title: Overexpression and characterization of a glucose-tolerant β-glucosidase from T. aotearoense with high specific activity for cellobiose.
abstract: Thermoanaerobacterium aotearoense P8G3#4 produced β-glucosidase (BGL) intracellularly when grown in liquid culture on cellobiose. The gene bgl, encoding β-glucosidase, was cloned and sequenced. Analysis revealed that the bgl contained an open reading frame of 1314 bp encoding a protein of 446 amino acid residues, and the product belonged to the glycoside hydrolase family 1 with the canonical glycoside hydrolase family 1 (GH1) (β/α)8 TIM barrel fold. Expression of pET-bgl together with a chaperone gene cloned in vector pGro7 in Escherichia coli dramatically enhanced the crude enzyme activity to a specific activity of 256.3 U/mg wet cells, which resulted in a 9.2-fold increase of that obtained from the expression without any chaperones. The purified BGL exhibited relatively high thermostability and pH stability with its highest activity at 60 °C and pH 6.0. In addition, the activities of BGL were remarkably stimulated by the addition of 5 mM Na(+) or K(+). The enzyme showed strong ability to hydrolyze cellobiose with a K m and V max of 25.45 mM and 740.5 U/mg, respectively. The BGL was activated by glucose at concentration varying from 50 to 250 mM and tolerant to glucose inhibition with a K i of 800 mM glucose. The supplement of the purified BGL to the sugarcane bagasse hydrolysis mixture containing a commercial cellulase resulted in about 20 % enhancement of the released reducing sugars. These properties of the purified BGL should have important practical implication in its potential applications for better industrial production of glucose or bioethanol started from lignocellulosic biomass.
authors.affiliation: Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, 510006, China.
authors.forename: Fang
authors.initials: F
authors.lastname: Yang
authors.affiliation: Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, 510006, China.|School of Energy and Environment, City University of Hong, Tat Chee Avenue, Kowloon, 999077, Hong Kong, China.
authors.forename: Xiaofeng
authors.initials: X
authors.lastname: Yang
authors.affiliation: Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, 510006, China.
authors.forename: Zhe
authors.initials: Z
authors.lastname: Li
authors.affiliation: School of Applied Sciences, The University of Huddersfield, Queensgate, Huddersfield, HD1 3DH, UK.
authors.forename: Chenyu
authors.initials: C
authors.lastname: Du
authors.affiliation: Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, 510006, China.
authors.forename: Jufang
authors.initials: J
authors.lastname: Wang
authors.affiliation: Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, 510006, China. shuangli@scut.edu.cn.|Guangzhou Higher Education Mega Center, Panyu District, Guangzhou, 510006, People's Republic of China. shuangli@scut.edu.cn.
authors.forename: Shuang
authors.initials: S
authors.lastname: Li
mesh_terms: D002475:Cellobiose / Q000378:metabolism*; D003001:Cloning, Molecular; D020536:Enzyme Activators / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005947:Glucose / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011188:Potassium / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000378:metabolism; D013696:Temperature; D046950:Thermoanaerobacterium / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D001617:beta-Glucosidase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D002475:Cellobiose; D012964:Sodium; D001617:beta-Glucosidase; D005947:Glucose; D011188:Potassium
keywords: Cellobiose degradation; Chaperones; Glucose tolerance; Thermoanaerobacterium aotearoense P8G3#4; β-Glucosidase