新型嗜碱产水型NADH氧化酶的基因挖掘及功能性质研究

在90%的工业反应过程中需要催化剂来促进反应，而作为生物催化剂的酶催化剂因为其绿色、高效等特性逐步成为催化剂发展的方向，氧化还原酶就是其中运用最广泛的一种类型。目前大部分氧化还原酶催化过程都需要NADH/NAD+（还原性辅酶Ⅰ）作为辅酶促进电子转移，而辅酶会在反应中逐渐消耗且不可逆，于是辅酶的再生成为氧化还原酶应用的关键。NADH氧化酶可以在氧气的作用下将NADH催化脱氢形成NAD+，可以与消耗NAD+的氧化还原反应形成循环促进反应。NADH氧化酶作为一种酶，需要在合适的温度与pH下才能发挥作用，目前大多数NADH氧化酶最适pH值都在中性或酸性范围，在碱性条件下的反应NADH氧化酶的活性就会大大降低，影响反应效率。本课题希望通过基因挖掘与蛋白质工程等手段获取一种碱性NADH氧化酶，其在碱性条件也能保持较高的活性，开拓NADH/NAD+辅酶在工业上的应用前景。

论文通过基因挖掘在嗜碱盐乳杆菌属微生物体内挖掘到一种新型的NADH氧化酶基因（HaNOX），利用蛋白质工程与基因工程体外克隆表达这种NADH氧化酶。纯化后酶学性质研究发现HaNOX的最适pH值为8.5左右，且在碱性条件下的比活力也保持在较高水平。并对HaNOX进行同源建模和分子对接，分析结合口袋中心关键氨基酸的相互作用。之后通过半理性设计对HaNOX进行定点突变改造，希望获得综合性能更优异的碱性NADH氧化酶。结果显示，通过构建F242A、F242V和F242S这3个突变体，发现F242S和F242A突变体的最适pH依旧保持在碱性范围，且F242S在碱性条件pH8.0下的比活力相较HaNOX有显著提高，特别是Kcat/Km 相比野生型提高了1.2倍，是性能更优异的突变体。HaNOX的F242S突变体在碱性条件下的高活力拓展了NADH氧化酶在碱性条件的应用，为更多利用氧化还原酶的工业生产提供了可能。

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