Purification of the key enzyme complexes of the anammox pathway from DEMON sludge,Biopolymers

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Purification of the key enzyme complexes of the anammox pathway from DEMON sludge
Biopolymers ( IF 3.2 ) Pub Date : 2021-04-02 , DOI: 10.1002/bip.23428
Mohd Akram ₁ , Andreas Dietl ₁ , Melanie Müller ₁ , Thomas R M Barends ₁

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Anaerobic Ammonium Oxidation (“anammox”) is a bacterial process in which nitrite and ammonium are converted into nitrogen gas and water, yielding energy for the cell. Anammox is an important branch of the global biological nitrogen cycle, being responsible for up to 50% of the yearly nitrogen removal from the oceans. Strikingly, the anammox process uniquely relies on the extremely reactive and toxic compound hydrazine as a free intermediate. Given its global importance and biochemical novelty, there is considerable interest in the enzymes at the heart of the anammox pathway. Unfortunately, obtaining these enzymes in sufficiently large amounts for biochemical and structural studies is problematic, given the slow growth of pure cultures of anammox bacteria when high cell densities are required. However, the anammox process is being applied in wastewater treatment to remove nitrogenous waste in processes like DEamMONification (DEMON). In plants using such processes, which rely on a combination of aerobic ammonia-oxidizers and anammox organisms, kilogram amounts of anammox bacteria-containing sludge are readily available. Here, we report a protein isolation protocol starting from anammox cells present in DEMON sludge from a wastewater treatment plan that readily yields pure preparations of key anammox proteins in the tens of milligrams, including hydrazine synthase HZS and hydrazine dehydrogenase (HDH), as well as hydroxylamine oxidoreductase (HAO). HDH and HAO were active and of sufficient quality for biochemical studies and for HAO, the crystal structure could be determined. The method presented here provides a viable way to obtain materials for the study of proteins not only from the central anammox metabolism but also for the study of other exciting aspects of anammox bacteria, such as for example, their unusual ladderane lipids.

中文翻译：

DEMON污泥中厌氧氨氧化途径关键酶复合物的纯化

厌氧氨氧化（“anammox”）是一种细菌过程，其中亚硝酸盐和铵转化为氮气和水，为细胞提供能量。厌氧氨氧化是全球生物氮循环的一个重要分支，每年从海洋中去除高达 50% 的氮。引人注目的是，厌氧氨氧化工艺独特地依赖于反应性极强且有毒的化合物肼作为游离中间体。鉴于其全球重要性和生化新颖性，人们对位于厌氧氨氧化途径核心的酶有相当大的兴趣。不幸的是，在需要高细胞密度时，考虑到厌氧氨氧化细菌的纯培养物生长缓慢，获得足够大量的这些酶用于生化和结构研究是有问题的。然而，厌氧氨氧化工艺正用于废水处理，以去除诸如 DEamMONification (DEMON) 等工艺中的含氮废物。在使用这种工艺的工厂中，依赖于好氧氨氧化剂和厌氧氨氧化生物的组合，很容易获得千克数量的含有厌氧氨氧化细菌的污泥。在这里，我们报告了一种蛋白质分离方案，该方案从废水处理计划中 DEMON 污泥中存在的厌氧氨氧化细胞开始，该方案很容易产生数十毫克的关键厌氧氨氧化蛋白质的纯制剂，包括肼合酶 HZS 和肼脱氢酶 (HDH)，以及羟胺氧化还原酶（HAO）。HDH 和 HAO 是活性的，质量足够用于生化研究，对于 HAO，可以确定晶体结构。

更新日期：2021-04-02

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