title: Cloning and characterization of a new β-glucosidase from a metagenomic library of rumen of cattle feeding with Miscanthus sinensis.
abstract: BACKGROUND
  The study on the second generation bio-fuel is a hot area of current research of renewable energy. Among series of key points in this area, the role of β-glucosidase in the degradation of intermediate gluco-oligosaccharides limits the rate of the complete saccharification of lignocellulose.
  
  
  RESULTS
  In this study, a new β-glucosidase gene, unglu135B12, which was isolated from a metagenomic library of rumen of cattle feeding with Miscanthus sinensis by the function-based screening, encodes a 779 amino acid polypeptide that contains a catalytic domain belonging to glycoside hydrolase family 3 (GH3). It was recombinantly expressed, purified and biochemically characterized. The recombinant β-glucosidase, unglu135B12, displayed optimum enzymatic activity at pH 5.0 at 38°C, and showed the highest specific activity of 2.5 × 10(3) U/mg under this optimal condition to p-nitrophenyl-β-D-glucopyranoside (pNPG), and its Km and Vmax values were 0.309 mmol/L and 7.292 μmol/min, respectively. In addition, the presence of Ca2+, K+, Na+ slightly improved β-glucosidase activity of unglu135B12 by about 5%, while about 10~85% loss of β-glucosidase activity was induced by addition of Mn2+, Fe3+, Zn2+, Cu2+. Interestingly, unglu135B12 was activated by glucose at the concentration lower than 40 mM.
  
  
  CONCLUSIONS
  Our findings indicate that unglu135B12 is a new β-glucosidase derived from rumen of cattle, and it might be a potent candidate for saccharification of lignocellulose in industrial application.
authors.forename: Yadan
authors.initials: Y
authors.lastname: Li
authors.forename: Ning
authors.initials: N
authors.lastname: Liu
authors.forename: Hui
authors.initials: H
authors.lastname: Yang
authors.forename: Fei
authors.initials: F
authors.lastname: Zhao
authors.forename: Ye
authors.initials: Y
authors.lastname: Yu
authors.affiliation: College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha 410128, China. tianyun79616@163.com.
authors.forename: Yun
authors.initials: Y
authors.lastname: Tian
authors.forename: Xiangyang
authors.initials: X
authors.lastname: Lu
mesh_terms: D000595:Amino Acid Sequence; D000821:Animal Feed / Q000032:analysis; D000818:Animals; D001419:Bacteria / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D002417:Cattle / Q000378:metabolism / Q000382:microbiology; D003001:Cloning, Molecular; D004795:Enzyme Stability; D015723:Gene Library; D007700:Kinetics; D054892:Metagenome; D008969:Molecular Sequence Data; D010802:Phylogeny; D006109:Poaceae / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012417:Rumen / Q000378:metabolism / Q000382:microbiology*; D001617:beta-Glucosidase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D001617:beta-Glucosidase