Study On The Metabolism Of Benzonitrile, 3-cyanopyridine And 3-indoleacetonitrile By The Nitrogen-fixing Bacteria Ensifer Meliloti 1021

We previously found that Ensifer meliloti CGMCC 7333 can degraded nicotinoid insecticides acetamiprid and thiacloprid and proved that nitrile hydratase(NHase)is mediated the conversion of acetamiprid and thiacloprid to its amide metabolite.In present study,the geomic DNA sequencing E.meliloti 1021 was used to transform benzonitrile(BN),3-cyanopyridine(3-CP)and indole-3-acetonitrile(IAN)to explore its nitrile-converting enzymes and evaluate its potenial for biodegradation and biotransformation of nitrile compounds.HPLC analysis showed that E.meliloti 1021 resting cells degraded BN and produced two metabolites,which have the same retention times with the standard benzamide(BAM)and benzoic acid(BAC)at all tested concentration of BN.E.meliloti 1021 resting cells transformed 3-CP with appearing different product peak set different concentrations of 3-CP.Nicotinamide(NAM)and nicotinic acid(NAC)were produced at the initial 200 mg/L 3-CP,while NAM was the sole product at the intial concentraion of 3-CP beyond 1g/L.E.meliloti 1021 resting cells transformed IAN into the only product IAM at all tested concentration of IAN.Whole-genome analysis showed that E.meliloti 1021 lacks of nitrilase gene and has NHase and amidase gene.There are a NHase gene and twelve amidase genes in E.meliloti 1021.Phylogenetic tree analysis showed that 4 amidases had homology with benzamide amidohydrolase in Genbank database.Gene cloning,expressing and enzymatic activity test showed that only the amidase with the accession number of CAC47672.1 had benzamidase activity with conversion of BAM into BAC.The NHase of E.meliloti 1021 has three subunits and similarity of 97%to the nucleic acid sequence of E.meliloti CGMCC 7333 NHase.Benzamidase has length of 434 amino acids,the molecular weight is 47 kDa and PI is 5.37.The benzamidase over-expressed into Escherichia coli Rosetta has poor protein solubility and therefore showed low BAM activitiy,The over-expression conditions were optimized,but the protein solubility could not be improved.The nicotinamidase coding gene was also cloned and this 636 bp nicotinamidase gene encodes a protein with a molecular weight of 22.6 KD,and its PI is 5.5.There has maximum activity of nicotinamidase at 30? towards 3-CP,The purity of the protein was conducted by using 6x His-Tagged Protein Purification Kit,and the concentration of nicotinamidase measured by Commassie Blue Staining Kit was 1.13 mg/ml?Enzymatic activity analysis showed that the optimum pH of nicotinamidase is 7,which decreases rapidly at pH<6 and pH>8.The Kcat/Km was 0.42 mmol/L/s,the Kmwas 2.28mmol/L and the Vmax was 142.86 U/mg protein.Nicotinamidase hashighly active between the temperature from 30 to70? with the relative enzyme activity above 97%.The results showed that Ag2+ had the highest inhibitory effect on nicotinamidase and the inhibition was 72.2%.Iron also inhibited the activity of the enzyme activity with the inhibitory rate of Fe2+ was 13.1%.The organic solvents also have a significant effect on the activity of nicotinamidase.For example,isoamyl alcohol has a 98.4%inhibition rate of nicotinamide activity and 38.1%of trichloromethane.In addition,nicotinamidase substrate spectrum is very narrowand it only transformed nicotinamide and its structural analog pyrazinamide.E.meliloti 1021 resting cells transformed 3-CP,no NAC was found at high concentrations of 3-CP,suggesting that 3-CP may have an effect on nicotinamide expression.Quantitative PCR showed that there is no difference at the transcriptional level between the control group without 3-CP induction andthe experimental group by adding 3-CP to LB culture broth.The immobilized nicotinamidase with sodium alginate and found that the activity of nicotinamide immobilized in E.coli did not decrease.In order to further improve the activity of nicotinamide enzyme,we used site-directed mutagenesis to change the conserved amino acids of nicotinamidase nucleic acid sequence.The results showed that the nicotinic acid production of mutant S169T and S100T increased by 1.18 and 1.2 times respectivly.In conclusion,three typical nitrile metabolic pathways were identified and the corresponding enzymes in this metabolic pathway were described in this paper,using BN,3-CP and IAN as the research objects.These studies provide a theoretical basis for the metabolism of other nitriles by E.meliloti sp.