【作者】 王小行；

【导师】 陈放；

【作者基本信息】 四川大学 ， 植物学， 2006， 博士

【摘要】 生物催化剂是生物柴油转酯反应研究中重要的研究对象，目前新酶种的发现和全细胞催化剂的开发是这个领域中的研究热点。麻疯树(Jatropha curcas)是重要的植物油脂资源，对生物柴油的开发具有重要价值，为此本文研究了麻疯树油脂代谢的重要代谢酶——脂肪酶，通过粉状毕赤酵母表达系统初步开发了该酶的全细胞催化剂，并且研究了该催化剂对麻疯树油脂的催化效率，为麻疯树资源的深层次开发奠定了基础。 本文通过基于生物信息学的基因克隆方法首次克隆了麻疯树脂肪酶(JcLIP3)，通过RACE实验获得了该基因全长序列，基因长1582bp，其最长读框编码356个氨基酸蛋白质，在大肠杆菌中表达实验表明，麻疯树脂酶具有活性0.8U／mL，同时运用生物信息学手段对JcLIP3基因核酸以及蛋白质序列做出了分析，预测了蛋白质的活性中心，并且发现蛋白质在“lid”结构上与毛霉脂酶存在较大差异。 本文首次构建了粉状毕赤酵母的表达系统，确定了转化系统的整合载体结构，感受态制备方案以及电击转化的参数，最终证明该表达系统在20天的连续培养中不会丢失，并且将麻疯树脂酶基因在该酵母中成功地表达，通过RT-PCR、蛋白质电泳以及酶活测定等实验证明了麻疯树脂酶能够在该酵母正确表达出具有活性的蛋白质。 在生物柴油制备研究中，我们比较了本文全细胞催化剂和不同公司的固定化脂肪酶对麻疯树油脂和大豆油脂的作用效果，结果表明麻疯树油脂中油酸甲酯和亚油酸甲酯含量相当且较多，而棕榈酸甲酯和亚麻酸甲酯略少。麻更多还原

【Abstract】 Biocatalysis is the major research field in the transesterification of biodiesel. Discoverying new lipase and developing the whole-cell biocatalysis may be the key problems in biocatalysis of biodiesel. Jatropha curcas is an important lipids resourse of plant, which is valuable in biodiesel. In this paper the lipase of J. curcas was cloned and was expressed in Pichia farinosa to make a novel whole cell biocatalysis. The effect of the biocatalysis was then studied. This will provide a practical performance for the exploitation of Jatropha.The lipase of J. curcas (JcLIP3) was cloned for the first time by gene cloning methods based on bioinformatics. The full-length cDNA was obtained by RACE experiment, which was 1582bp and encoded 356aa protein in its fullest ORF. The gene was expressed in E. coli to characterize the function. The enzyme activity was 0.8U/mL in E. coli. The bioinformatics analysis results showed that JcLIP protein hads the same activity centre as that of Rhizomucor miehei, but a different lid structure from the known structure.In this paper the expression system of Pichia farinosa was first constructed. The structure of integration vector, method of preparation of competent cells and parameters of electroporation was determined. And the genetic stability of this system was tested and results showed that it could be stable in 20 days’ continuous culture. The JcLIP3 gene was then expressed in this system. The results of RT-PCR, protein expression analysis and determination of enzyme更多还原