, P. R. China [2]Affiliated Hospital, Changchun University of Chinese Medicine Changehun 130117, P. R. China [3]Jilin Agricultural University, Changchun 130118, P. R. China [4]Changchun Center of Mass Spectrometry, Changehun Institute of Applied Chemistry, Chinese Academic of Science, Changchun 130022, P. R. China

出　　处：《Chemical Research in Chinese Universities》2014年第5期778-784,共7页高等学校化学研究（英文版）

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.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：Geobacillus kaustophilus HTA426 Β-GALACTOSIDASE Thermostability Glycoside hydrolase 

分 类 号：Q78[生物学—分子生物学] Q503