【作者】 刘文山；

【导师】 闫云君；

【作者基本信息】 华中科技大学 ， 生化与分子生物学， 2010， 博士

【摘要】 随着石化能源的日益枯竭,生物柴油作为一种清洁环保、性能优良的绿色可替代能源受到广泛的关注。脂肪酶法制备生物柴油技术由于具备反应条件温和、无污染物排放等优点,发展前景广阔。但脂肪酶价格高、在有机溶剂中易失活等缺点,限制了酶法制备生物柴油工艺的推广和工业化。开发脂肪酶表面展示技术则能较好解决上述问题。表面展示脂肪酶除可回收重复利用、增强酶的稳定性外,还能省去传统固定化酶复杂而耗时的分离纯化工作,节约成本；而且酶被固定在细胞表面,底物易于进入,产物易于脱离。此外,表面展示还可作为脂肪酶定向进化的高通量筛选手段,为后续脂肪酶的分子改造提供技术平台。本论文主要研究工作和结果摘要如下：(1)利用生物信息学方法预测了解脂耶氏酵母脂肪酶Lip2的疏水性、柔性、二级结构、三级结构等结构特征,确定了催化三联体S162、D230、H289及阴离子氧洞T88和L163,明晰了活性中心与其N端和C端的位置关系。(2)首次以C端锚定的Cwp2作为锚定蛋白展示了Lip2。利用免疫荧光分析及平板活性检测确定Lip2成功展示在酿酒酵母INVScl细胞表面。尽管添加了不同连接序列,但所得展示脂肪酶活性仍较低,连接序列(G4S)3可在一定程度上提高脂肪酶的活性,使其酶活从4.3 U/g干细胞提高为7.6 U/g干细胞,但仍无水解橄榄油活性,结合其三维结构预测结果,我们推测Cwp2可能在一定程度上影响了脂肪酶活性中心的构象。展示脂肪酶最适温度和最适pH分别为40°C、pH 8.0；Ca2＋离子对其酶活有明显促进作用,表面活性剂Triton X-100与Tween 80对其酶活基本没有影响,其DMSO稳定性较好。此外,展示的Lip2还表现出比游离态Lip2更好的温度稳定性。(3)以仅469个氨基酸残基的Flo1片段作为锚定蛋白,并在其与Lip2之间加入(G4S)3链接序列,成功地展示了Lip2。免疫荧光分析和平板活性检测均确认了Lip2展示在酿酒酵母细胞表面,其水解pNPC活力最高达65.2 U/g干细胞,高于以Cwp2作为锚定蛋白的展示酶活,但仍无水解橄榄油活性。其最适温度和最适pH分别为40°C、pH 8.0；相对于以Cwp2为锚定蛋白展示的Lip2,而SDS以及DMSO稳定性有所下降。上述结果说明N端锚定的Flo1片段对表面展示Lip2的活性抑制相对较小。(4)首次利用a凝集素小亚基Aga2作为锚定蛋白展示了Lip2。利用免疫荧光检测确定Lip2成功展示在酿酒酵母细胞表面,利用平板检测证实展示Lip2具备水解橄榄油活性。表面展示Lip2最高水解pNPC酶活可达634.9U/g干细胞,远高于目前已报道的酵母表面展示脂肪酶的酶活。其温度稳定性比以Cwp2和Flo1片段作为锚定蛋白展示的Lip2为高,尤其具备良好的甲醇、乙醇耐受性,20%的甲醇、乙醇处理0.5 h后,酶活分别为初始酶活的109.4%和98.5%,远高于游离态Lip2,更适用于生物柴油的制备。(5)通过比较三种不同的锚定蛋白对表面展示Lip2活性的影响,初步了解了不同锚定蛋白可能对脂肪酶结构造成的影响,从而粗略总结出一般脂肪酶表面展示时锚定蛋白的选择原则,即先对脂肪酶进行结构预测了解其三维结构特征,然后选择锚定结构域远离脂肪酶活性中心的细胞壁蛋白作为锚定蛋白。遵循该原则对解脂耶氏酵母脂肪酶Lip7、Lip8以及黑曲霉脂肪酶ANL进行了表面展示研究。结果表明,展示的Lip7、Lip8表现出较高活力,分别为282.9 U/g干细胞、121.3 U/g干细胞,且温度稳定性较高,50°C温浴5h后残余酶活仍保持在初始酶活的90%以上；展示的ANL酶活为43.8 U/g干细胞,也同样具备较好的温度稳定性,50°C温浴4h仍保持95%以上的残余酶活。更多还原

