扩展青霉脂肪酶基因克隆、密码子优化及表达

Cloning, codon optimization and expression of mature lipase gene Penicillum expansum

摘要:

摘要：【目的】克隆扩展青霉脂肪酶基因，实现具强催化活性的脂肪酶的异源高效表达。【方法】利用 RT-PCR 扩增扩展青霉CICC 40356脂肪酶（PEL）cDNA序列，利用重叠延伸 PCR（Over-lap extension PCR）技术对 PEL 的 10 个稀有氨基酸密码子和表达载体 pPIC9K α 信号肽的 9 个氨基酸密码子进行了优化，获得了改造过的脂肪酶基因 PELM 和表达载体pPIC9KM。并构建了带有脂肪酶自身信号肽的 pPIC9K-PEL1、pPIC9KM-PELM1、pPIC3.5K-PEL1、pPIC3.5K-PELM1 和不带有脂肪酶自身信号肽的 pPIC9K-PEL2、pPIC9KM-PELM2 六个重组质粒。利用对硝基苯酚棕榈酸酯（pNPP）为底物检测工程菌脂肪酶的酶活。在此基础上，对工程菌的酶学性质进行了研究。【结果】扩展青霉脂肪酶基因cDNA序列分析结果表明该序列与已报道PEL cDNA序列仅相差3 个碱基，同源性高达99 %。6个重组工程菌在甲醇诱导下，均表现出pNPP水解活性，28℃诱导100h时酶活达到最高，发酵上清的酶活分别为 3.65 U/mL、30.49 U/mL、90.85 U/mL、212.05 U/mL、15.29 U/mL、76.32 U/mL。SDS-PAGE 结果表明重组脂肪酶分子量均约28 kDa。酶学性质研究表明，重组脂肪酶PELM最适温度为35℃，最适pH为9.5，在 pH 7.0 ?10.0 范围内该脂肪酶均较稳定，Ca2+ 和 Mg2+ 对其有激活作用，Fe2+、Zn2+、Cu2+ 则有抑制作用，EDTA能使之快速失活。以不同碳链长度的对硝基苯酚酯为底物检测其底物特异性，结果显示其对中链酯（C8-C12）有较强的水解能力，最适底物为为C8 的 pNP 酯。【结论】密码子优化后的扩展青霉脂肪酶基因在毕赤酵母中获得理想的表达，其酶活力比未优化的野生脂肪酶的提高了2.3-2.5倍，表明定点突变对其基因本身更改特有稀有密码子是实现 PEL功能蛋白的异源高效表达的有效策略之一。

关键词:关键词：扩展青霉脂肪酶;基因克隆;密码子优化;表达

Abstract:

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 α-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 ℃ 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.

Key words:Keywords: Penicillium expansum lipase; gene cloning; codon optimization;