基于肌红蛋白构筑新型生物催化剂

The development and preparation of environmentally friendly and green new biocatalysts is not only the frontier and hot topics of current biochemical research, but also the core of the new generation industrial technology, which is of great significance to the sustainable economic and social development. Indigo is a dye of a wide range of application. The industrial method for indigo production involves complicated operations, using and producing highly toxic chemicals, and involves messy, potentially hazardous processes, including cyanylation, saponification, and sodium-ammonia reactions that has brought a great deal of threat to the healthy of production personnel and the ecological environment. .In this study, we try to construct biocatalysts based on myoglobin scaffold for indigo production by rational design of the active site of myoglobin mutants at atomic level through a combination of computer simulation and site-directed mutagenesis. We will identify the reaction metabolites, elucidate the reaction mechanism, prepare the crystals and clarify the relationship between the arrangement of amino acids in the active site and the catalytic ability..Relative to the traditional enzymatic system (cytochrome P450), the biocatalytic system employed in this research has several advantages such as: high expression level of protein, good stability, economical catalyst (hydrogen peroxide), simplicity of the system, easy separation and purification of the products, which is expected to become a green alternative method for indigo production and has strong prospects for industrial application.

环保、绿色的新型生物催化剂的开发与制备是当前生物化学研究的学科前沿与热点，是新一代工业技术的核心，对经济社会的可持续发展具有重要意义。靛蓝是一种适用范围广泛的染料，其工业生产方法步骤繁琐，需要大量有机溶剂，污染严重，并且包括氰基化、皂化、钠氨反应等很多高毒步骤，对生产人员的健康以及生态环境都带来了极大的威胁。本研究通过计算机模拟优化及基因定点突变技术相结合的方式，在原子水平上对肌红蛋白（Myoglobin）活性部位结构进行理性优化设计，开发具有优异催化性能的肌红蛋白变体，催化吲哚制取靛蓝，鉴定反应代谢物，阐明反应机制，培养蛋白晶体，探讨肌红蛋白变体活性部位氨基酸排布与其催化性能的关系。相对于传统酶催化系统（细胞色素P450等），本生物催化系统具有蛋白表达量高、稳定性好、催化剂（过氧化氢）价格低廉，体系简单、产品易于分离纯化等优点，有望成为工业成产靛蓝的绿色替代方法，具有很强的工业应用前景。

环保、绿色的新型生物催化剂的开发与制备是当前生物化学研究的学科前沿与热点，是新一代工业技术的核心，对经济社会的可持续发展具有重要意义。靛蓝是一种适用范围广泛的染料，其工业生产方法步骤繁琐，需要大量有机溶剂，污染严重，并且包括氰基化、皂化、钠氨反应等很多高毒步骤，对生产人员的健康以及生态环境都带来了极大的威胁。本研究通过计算机模拟优化及基因定点突变技术相结合的方式，在原子水平上对肌红蛋白（Myoglobin）活性部位结构进行理性优化设计，开发了15个突变体，催化吲哚制取靛蓝，鉴定了反应代谢物，阐明了反应机制，探讨了肌红蛋白变体活性部位氨基酸排布与其催化性能的关系。相对于传统酶催化系统（细胞色素P450等），本生物催化系统具有蛋白表达量高、稳定性好、催化剂（过氧化氢）价格低廉，体系简单、产品易于分离纯化等优点，有望成为工业成产靛蓝的绿色替代方法，具有很强的工业应用前景。