CreiLOV 催化过氧化氢生成能力的表征与工程改造

过氧化氢与氧化酶结合在基于生物催化实现选择性氧化反应方面具有 潜力，但其高浓度下导致生物酶失活是走向工业应用场景面临的主要问题 之一。酶催化原位生成过氧化氢有望避免外源添加过氧化氢带来的问题， 因此本项目希望重组表达核黄素荧光蛋白 CreiLOV 并表征其光照下催化产 生过氧化氢的能力。 本项工作构建了含 CreiLOV 基因的重组表达质粒，成功进行了目的蛋 白的表达及纯化，探究了不同构建的组氨酸标签的 CreiLOV 荧光蛋白在不 同容器中进行光反应产过氧化氢的能力；构建了 CreiLOV 荧光蛋白单点饱 和突变扫描文库，比较了野生型和荧光强度改变突变体蛋白产过氧化氢能 力；为了能够高通量筛选蛋白质突变体文库，探索了不同裂解方式，以实 现在细胞裂解粗酶液中对 CreiLOV 的催化性能进行检测。 经过文中的工作，得到了纯度较高的带组氨酸标签的 CreiLOV。我们 确定了光反应的容器 10 mL 螺纹口透明玻璃瓶和最适的构建 N-His CreiLOV 以进行批量蛋白样品光反应实验。基于此反应体系可以较好地表征蛋白样 品在光照下催化产生过氧化氢的能力，并确定了其与荧光改变没有相关关 系。建立了用生物酶裂解方法直接制备包含无组氨酸标签 WT CreiLOV 蛋 白的细菌粗提物并进行批量、快速检测的体系。我们确立了 CreiLOV 突变 体文库高通量筛选的方法，为将来基于蛋白质工程提高 CreiLOV 产过氧化 氢能力、应用于酶法选择性氧化催化的研究奠定了基础。

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