脂肪酸ω-羟化酶催化特异性研究

项目名称： 脂肪酸ω-羟化酶催化特异性研究

项目编号： No.21202179

项目类型： 青年科学基金项目

立项/批准年度： 2013

项目学科： 有机化学

项目作者： 曹玉锦

作者单位： 中国科学院青岛生物能源与过程研究所

项目金额： 25万元

中文摘要： ω-羟基脂肪酸是合成生物可降解聚合材料的重要单体，由于其羟基位于不活泼的碳链端，传统的化学工艺很难特异性的合成此类化合物，利用微生物转化长链脂肪酸制备ω-羟基脂肪酸已成为国际上的研究热点。催化ω-羟基脂肪酸生成的酶为细胞色素P450单加氧酶，该酶已经在多种微生物中得到克隆。这些不同来源的脂肪酸ω-羟化酶对不同链长、不同饱和度的脂肪酸具有不同的催化活性，但是普遍存在效率不高、产物特异性不好的问题。本研究拟对不同来源的脂肪酸ω-羟化酶基因进行克隆、异源表达和功能鉴定，并通过生物信息学的手段分析决定其底物链长特异性和产物羟基位置特异性的关键位点，进而采用定点突变的方法改造决定其催化特异性的活性结构域，以期获得改性的脂肪酸ω-羟化酶，并构建异源转化脂肪酸生成ω-羟基脂肪酸的体系。本研究有望为微生物合成ω-羟基脂肪酸打下基础。

中文关键词： 羟基脂肪酸；ω-羟化酶；催化特异性；定点突变；

英文摘要： ω-Hydroxy fatty acids are important precursors for biodegradable polymer materials. Due to the hydroxyl group in the inactive terminal of the carbon chain, it is difficult to specifically synthesize these compounds using traditional chemical processes. Therefore, microbial transformation of long-chain fatty acids to prepare ω-hydroxy fatty acids has become a research hotspot during the last few years. The enzymes catalyzing the biosynthesis of ω-hydroxy fatty acid are cytochrome P450 monooxygenases, which have been identified from many different microorganisms. These ω-hydroxylases show diverse catalytic activity towards fatty acids of different chain lengths and different saturation levels. However, their catalytic efficiency is not high enough and the product specificity is still very poor. In this study, fatty acid ω-hydroxylases of different origins will be cloned, heterologously expressed and enzymatically analyzed. Special attentions will be paid on the chain-length specificity of the substrates and the hydroxyl group position of the products. Site-directed mutagenesis strategy to modify the active domain of ω-hydroxylase will also be employed to enhance its catalytic activity. Finally, a heterologous system will be constructed to convert fatty acids to their ω-hydroxyl counterparts. This study is expected

英文关键词： hydroxy fatty acid；ω-hydroxylase；catalytic specificity；site-specific mutagenesis；