【Abstract】 With the gradual depletion of fossil oil, biodiesel, a renewable, non-toxic and biodegradable fuel, has gained more attentions in recent years for its ability to replace fossil fuels. The use of lipase instead of acid or alkaline as catalyst for biodiesel production has great market potentiality due to its environment-friendly property and mild reaction conditions. However, lipases have some disadvantages, such as high price and easily being inactivated in organic solvent, which restrict industrialization of this technology. The above problem could be solved by surface display of lipase, because surface display of lipases as a whole-cell catalyst can be recycled and the stability of lipase can be greatly improved in comparison with traditional immobilization of lipases. Compared with conventional immobilized enzymes, the whole-cell catalysts have unique advantages, such as a simpler product purification and a more cost-effective downstream processing. Furthermore, because the lipases are immobilized on the surface of cells, the substrates can be simply access to the enzyme and the products can be easily released. Additionally, surface display of lipase can also serve as an efficient high-throughput screening method for protein engineering of lipase. The main results of this study were summarized as follows:1. Through bioinformatic analysis, we have learnt many characteristics of Yarrowia lipolytica lipase Lip2, such as hydrophobicity, flexibility, secendary structure and tertiary structure. Its catalytic triad consists of S162, D230 and H289. T88 and L163 form the oxyanion hole. Its activity centre is close to the C-terminal.2. Lip2 from Y. lipolytica was displayed on the cell surface of Saccharomyces cerevisiae using Cwp2 as an anchor protein for the first time. Successful display of the lipase on the cell surface was confirmed by immunofluorescence microscopy and halo assay. The length of linker sequences was further examined to confirm whether the correct conformation of Lip2 was maintained. The results showed that the displayed Lip2 exhibited the highest activity at 7.6 U/g-dry cell when using (G4S)3 sequence as the linker, with an optimal temperature and pH at 40℃and pH 8.0. The displayed lipase did not lose any activity after being treated with 0.1% Triton X-100 and 0.1% Tween 80 for 30 min, and it retained 92% of its original activity after incubation in 10% DMSO for 30 min. It also exhibited better thermostability than free-form Lip2 as reported previously.3. A Flol fragment consisting of 469 amino acid residues was used as an anchor protein for surface display of Lip2 in S. cerevisiae. Successful surface display of Lip2 was also confirmed by immunofluorescence microscopy and halo assay. The results showed that the highest activity of displayed Lip2 was 65.2 U/g-dry cell, much higher than that of using Cwp2 as anchor protein. Compared with the displayed Lip2 using Cwp2 as an anchor protein, the stability against SDS and DMSO was slightly decreased. The optimal temperature and pH, as well as the thermostability were consistent with that of using Cwp2 as anchor protein.4. It is also for the first time that the a-agglutinin was used as an anchor protein for surface display of Lip2. From immunofluorescence microscopy and halo assay was successful surface display of Lip2 in S. cerevisiae confirmed. The activity of displayed Lip2 reached 634.9 U/g-dry cell, much higher than those of using Cwp2 and Flol fragment as anchor proteins. The optimal temperature and pH was 40℃and pH 8.0. Compared with the displayed Lip2 using Cwp2 and Flol fragment as anchor proteins, the thermostability and the stability against organic solvent were much improved. Especially, the displayed Lip2 remained 109.4% and 98.5% of its original acitivity after being treated with 20% methanol and ethanol, much better than free-form Lip2, which suggests that the displayed Lip2 will be more suitable for biodiesel production.5. By comparing the results of surface display of Lip2 using three different anchor proteins, we have preliminarily learned the influence of different anchor proteins on the conformation of displayed Lip2. Based on this, we bring forward a rough principle on how to select a suitable anchor protein for surface display of a target lipase. It says that the structure feature of the target protein should first be assayed through bioinformatics and then the cell wall protein with its anchor domain far away from the activity centre of the target protein should be chosen as the anchor protein. By following this principle, we successfully displayed Y. lipolytica lipases Lip7, Lip8 and Aspergillus niger lipase (ANL) on the surface of S. cerevisiae. The displayed Lip7 and Lip8 showed high activities, which reached 282.9 U/g-dry cell and 121.3 U/g-dry cell, respectively. The thermostability of displayed Lip7 and Lip8 were much better than that of the displayed Lip2. Each displayed lipase retained above 90% of its original activity after incubation at 50℃for 5 h. However, the surface displayed ANL exhibited a relatively low activity of 43.8 U/g-dry cell. It could also remain more than 95% of its original activity after incubation 50℃for 4 h.更多还原