title: Characterization and application of an acidophilic and thermostable β-glucosidase from Thermofilum pendens.
abstract: The gene encoding a β-glucosidase from the archaeon Thermofilum pendens (Tpbgl) was cloned and expressed in Escherichia coli. The purified recombinant enzyme had a molecular mass of 77.8 kDa and released glucose or mannose from p-nitrophenyl-β-d-glucopyranoside (pNPG), cellobiose, mannobiose, and genistin. Peak Tpbgl activity was detected at 90°C, and 50% activity remained after incubation for 60 min at 95°C. The optimal pH for pNPG hydrolysis was 3.5. When the enzyme was incubated with pNPG in the presence of ethanol and propanol, the glucose moiety was transferred to acceptor alcohols. Tpbgl is the archaeal β-glucosidase from glucoside hydrolase family 3 and found to be most heat stable under extremely acidic conditions (pH 3.5). The kinetic parameters revealed that Tpbgl had the highest catalytic efficiency toward pNPG (kcat/Km = 3.05) with strong substrate affinity for such natural substrates as cellobiose (Km = 0.149) and mannobiose (Km = 0.147). Genistin solubilized in 10-40% DMSO was hydrolyzed to genistein with nearly 99% conversion, indicating that high concentrations of the water-insoluble isoflavone glycoside can be treated by the enzyme. Our results indicate that Tpbgl has great potential in cellulose saccharification and the glucoside hydrolysis of natural compounds.
authors.affiliation: Key Laboratory of Agricultural Products Processing in Jilin Provincial Universities, Changchun University, Satellite Road 6543, Changchun 130022, People's Republic of China. drlidan@yahoo.com
authors.forename: Dan
authors.initials: D
authors.lastname: Li
authors.forename: Xiaolei
authors.initials: X
authors.lastname: Li
authors.forename: Wei
authors.initials: W
authors.lastname: Dang
authors.forename: Phuong Lan
authors.initials: PL
authors.lastname: Tran
authors.forename: Sung-Hoon
authors.initials: SH
authors.lastname: Park
authors.forename: Byung-Chul
authors.initials: BC
authors.lastname: Oh
authors.forename: Wan-Soo
authors.initials: WS
authors.lastname: Hong
authors.forename: Jin-Sil
authors.initials: JS
authors.lastname: Lee
authors.forename: Kwan-Hwa
authors.initials: KH
authors.lastname: Park
mesh_terms: D000595:Amino Acid Sequence; D002475:Cellobiose / Q000378:metabolism; D004795:Enzyme Stability; D005960:Glucosides / Q000378:metabolism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007529:Isoflavones / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D041561:Thermofilaceae / Q000201:enzymology*; D001617:beta-Glucosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D005960:Glucosides; D007529:Isoflavones; D002475:Cellobiose; C040641:genistin; C025193:4-nitrophenyl beta-D-glucoside; D001617:beta-Glucosidase