Gene Cloning,Expression,and Reducing Property Of Nitric Oxide Reductase From Achromobacter Sp.TB

Nitric oxide?NO?is a major atmospheric pollutant,which can easily cause secondary environmental pollution such as acid rain or ozone depletion.Biological purification has proven to be an effective method for green control of NO.The main principle of this method to remove NO is microbial-dominated denitrification,that is,the key reductase in the microorganism can bind NO,and reduce NO to non-toxic and harmless N₂ by enzymatic reaction.In this paper,a denitrifying bacteria Achromobacter denitrificans strain TB was used as the experimental strain.The genomic sequence of the strain was obtained by De Novo sequencing,and the denitrifying gene in the genome was identified.Based on the coding sequence of nitric oxide reductase?NOR?obtained by De Novo sequencing,the norB gene in strain TB was successfully obtained by PCR amplification,and the genetically engineered bacteria E.coli BL21?DE3?-pET28a-norB was generated by the construction of expression vector.The expression of NorB protein in recombinant E.coli was induced by IPTG,and then the transcription of norB gene in strain was detected from the mRNA level.The NO reduction performance of strain was also investigated.The main conclusions are as follows:The whole genome sequence of Achromobacter sp.TB was obtained by bacterial De Novo sequencing.The total length of the genomic sequence was 6692590 bp and the GC content was 67.39%.There are five types of denitrification genes,namely narKKGHJIQL,napEDABC,nosRZDFYL gene cluster and nirK,norB single genes.nos,nor,nir,nar are arranged in sequence on the antisense strand of the genome,transcribed in the same direction;and nap is located solely on the genomic sense strand,transcribed in the opposite direction to other genes.Specific primers were designed according to the NOR coding sequence of Achromobacter sp.TB genome.The Achromobacter sp.TB genomic DNA was used as a template.After PCR amplification,TA clones were constructed and sequenced to obtain the target gene norB with a length of 1218 bp.Bioinformatics analysis found that norB encodes 405 amino acids and NorB protein has a calculated molecular mass of44.7 kDa,which contains ten transmembrane helices and is a hydrophobic protein.The phylogenetic analysis indicated that the confidence of homology with norB in Achromobacter sp.TB and Achromobacter denitrificans strain PR1 was 100.The gene expression vector pET28a-norB was constructed by EcoRI and XhoI double digestion reaction,and engineered bacteria E.coli BL21?DE3?-pET28a-norB was obtained by transforming E.coli.NorB protein was induced by IPTG,and the optimal induction conditions were as follows:IPTG was added to the logarithmic phase of the recombinant E.coli(OD₆₀₀ was about 0.6)to a final concentration of 1.0 mM,and induced at 30°C for 5 h.The results of mRNA transcription level experiments showed that the relative expression abundance of norB in engineered bacteria reached 0.83%.The results of NO degradation rate detection found that the engineering bacteria could reduce NO to N₂O,the reduction efficiency was 7.16%in 13 h.These results indicate that the norB gene was successfully transcribed in E.coli BL21?DE3?,while the expressed NorB protein has NO reducing activity.The NO reduction efficiency of strain TB?33.48%?was 4.68times that of engineering bacteria.However,the relative expression abundance of norB in strain TB was only 0.05%,suggesting that the norB gene cloned in the engineered bacteria may be only the core coding sequence that catalyzes NO reduction in NOR of strain TB,as well as some auxiliary sequences that are transcribed and expressed together with the norB gene,have not been cloned.These auxiliary sequences bear a key role in increasing the enzyme activity of NOR.The core peptide of the catalytic reduction structure in the NOR from Achromobacter sp.TB can independently exert NO reduction.