Isolation, expression, and biochemical characterization: nitrite reductase from Bacillus cereus LJ01

Abstract

Biological remediation of toxic oxygen-containing anions such as nitrate that are common in the environment is of great significance. Therefore, it is necessary to understand the specific role of nitrate and nitrite reductase in the bioremediation process. Bacillus cereus LJ01, which was isolated from traditional Chinese soybean paste, effectively degraded nitrite (such as NaNO₂) at 0–15 mmol L⁻¹ in LB medium. Moreover, the nitrite-degrading active substance (ASDN) was isolated and purified from B. cereus LJ01. The nitrite-degrading activity of nitrite reductase (named LJ01-NiR) was 4004.89 U mg⁻¹. The gene encoding the assimilation of nitrite reductase in B. cereus LJ01 was cloned and overexpressed in E. coli. The purified recombinant LJ01-NiR has a wide range of activities under temperature (20–60 °C), pH (6.5–8.0) and metal ions (Fe³⁺, Fe²⁺, Cu²⁺, Mn²⁺, and Al³⁺). Kinetic parameters of LJ01-NiR, including the values of K_m and V_max were 1.38 mM and 2.00 μmol g⁻¹ min⁻¹, respectively. The results showed that LJ01-NiR could degrade nitrite with or without an electron donor. In addition, sequence analysis revealed that LJ01-NiR was a ferredoxin-dependent nitrite reductase given the presence of conserved [Fe4–S4] cluster and heme-binding domain. The nitrite ion binds to the LJ01-NiR active site by forming three hydrogen bonds with the residues ASN72, ALA133 and ASN140. Due to its high nitrite-degrading activity, LJ01-NiR could potentially be used for environmental pollution treatment.