Characterization of a Thermophilic Monosaccharide Stimulated β-Giucosidase from Acidothermus cellulolyticus  

作　　者：LI Yuwei BU Mingwei CHEN Peng LI Xiaohong CHEN Changwu GAO Gui FENG Yan HAN Weiwei ZHANG Zuoming 

机构地区：[1]Key Laboratory for Molecular Enzymology & Engineering, Ministry of Education, School of Life Science, Jilin University, Changehun 130012, P. R. China [2]State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012, P. R. China [3]The Second Hospital ofdilin University, Changchun 130022, P R. China [4]College of Food Engineering, Jilin Engineering Normal University, Changchun 130052, P. R. China

出　　处：《Chemical Research in Chinese Universities》2018年第2期212-220,共9页高等学校化学研究（英文版）

摘　　要：The gene（ABK51908） from Acidothermus cellulolyncus encodes a mature protein of 484 residues with a calculated molecular mass of 53.0 kDa. Sequence analysis revealed that the protein had 59% identity to the β-glucosidases CAA82733, which belongs to glycoside hydrolase family I（GH1）. We cloned and expressed the gene in Escherichia coli BL21-Gold（DE3）. The recombinant protein（AcBg） had an optimal pH and temperature of 7.0 and 70℃, respectively. The specific activities of AeBg under optimal conditions were 290 and 33 U/rag for p-nitrophenyl-fl-D-glucopyranoside（pNPG） and cellobiose, respectively. AcBg hydrolyzed the oligosaccharide sequentially from the non-reducing end to produce glucose units according to the results of HPLC analysis. AcBg showed high salt tolerance and monosaccharide-stimulation properties. Its activity rose more than 2-fold when 5 mol/L NaC1/KC1 were added. The activity of the β-glucosidase was remarkably enhanced in the presence of 0.2 mol/L D-glucose（increased more than 1.9-fold）, 0.1 mol/L a-methyl-D-glucose（increased more than 1.4-fold） and 1.0 mol/L D-xylose（increased more than 1.9-fold）. The catalysis kinetics and structural changes in various concentra- tions of glucose were determined. The results indicate that glucose reduces substrate affinity and causes conforma- tional changes.The gene（ABK51908） from Acidothermus cellulolyncus encodes a mature protein of 484 residues with a calculated molecular mass of 53.0 kDa. Sequence analysis revealed that the protein had 59% identity to the β-glucosidases CAA82733, which belongs to glycoside hydrolase family I（GH1）. We cloned and expressed the gene in Escherichia coli BL21-Gold（DE3）. The recombinant protein（AcBg） had an optimal pH and temperature of 7.0 and 70℃, respectively. The specific activities of AeBg under optimal conditions were 290 and 33 U/rag for p-nitrophenyl-fl-D-glucopyranoside（pNPG） and cellobiose, respectively. AcBg hydrolyzed the oligosaccharide sequentially from the non-reducing end to produce glucose units according to the results of HPLC analysis. AcBg showed high salt tolerance and monosaccharide-stimulation properties. Its activity rose more than 2-fold when 5 mol/L NaC1/KC1 were added. The activity of the β-glucosidase was remarkably enhanced in the presence of 0.2 mol/L D-glucose（increased more than 1.9-fold）, 0.1 mol/L a-methyl-D-glucose（increased more than 1.4-fold） and 1.0 mol/L D-xylose（increased more than 1.9-fold）. The catalysis kinetics and structural changes in various concentra- tions of glucose were determined. The results indicate that glucose reduces substrate affinity and causes conforma- tional changes.

关 键 词：β-Glueosidase Monosaccharide-stimulation Salt tolerance Thermophilic enzyme 

分 类 号：O657.72[理学—分析化学] TS245.4[理学—化学]