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

作　　者：张正平[1] 杨江科[1] 徐莉[1] 刘云[1] 闫云君[1] 

机构地区：[1]分子生物物理教育部重点实验室,华中科技大学生命科学与技术学院,武汉430074

出　　处：《微生物学报》2010年第2期228-235,共8页Acta Microbiologica Sinica

基　　金：国家“863计划”(2006AA020203,2007AA05Z417,2009AA03Z232);武汉市攻关项目(200720422138)~~

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pPIC9Kα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pH7.0-10.0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。[ Objective] To clone Penicillum expansum CICC 40356 lipase （PEL） gene cDNA and to over-express active lipase in Pichia pastoris GSl15. [ Methods] Primers were designed according to the nucleotide sequence of reported lipase gene from Penicillum. Ten rare codons of PEL and nine of the a-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 Cs acyl-chains. It could be stimulated by Ca^2＋ and Mg^2＋ , but strongly inhibited by EDTA and slightly repressed by Fe^2＋ ,Zn^2＋ and Cu^2＋. [ 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.

关 键 词：扩展青霉脂肪酶 基因克隆 密码子优化 表达 

分 类 号：Q786[生物学—分子生物学]