Cloning, Expression, Purification And Characterization Of Nitrite Reductase Gene From Bacillus Cereus LJ01

Nitrites are potential carcinogens that do acute and chronic harm to human body.Thus,it is very important to strictly control the content of nitrites in food.In our previous study,a strain of Bacillus cereus sp.LJ01 with high nitrite degradation ability was screened from soybean paste.In this study,the full-length encoding gene of nitrite reductase?abbreviated NiR?was obtained successfully and analyzed by bioinformatics.Genetic engineered strains were constructed and some properties of the recombinant NiR were also studied.The gene encoding NiR from B.cereus LJ01 was composed of 1623 base pairs and its accession number was MG839504 in the GenBank Data Libraries.Bioinformatics analysis showed that NiR from B.cereus LJ01 was consisted of 540 amino acids.Its p I was 5.47 and theoretically predicted molecular weight was 60 ku.It was a kind of stable and hydrophilic protein without signal peptide and transmembrane structure,and was mainly composed of alpha-helix and random coil.Moreover,it was likely to belong to NiRA in Fd-NiR.The full-length coding sequence of NiR from B.cereus LJ01 was cloned to various vectors,and recombination strains pET-28a?+?-nir-BL21 and pET-32a?+?-nir-BL21 were constructed respectively.Experimental results indicated that the expression level of recombinant NiR was the highest at 16?,followed by 25?and at 37?.The expression level of the recombinant NiR in the strain of pET-28a?+?-nir-BL21 was obviously higher than that in the other strain.Therefore,the strain of pET-28a?+?-nir-BL21 was selected for the following research.The optimum medium of genetically engineered NiR strain was study and the conditions for the best biomass were obtained:Luria-Bertanimedia medium was used as basal mudium and 5 g·L^-11 glucose and 15 g·L^-1 bacteriological peptone were extra added.In addition,response surface method was used to obtain the highest NiR expression level,and the optimal condition was:the strain of p ET-28a?+?-nir-BL21 was induced with 0.2 mM?-D-Thiogalactoside?abbreviated IPTG?at 13?for 20 h.The recombinant NiR was primary purified through Ni-chelating affinity chromatography and was eluted by 25,50,75,250 and 500 mM imidazole buffer in turn.It was shown that the soluble NiR content was the highest in 250 mM imidazole elution fractions followed by 75 mM,and the lowest in 500 mM.The enzymatic activity of purified NiR exhibited little change whit the occurrence of cytochrome C,which indicated that the recombinant NiR was capable of nitrite degradation without cytochrome C.Enzymatic study of the recombinant NiR showed that its optimum reaction temperature was 35?while its optimum pH was 7.5.The K_m for nitrites was 1.38 mM.Metal ions'effects on the enzyme showed that:when ion concentration was 1 mM,the order of activation on NiR was Cu²⁺>Al³⁺>Fe³⁺>Mn²⁺>K⁺>Mg²⁺,and the order of inhibition on NiR was Hg²⁺>Ba²⁺>Pb²⁺>Ca²⁺>Fe²⁺>Zn²⁺;when ion concentration was 10 mM,the activation of Fe³⁺was strongest,followed by Cu²⁺,Fe²⁺,Al³⁺and Mn²⁺,while other ions showed inhibition,among which Hg²⁺showed the strongest inhibition.The recombinant NiR obtained by Ni-chelating affinity chromatography,ion-exchange chromatography and gel filtration was in high purity and stable and uniform state.The pre-crystallization test was carried out to determine the appropriate concentration for crystallization screening.So far single crystals of NiR haven't been obtained.