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Wei Sheng Wu Xue Bao. 2010 Feb;50(2):228-35.

[Cloning, codon optimization and expression of mature lipase gene Penicillium expansum].

[Article in Chinese]

Author information

1
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

Abstract

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+.

CONCLUSION:

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.

PMID:
20387466
[Indexed for MEDLINE]

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