摘要
脂肪酶(Lipase,EC 3.1.1.3)即三酰甘油酯酰基水解酶,广泛存在于各种动植物和微生物中,是一类具有多种催化能力的酶,可以催化三酰甘油酯及其他一些水不溶性酯类的水解、酯化、醇解、酯交换以及酯类的逆向合成反应等。广泛应用于油脂加工、食品、纺织、医药、日化、皮革脱脂、洗涤以及生物传感器等行业,受到广泛的关注。但目前脂肪酶成本较高,因而开发高效、低成本的脂肪酶成为研究的焦点。本论文以解脂耶氏酵母(Yarrowia lipolytica)为出发菌株,根据NCBI中报道的脂肪酶家族基因序列,扩增其脂肪酶基因,并首次实现脂肪酶基因lip1在巴斯德毕赤酵母GS115(Pichia pastoris)中的功能性表达。具体工作和研究结果如下:
1.根据GeneBank中已经提交的Y. lipolytica脂肪酶家族基因序列,运用生物信息学设计引物,以Y. lipolytica基因组DNA为模板,扩增脂肪酶基因lip1-lip8,其中除lip2、lip7、lip8之外的其它脂肪酶基因的表达,到目前为止均未见文献报道。本论文首次实现了脂肪酶基因lip1在P. pastoris GS115中的功能性表达,丰富了脂肪酶基因资源。
2.由于P. pastoris表达系统存在密码子偏好性,而脂肪酶基因lip1中含有P. pastoris低频率使用密码子,因而运用重叠延伸PCR(Overlap extension PCR)技术对Lip1的8个氨基酸位点进行优化,改造成为P. pastoris偏爱性密码子,实现了Lip1的高效表达。根据生物信息学预测,Lip1不含有信号肽,是一种细胞内表达脂肪酶。因此,本论文构建了含有GAP强启动子的组成型表达载体pGAP9K,使Lip1实现胞外分泌表达,为进一步研究该脂肪酶的性质奠定技术基础。
3.将原始脂肪酶基因lip1以及经密码子优化后的基因Mlip1分别克隆至诱导型分泌载体pPIC9K和新构建的组成型分泌载体pGAP9K上,构建了4个表达质粒:pPIC9K-lip1、pPIC9K-Mlip1、pGAP9K-lip1和pGAP9K-Mlip1,表达载体电转至P.pastoris GS115中进行功能性表达,发酵测酶活,结果表明:密码子优化后基因表达水平相对于出发基因有较大提高,且优化后组成型表达水解酶活达8.07 U/mL,明显优于诱导型表达。同时,通过SDS-PAGE电泳验证脂肪酶分子量大小,在53 kDa处有一特异条带。对酵母工程菌GS115-pGAP9K-Mlip1发酵条件进行初步优化,确定其最优发酵培养基,优化结果为最适YP(酵母膏+蛋白胨)含量为3(YP),最适碳源为油酸。
4.对Lip1酶学性质进行了初步研究,结果表明:Lip1对短链的对硝基苯酚酯有较强的水解活性,其最适底物为pNPB(C4),最适pH为8.5,最适温度为45℃。在60℃的恒温水浴条件下温浴2 h后残余酶活为35.88%;金属离子对脂肪酶Lip1活力影响实验表明,Mg~(2+)、Fe~(2+)和Zn~(2+)对其活力有一定的激活作用,而Cu~(2+)、Ba~(2+)、Mn~(2+)和EDTA则抑制其水解酶活力。此外,运用生物信息学预测和分析脂肪酶Lip1的一级结构,二级结构和三级结构,为下一步对Lip1进行蛋白质纯化及结构生物学研究奠定了技术基础。
Triacylglycerol hydrolases or lipases (EC3.1.1.3.), exists widely in various plant,animal and microorganisms, can catalyse hydrolysis, esterification, alcoholysis, transesterification and ester of reverse synthesis reactions, etc. Widely used in oil processing, foodstuff, textile, pharmaceutical, cosmetic, leather degreasing, washing and biological sensors, etc, has attracted a lot of attention. However, the catalyst---lipases have high price owing to its diffcult derivation. Therefore, the development of high efficiently expression lipase gene resources have become one of the research hot spots. In this paper, by following the information in NCBI, the lipase gene family lip1 - lip8 of Yarrowia lipolytica were amplified, and lip1 was successfully realized functional expression in Pichia pastoris for the first time. The main work and results were as follows:
1. According to the lipase gene sequences of Y. lipolytica submitted to GeneBank, by applying bioinformatics to design primers, using Y. lipolytica genomic DNA as template, lipase genes lip1-lip8 were amplified. Axcept lip2、lip7 and lip8, there were no report on expression of other lipases so far. In this paper, we first realized functional expression of lip1 in P. pastoris GS115, which greatly enriched lipase gene resources.
2. Because P. pastoris expression system exits codon bias, while lip1 contains low frequency usage codons, the eight amino acid sites of lip1 were optimized using overlap extensions PCR technology for the first time. The optimized gene Mlip1 was realized efficiently expression in P. pastoris. According to the bioinformatics analysis, lip1 contained no signal peptide, suggesting it is an intracellular lipase. Thus, in this paper we constructed constitutive expression vector pGAP9K with a strong promoter GAP so as to realize exocellular expression of lip1, which layed a solid foundation for further research of this lipase.
3. Four expression vectors: pPIC9K-lip1、pPIC9K-Mlip1、pGAP9K-lip1 and pGAP9K-Mlip1 were built, and then were electrotransformated into P. pastoris GS115 to implement functional expression of lip1. High copy transformation recombinants were gained through lipase activity functional verification and G418 resistances screening. After shake flask fermentation, PNP ester was used as substrate for assay the activities of the supernatant. The results showed that the optimized gene had a higher expression level than the original one, and activity of the constitutive expression was superior to that of the inducible expression. Fermentation conditions of engineering strain GS115-pGAP9K-Mlip1 was preliminarily optimized the optimal YP (yeast extract + tryptone) content is 3 (YP), and the optimal carbon source is oleic acid.
4. Preliminary analysis showed that the optimum substrate of Lip1 was p-nitrophenyl butyrate (C4), the optimal temperature and pH was 45℃and 8.5, respectively. After warm bath for 2 h at a constant temperature of 60℃, the residual activity of the lipase was 35.88%. Metal ion had significant influence on the lipase. Mg~(2+), Fe~(2+) and Zn~(2+) could stimulate lipase, while Cu~(2+)、Ba~(2+)、Mn~(2+) and EDTA inhibit its hydrolysis activity. In addition, the primary, secondary and tertiary structures of Lip1 were predicted and analyzed through bioinformatics, which provided a theoretical basis for the following study on protein purification and structure biology of Lip1.
引文
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