[Cloning, expression and characterization of a novel lipase gene lipB from Aspergillus niger F044].
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.
Yang
Jiangke
J
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
Zhang
Zhengping
Z
Liu
Liyin
L
Yan
Yunjun
Y
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
D001426:Bacterial Proteins; C088618:lipase activator protein, Bacteria