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Cloning, Codon Optimization and Expression of Lipase Gene Penicillum Expansum

Author: ZhangZhengPing
Tutor: ZuoYunJun；XuLi
School: Huazhong University of Science and Technology
Course: Biochemistry and Molecular Biology
Keywords: Penicillium expansum Lipase Cloning Codon Optimization Expression
CLC: Q78
Type: Master's thesis
Year: 2009
Downloads: 59
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Abstract


Lipase is an important industrial enzymes commonly found in nature, many lipase gene has been cloned, and the corresponding high-level expression of the engineered bacteria. Lipase from Penicillium expansum is a medium-temperature alkaline lipase, triacylglycerol and can be decomposed under alkaline conditions have a wide range of applications in industry. In this study, Penicillium expansum (Penicillium expansum) CICC 40356 starting fungus, Penicillium expansum lipase gene (PEL) and its open reading frame (ORF) by PCR and RT-PCR cloning and gene low frequency of use password sub-optimized, making it ideal expression in a heterologous host Pichia pastoris GS115, and the enzymatic properties of the recombinant lipase were studied. The main work is as follows. (1), respectively, by the use of PCR and RT-PCR cloning of Penicillium expansum (P. expansum) CICC 40356 lipase gene (PEL) and its open reading frame (ORF). Sequence analysis showed that the gene consists of six exons and five introns, the gene is 1140 bp ORF to 858 bp, encoding 286 amino acid residues, have been reported in GenBank PEL gene sequence homology was 99%. (2) exogenous protein heterologous expression levels of a variety of factors, including codon usage is an important factor. Lipase from Penicillium expansum in order to obtain high-level expression, the use of overlap extension PCR (Over-LAP Extension PCR) technology PEL of 10 amino acid codon and expression vector pPIC9Kα signal peptide of 9 amino acid codon optimized transformation The over the lipase gene PELM and expression vector pPIC9KM. And constructed with a lipase signal peptide pPIC9K-PEL1 pPIC9KM-PELM1 pPIC3.5K-PEL1 pPIC3.5K-PELM1 and not with the lipase signal peptide pPIC9K-PEL2, pPIC9KM-PELM2 six recombinant plasmid. Transformed into the expression vector by Sal I linearized to P.pastoris GS115 expression. After MD, MM, active function tests tablet and G418 selection, the best Pichia pastoris GS115 transformants in shake flask methanol induction fermentation 100h pNPP colorimetric fermentation supernatant enzyme activity to 3.65 U / ml, 30.49 U / ml, 90.85 U / ml, 212.05 U / ml, 15.29 U / ml, 76.32 U / ml. The fermentation supernatant by ammonium sulfate precipitation, dialysis, dialysis product was detected by SDS-PAGE and found that the specific protein band of about 28 kDa. (3) the recombinant Lipase from Penicillium expansum enzymatic properties were studied. The results showed that: Lipase from Penicillium expansum optimum temperature is 35 ℃, the optimum pH was 9.5, the lipase were stable at pH 7.0 -10.0 range; optimal substrate the C 8 strong hydrolysis of medium-chain ester (C 8 -C 12 ); the Ca 2 and the Mg 2 its activation of the Fe 2 , the Zn 2 , Cu 2 inhibition, EDTA can be fast inactivation.

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