鱼腥藻脂肪氧合酶热稳定性提高及其分子机理研究

项目名称： 鱼腥藻脂肪氧合酶热稳定性提高及其分子机理研究

项目编号： No.31470095

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 农业科学

项目作者： 吕凤霞

作者单位： 南京农业大学

项目金额： 30万元

中文摘要： 脂肪氧合酶(LOX)对小麦面粉具有强筋和漂白双重作用，在面粉品质改良中起重要的作用，是一种潜在的替代溴酸钾及过氧化苯甲酰的绿色面粉改良剂。在前期研究中发现重组鱼腥藻脂肪氧合酶(Ana-LOX)在面粉中应用热稳定性还不够理想。本项目在已克隆表达Ana-LOX基因的基础上，旨在通过蛋白质工程技术提高Ana-LOX热稳定性和活性，并阐明其热稳定性机理。首先采用定向进化方法对Ana-LOX热稳定性进行改造，建立Ana-LOX的高通量筛选方法和突变体文库，以及利用分子动力学模拟和B-factor值分析方法等计算机模拟的理性设计对Ana-LOX热稳定性改造，获得热稳定性与酶活同时提高的突变体；其次，通过结合同源建模和分子动力学模拟以及突变体晶体结构和热稳定性的关系分析，阐明提高Ana-LOX热稳定性分子机理，揭示Ana-LOX热稳定性与酶的结构之间的关系，为酶的热稳定性分子改良和酶学理论提供理论依据。

中文关键词： 脂肪氧合酶；定点突变；定向进化；热稳定性；机制

英文摘要： Lipoxygenase (LOX), used in gluten strength and bleaching, plays an important role in improving flour quality, which is as a potential flour additive to substitute potassium bromate and benzoyl peroxide in flour industry. Our previous study found that the thermal stability of the recombinant Anabaena lipoxygenase (Ana-LOX) was not satisfied. As a result, based on the expression of the Ana-LOX genes, the current study was focused on improving the thermal stability and activity of Ana-LOX as well as the mechanism of its thermal stability by protein engineerng technology. We first modified the thermal stability of Ana-LOX by using directed evolution, and also estabilished the high-throughput screeing method and mutant geng library. In order to obtain mutants with advanced thermal stability and activity, we applied rational strategy of computer simulation, such as molecular dynamics simulations and B-factor value analysis. In the meantime, by combining homology modeling and molecular dynamics simulations, as well as mutant protein crystal structure and thermal stability of the relationship analysis, we illustrated molecular mechanism of improving the thermal stability of Ana-LOX. Moreover, we uncovered the relationship between the thermal stability of Ana-LOX and the corresponding enzyme structure. All these results

英文关键词： Lipoxygenase；Site-directed mutagenesis；Directed evolution；Thermal stability；mechanism