Zhang Zhengping, Yang Jiangke, Xu Li, Liu Yun, Yan Yunjun
Key Laboratory of Molecular Bio-physics, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China.
Wei Sheng Wu Xue Bao. 2010 Feb;50(2):228-35.
To clone Penicillum expansum CICC 40356 lipase (PEL) gene cDNA and to over-express active lipase in Pichia pastoris GS115.
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.
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+.
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.
克隆扩展青霉CICC 40356脂肪酶（PEL）基因cDNA，并在毕赤酵母GS115中实现活性脂肪酶的过量表达。
根据已报道的青霉脂肪酶基因的核苷酸序列设计引物。通过PCR对PEL的10个稀有密码子和α信号肽的9个稀有密码子进行优化。将天然的和密码子优化后的PEL基因分别克隆到pPIC9K、pPIC9KM和pPIC3.5K载体中。同时测定重组脂肪酶的性质。
核苷酸序列分析表明，PEL cDNA包含一个858 bp的开放阅读框。推导的氨基酸序列对应于286个氨基酸残基，包括一个20个氨基酸残基的潜在信号肽序列。密码子优化后PEL的水解活性增强。其最适温度和pH分别为35℃和9.5。它偏好中链酯（C8 - C12），对C8酰基链表现出最大活性。它能被Ca2+和Mg2+刺激，但被EDTA强烈抑制，被Fe2+、Zn2+和Cu2+轻微抑制。
与野生型相比，PEL的活性提高了2.3 - 2.5倍，表明密码子优化是在毕赤酵母系统中产生活性PEL的有效措施。
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