title: [Cloning, expression and characterization of a novel lipase gene lipB from Aspergillus niger F044].
abstract: OBJECTIVE
  We cloned, expressed and characterized a novel lipase gene lipB from Aspergillus niger F044, to facilitate the large scale production and application of that enzyme.
  
  
  METHODS
  We cloned lipB gene and the cDNA sequence by PCR and RT-PCR, and then cloned the open reading frame of lipB into pET28a vector and expressed by isopropyl beta-D-1-thiogalactopyranoside (IPTG) induction. After Ni-agarose purification, the characteristics were determined and the conformation change was checked by circular dichroism methods.
  
  
  RESULTS
  The novel lipase genes cDNA of lipB were cloned from Aspergillus niger F044 (GenBank: FJ536287, FJ536288) and expressed in Escherichia coli. The molecular weight of LipB was about 43 kDa. The optimal substrate of this enzyme is 4-nitrophenyl octanoate (pNPC-C8) with Km = 5.98 mmol/L. The optimal temperature and pH was 50 degrees C and pH 6.0. The enzyme was stable below 40 degrees C. After incubated at 60 degrees C for 1 h, only 18.8% activity remained. After treated by 2 mmol/L Ca2+ for 1 h, the activity improved 2.6-fold.
  
  
  CONCLUSIONS
  Enzymatic characteristics of LipB determined showed this enzyme might have potential in industrial applications.
authors.affiliation: Laboratory of Molecular Biophysics of Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China. jiangke.yang@hotmail.com
authors.forename: Jiangke
authors.initials: J
authors.lastname: Yang
authors.forename: Zhengping
authors.initials: Z
authors.lastname: Zhang
authors.forename: Liyin
authors.initials: L
authors.lastname: Liu
authors.forename: Yunjun
authors.initials: Y
authors.lastname: Yan
mesh_terms: D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000737:chemistry / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D015870:Gene Expression; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010802:Phylogeny; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; C088618:lipase activator protein, Bacteria