Heterologous expression, enzymatic properties, product analysis and molecular docking of assimilative nitrite reductase (NiR) in Bacillus velezensis GXMZU-B1 derived from mariculture

Int J Biol Macromol. 2024 Dec 19:139047. doi: 10.1016/j.ijbiomac.2024.139047. Online ahead of print.

Abstract

High concentrations of nitrite stress aquatic animals, leading to significant fish and shrimp deaths as well as environmental pollution. Reducing nitrite levels in high-density aquaculture is crucial for both aquaculture safety and environmental protection. Nitrite reductase (NiR) can rapidly reduce nitrite in water, offering potential applications in aquaculture and water treatment. In this study, a novel NiR gene (nasD) was isolated from Bacillus velezensis GXMZU-B1, a highly effective nitrite-degrading bacterium, and expressed heterologously in Escherichia coli. The recombinant NASD was purified using Ni-NTA affinity chromatography, and its physicochemical properties and reaction products were analyzed. The enzyme showed optimal activity at 30 °C and pH 6.5. Metal ions such as Fe3+, Zn2+, and Ba2+ enhanced enzyme activity, whereas Cu2+, K+, Mg2+, and Mn2+ reduced it. The best electron donors was NADPH. NASD converts nitrite (NO2-) into ammonium (NH4+), making it environmentally friendly and potentially valuable for aquaculture and water pollution control. Bioinformatics analysis indicated that the enzyme is stable, with a conserved sequence and a Pyr_redox_2 domain. Using NADPH as a coenzyme, AlphaFold3 modeling and molecular docking with nitrite identified 14 potential catalytic sites. These findings highlight the potential of recombinant NASD as a promising candidate for nitrite degradation in aquaculture. SUBJECTS: Biochemistry, Biotechnology, Microbiology.

Keywords: Bacillus velezensis; Characterization; Heterologous expression; Molecular docking; Nitrite reductase.