Hu Liyong, Wang Xiaoping, Chen Cong, Chen Jianmeng, Wang Zeyu, Chen Jun, Hrynshpan Dzmitry, Savitskaya Tatsiana
College of Environment, Zhejiang University of Technology Hangzhou 310014 China.
Key Laboratory of Pollution Exposure and Health Intervention of Zhejiang Province, Interdisciplinary Research Academy, Zhejiang Shuren University Hangzhou 310015 China
RSC Adv. 2022 Jan 19;12(5):2549-2557. doi: 10.1039/d1ra09008a. eCollection 2022 Jan 18.
Nitrous oxide reductase (NOR) is the only known enzyme that can reduce the powerful greenhouse gas nitrous oxide (NO) to harmless nitrogen at the final step of bacterial denitrification. To alleviate the NO emission, emerging approaches aim at microbiome biotechnology. In this study, the genome sequence of facultative anaerobic bacteria WXP-4, which efficiently degrades NO, was obtained by sequencing for the first time, and then, four key reductase structure coding genes related to complete denitrification were identified. The single structural encoding gene Z with a length of 1914 bp from strain WXP-4 was cloned in BL21(DE3), and the NOR protein (76 kDa) was relatively highly efficiently expressed under the optimal inducing conditions of 1.0 mM IPTG, 5 h, and 30 °C. Denitrification experiment results confirmed that recombinant had strong denitrification ability and reduced 10 mg L of NO to N within 15 h under the optimal conditions of pH 7.0 and 40 °C, its corresponding NO reduction rate was almost 2.3 times that of strain TB, but only 80% of that of wild strain WXP-4, meaning that gene cluster auxiliary gene deletion decreased the activity of NOR. The 3D structure of NOR predicted on the basis of sequence homology found that electron transfer center CuA had only five amino acid ligands, and the S2 of the catalytically active center CuZ only bound one Cu atom. The unique 3D structure was different from previous reports and may be closely related to the strong NO reduction ability of strain WXP-4 and recombinant . The findings show a potential application of recombinant in alleviating the greenhouse effect and provide a new perspective for researching the relationship between structure and function of NOR.
一氧化二氮还原酶（NOR）是已知唯一能在细菌反硝化作用的最后一步将强大的温室气体一氧化二氮（N₂O）还原为无害氮气的酶。为了减少N₂O排放，新出现的方法旨在微生物组生物技术。在本研究中，首次通过测序获得了能高效降解N₂O的兼性厌氧细菌WXP-4的基因组序列，然后鉴定了与完全反硝化相关的四个关键还原酶结构编码基因。将来自菌株WXP-4的长度为1914 bp的单个结构编码基因Z克隆到BL21(DE3)中，在1.0 mM IPTG、5 h和30°C的最佳诱导条件下，NOR蛋白（76 kDa）得到了相对高效的表达。反硝化实验结果证实，重组体具有很强的反硝化能力，在pH 7.0和40°C的最佳条件下，15小时内可将10 mg/L的N₂O还原为N₂，其相应的N₂O还原率几乎是TB菌株的2.3倍，但仅为野生菌株WXP-4的80%，这意味着基因簇辅助基因缺失降低了NOR的活性。基于序列同源性预测的NOR三维结构发现，电子传递中心CuA只有五个氨基酸配体，催化活性中心CuZ的S2仅结合一个Cu原子。这种独特的三维结构与先前的报道不同，可能与菌株WXP-4和重组体强大的N₂O还原能力密切相关。这些发现显示了重组体在缓解温室效应方面的潜在应用，并为研究NOR的结构与功能关系提供了新的视角。
