硝化酶的研究进展

doi:10.13560/j.cnki.biotech.bull.1985.2020-0065

摘要/Abstract

摘要：

有机化合物尤其是芳香族化合物的硝化反应在化工生产中具有重要作用,芳香族硝基化合物以其能量高、结构稳定的优势成为一种高附加值的化工中间体及工业产品。目前,化学合成法由于操作简便、投资小、生产条件可控成为最常用的硝化方法,但该生产方法转化效率低,废水、废酸产量大,造成环境污染,这些问题限制了硝基化合物的生产及应用。因此,需要寻找更加高效环保的合成方法。生物酶催化的应用日益广泛,且自然界中有许多微生物可通过转氨或氧化作用进行含氮基团的转化,为高能材料的酶促合成提供了资源库。本文总结了硝基化合物的种类和主要功能,归纳了当前已有硝基化合物合成方法以及存在的问题,综述了P450超家族、过氧化物酶和N-加氧酶中主要硝化酶的种类及特点,并对硝化酶的蛋白结构、催化机理及作用方式进行了深入分析,列举了硝化酶的定向进化研究,最后对利用硝化酶制备硝基化合物的研究方向进行了展望。

关键词: 硝化反应, 硝基化合物, 硝化酶, 生物合成

Abstract:

The nitration of organic compounds especially aromatic nitro-compounds play an important role in chemical production. Aromatic nitro-compounds have become high-value chemical intermediates and industrial products due to their high energy and stable structure. At present,chemical synthesis is the most commonly used nitrification method due to its simple operation,low cost and controllable production conditions. However,this method is of low conversion efficiency and of a lot of waste acid and waste water are produced,and thus causes environmental pollution. These drawbacks restrict the production and application of nitro-compounds. Therefore,it is necessary to seek more efficient and environmental synthesis methods. The application of enzyme catalysis is becoming more and more extensive,and there are abundant microorganisms in nature that can transform nitrogen-containing groups through ammonia or nitrification,providing a repository for enzymatic synthesis of high-energy materials. In this article,we first summarized the types and main functions of nitro-compounds as well as available methods and their deficiencies for synthesizing nitro-compounds. Then we reviewed the types and characteristics of the main nitrifying enzymes in the P450 superfamily,peroxidases and N-oxygenases,analyzed the protein structure and catalytic mechanism of nitrifying enzymes in depth,and listed the directed evolution of nitrifying enzymes. Finally we prospected the research direction of preparing nitro-compounds by using nitrifying enzyme.

Key words: nitration, nitro-compounds, nitrifying enzyme, biosynthesis

赵璐瑶, 陈振娅, 霍毅欣. 硝化酶的研究进展[J]. 生物技术通报, 2020, 36(11): 164-172.

ZHAO Lu-yao, CHEN Zhen-ya, HUO Yi-Xin. Advances in Nitrifying Enzymes[J]. Biotechnology Bulletin, 2020, 36(11): 164-172.

图/表 6

图1 硝基化合物的制备及转化

图2 对、邻二硝基苯的合成[22,23]

图3 AurF结构分析[46] （a）由蓝到红（氨基）表示的二铁AurF同源二聚体的完整结构图,红色球体为铁原子;（b）二铁AurF（蓝色）及二锰AurF（粉色）活性部位的示意图,红色球体为铁原子,绿色球体为桥联的μ-氧原子,黄色球体为锰原子;（c）AurF活性中心,红球为铁离子,绿球为桥接的氧原子

表1 硝化酶的改造及效果

蛋白	氧化还原结构	原始产物	突变位点	效果	参考文献
PrnD	2Fe-2S	吡咯尼特林	F312A L277A	底物转化效率提高4.89倍底物转化效率提高3.28倍	[53] [53]
TxtE	血红素	4-硝基色氨酸	融合BM3R和14氨基酸连接肽 R59X H176F/H176Y	增加C5催化位点产量提高3.5倍催化位点由C4转换为C5	[54] [55] [55-56]
AurF	2Fe-2S	p-硝基苯甲酸	T100L L202F	相对活性提高3.14倍相对活性提高3.57倍	[57] [57]

图4 含有长度可变的连接肽或交换环（黄色）的TxtE-BM3R嵌合体结构示意图[54]

图5 使用已建模的底物pABA（绿色）观察AurF亚基活性中心的立体结构[57]

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