[Cloning, codon optimization and expression of mature lipase gene Penicillium expansum].
OBJECTIVE
To clone Penicillum expansum CICC 40356 lipase (PEL) gene cDNA and to over-express active lipase in Pichia pastoris GS115.


METHODS
Primers were designed according to the nucleotide sequence of reported lipase gene from Penicillum. Ten rare codons of PEL and nine of the alpha-signal peptide were optimized by PCR. The native and codon-optimized PEL genes were respectively cloned into pPIC9K, pPIC9KM, and pPIC3.5K vectors. The properties of recombinant lipase were also determined.


RESULTS
Nucleotide sequence analysis revealed that the PEL cDNA contained an 858 bp open reading frame. The deduced amino acid sequence corresponds to 286 amino acid residues, including a potential signal peptide sequence of 20 amino acid residues. The hydrolysis activity of PEL was enhanced with codon-optimization. Its optimal temperature and pH were 35 degrees C and 9.5. It favored medium chain esters (C8-C12) and showed the maximal activity toward C8 acyl-chains. It could be stimulated by Ca2+ and Mg2+, but strongly inhibited by EDTA and slightly repressed by Fe2+, Zn2+ and Cu2+.


CONCLUSIONS
The activity of PEL was improved 2.3-2.5 folds compared to that of the wild type, suggesting that the codon optimization is an efficient measure to produce the active PEL in P. pastoris system.
Zhang
Zhengping
Z
Key Laboratory of Molecular Bio-physics, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China. zhengping_zhang@yahoo.cn
Yang
Jiangke
J
Xu
Li
L
Liu
Yun
Y
Yan
Yunjun
Y
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003062:Codon; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D008049:Lipase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010407:Penicillium / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
D003062:Codon; D005656:Fungal Proteins; D008049:Lipase