脂肪氧合酶(LOX)对小麦面粉具有强筋和漂白双重作用，可替代化学合成的面粉改良剂。前期研究中，通过定向进化鱼腥藻脂肪氧合酶（Ana-LOX）获得热稳定性和催化活性同时提高的突变体，但其催化机制尚不明确。本项目旨在通过制备Ana-LOX晶体及其与底物复合物的晶体，利用X-射线衍射法解析Ana-LOX晶体结构，研究其底物进入和产物释放通道的特点，确定酶与底物结合模式，阐明Ana-LOX催化机制，填补了国内外Ana-LOX结构信息和催化机制研究的空白。在此基础上，采用理性和半理性设计Ana-LOX催化活性改造策略，通过分子对接、分子动力学模拟等计算机辅助方法，探究酶活性中心氨基酸残基与底物之间的相互作用，确定关键催化氨基酸位点，并利用定点突变和组合活性位点饱和突变技术，获得催化活性显著提高的突变体，进一步揭示影响其催化效率的分子机制，为LOX的分子改造和酶学理论提供科学依据。

Lipoxygenase (LOX) plays a dual role as both bleaching and gluten strengthening, so that it could replace chemical flour improver. In previous work, we selected mutants with increased thermos-stability and catalytic activity through directed evolution of lipoxygenase from Anabaena sp. PCC 7120 (Ana-LOX), however, the catalytic mechanism is not clear. The projects aims to study characteristics of substrate entrance and product release, determine the binding mode of enzyme and substrate by preparation crystals of Ana-LOX and its enzyme-substrate complex, and analyze crystal structure through X-ray diffraction., and finally elucidate the catalytic mechanism of Ana-LOX and fill in the blanks of its structural information and catalytic mechanism at home and abroad. Moreover, we use molecular docking and molecular dynamics simulation methods to explore the interaction between amino acid residues around active site and substrate and analyze related amino acid residues of active site in catalytic channel. To verify the function of target sites, we employ rational and semi-rational strategy, especially site-directed and combinatorial active-site saturation test (CAST) to obtain mutants which catalytic ability significantly improved. In the meantime, we analyze structure activity relationship based on variation of mutants. All these results can be used to investigate the molecular mechanism which affect Ana-LOX catalytic efficiency, and provide scientific theory basis of LOX molecular engineering and enzymology theory.