Identification And Enzymatic Characteristics Of A Novel Nitrite Degrading Bacterium

Nitrite reductase,as a key enzyme for nitrogen cycle in the nature,plays an important role for reducing nitrite in food industry and environmental remediation.The study of its catalytic properties will help to improve its application in industry.In this paper,a strain capable of significantly degrading nitrite was screened and identified as Acinetobacter haemolyticus.The conditions and enzymatic characteristics of its nitrite reductase production were studied,and the nitrite reductase protein was successfully expressed in E.coli using genetic engineering technology.The specific research contents were as follows:（1）A highly efficient nitrite-degrading strain was examined.After 16S r DNA sequencing and physiological and biochemical characteristics analysis,the strain was identified as Acinetobacter haemolyticus WUST01.The nitrite reductase production conditions for the A.haemolyticus WUST01 strain were optimized using single factor and response surface optimization,and the ideal conditions were reached.Under these conditions,Ah-Nir activity increased to 12.51 U/m L,a 96.7%increase over 6.36 U/m L prior to optimization.（2）The enzymatic characteristics of Ah-Nir produced were investigated.The ideal reaction conditions were 30°C,p H7.5,high stability at room temperature,and weak acidity.Mg²⁺and Fe²⁺promoted the activity of Ah-Nir,while Zn²⁺,Mn²⁺,Ca²⁺and K⁺showed inhibition,with the inhibition size of K⁺>Ca²⁺>Zn²⁺>Mn²⁺.Using dynamic simulation,the Ah-Nir Vmax was determined to be 6.45×10^-6 mmol/min and the Km to be 1.52×10^-3mmol/L.The storage stability of Ah-Nir crude enzyme solution was greatly improved after adding glycerol and DTT solution.After adding glycerol solution,the enzyme activity remained about 80%after 72 hours storage at 0°C.（3）The target gene of Ah-Nir was obtained after comparison,the expression vector p ET-28a-Ah-Nir was created,and was introduced into E.coli BL21.The recombinant strain E.coli BL21/p ET-28a-Ah-Nir was obtained,and the activity of Ah-Nir was 149%higher than that of the control strain E.coli BL21/p ET-28a.Ah-Nir protein has[Fe-S]clusters,as determined by DDTC tests and amino acid sequence analysis.The molecular docking method was used to model the Ah-Nir catalytic mechanism,and the outcomes demonstrated that Tyr-282 and Ala-289 were the catalytically active residues.In this investigation,nitrite reductase-producing Acinetobacter haemolyticus was discovered for the first time.The production conditions for nitrite reductase were improved,and its enzymatic properties and catalytic mechanism were thoroughly investigated,resulting in the creation of a new substitute enzyme for the biodegradation of nitrite.