In vitro Nitrate Reductase Activity Assay of Mycolicibacterium smegmatis Crude Extract

Wei Tan; Zhi-Hui Shao; Guo-Ping Zhao

doi:10.21769/BioProtoc.4098

In vitro Nitrate Reductase Activity Assay of Mycolicibacterium smegmatis Crude Extract

Bio Protoc. 2021 Jul 20;11(14):e4098. doi: 10.21769/BioProtoc.4098.

Authors

Wei Tan^{1

2}, Zhi-Hui Shao³, Guo-Ping Zhao^{1

2

3}

Affiliations

¹ Department of Microbiology, The Chinese University of Hong Kong, Hong Kong, China.
² CAS Key Laboratory of Quantitative Engineering Biology, Guangdong Provincial Key Laboratory of Synthetic Genomics and Shenzhen Key Laboratory of Synthetic Genomics, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China.
³ CAS Key Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, China.

Abstract

Nitrate is one of the major inorganic nitrogen sources for microorganisms. Many bacterial and archaeal lineages can express cytoplasmic assimilatory nitrate reductase (NAS), which catalyzes the rate-limiting reduction of nitrate to nitrite in the nitrate assimilation pathway. Here, we present a detailed protocol for measuring in vitro nitrate reductase (NaR) activity of NAS enzymes from Mycolicibacterium smegmatis crude extract using both physiological and non-physiological electron donors.

Keywords: Assimilatory nitrate reductase; In vitro nitrate reductase activity assay; Methyl viologen; Mycolicibacterium smegmatis; NADH; NADPH.