Discovey Of Nitrile Hydratase Genes And Their Recombinant Expression For The Production Of Nicotinamide

Hydration of nitriles to corresponding amides is an important reaction in the organic synthesis and pharmaceutical industries. However, such an application is restricted because of the harsh conditions, such as the need for strong acids or bases at a high temperature. By contrast, biotransformation of nitriles to amides is of great interest because of its mild conditions and high conversion, and showing unique chemoselectibity, regioselectivity and enantioselectivity. Microbial nitrile hydratase （NHase, EC 4.2.1.84） is a key enzyme in the bienzymatic pathway of nitrile degradation. NHase can catalyze the hydration reaction to produce high value-added amides, and has been successfully used to synthesize some important intermediates and fine chemicals in the field of pesticides and pharmaceuticals. Currently, some microbes with NHase activity have been successfully used to produce acrylamide, nivotinamide and 5-cyanovaleramide. However, these microbes can also transform amides further into carboxylic acids using amidase, which is usually expressed along with NHase in the same operon. Consequently, the yield and purity of the target product are reduced.In this study, we tried to screen some novel NHase genes using genome mining, and achieve their functional expression in E. coli cells. Recombinant expression of NHase appears to be an alternative way to produce some high value-added amides of high quality.So far, the experimentally confirmed NHase genes are less. As we know, the gene number is not more than 40, and the sources of NHase genes are also not very diverse. Currently, only one Fe-type NHase was reported from Pseudomonas chlororaphis B23, and other all were from Rhodococcus erythropolis. The protein sequences of NHases from R. erythropolis also shared high homology,>95% identity. Therefore, we firstly constructed a genome mining method to find novel NHase genes based on the conserved amino acid sequence including the metal binding motif in NHase. A set of putative NHase sequences was assembled by searching GenBank using this method. These seguences were all from the sequenced genome from different microbes with comprehensive information on gene data. The results showed that number of putative Fe-type NHase genes was relatively few in database, and only twenty abailable Fe-type NHase genes was found with percent identities of 45%～99%. Besides Rhodococcus, Pseudomonas was another main cluster. Other genuses containing putative Fe-type NHase genes were found in databased, such as Burkholderia, Acinetobacter, Variovorax and Acidovorax. To Co-type NHase, there are a number of putative genes in database, and only fifity-six putative gene sequences were screened and further analyzed. These Co-type NHase genes were from different genuses, such as Rhodococcus, Streptomyces, Bacillus, Pseudonocardia, Bradyrhizobium, and so on.To study the functional expression of Fe-type NHase, we cloned and expressed the putative NHase gene from Pseudomonas putida F1 in E. coli cells, and then characterized the enzymatic properties. The homology of NHase_F1 aligned with NHases from R. erythropolis was relatively low, only identity less than 60%. The results showed that the gene Pput₂730 adjacent to β-subunit region was necessary for active expression of the NHase. Maximal enzyme activity was obtained when the structural and Pput₂730 genes were transcribed as one unit in plasmide pPCDFDuet-1, and the expressed product was in soluble form. Therefore, the Pput₂730 gene was considered as NHase activator, and its partial function may be involved in protein folding of the α-and β-subunits of NHase. In addition, the Fe-type NHase exhibited broad substrate specificity. The enzyme can efficiently hydrate some aromatic nitriles, such as 3-cyanopyridine,4-cyanopyridine, and benzonitrile, asides from aliphatic nitriles preferentially. Therefore, the recombinant NHase_F1 shows potential applications in some amides preparation.Generally, Co-type NHases were reported to possess board substrate specificity, particularly exhibiting a high affinity for aromatic nitriles. In this work, we cloned and expressed putative six Co-type NHase genes from different microbes, Silicibacter pomeroyi DSS-3, Aurantimonas manganoxydans SI85-9A1, Bradyrhizobium japonicum USDA110, Pseudomonas putida 5B, Mesorhizobium loti JCM 21590 and Sinorhizobium meliloti NRRL L-45, and all of them can hydrate 3-cyanopyridine to produce nicotinamide. The NHase from A. manganoxydans SI85-9A1 showed higher activity to hydrate 3-cyanopyridine after a series of comparisons, defined as NHase₀8. The enzyme is a member of the Co-type NHase family, but its structural gene order wasa-subunit beyond β-subunit, different from the order typically reported for Co-type NHase genes. A downstream gene agdacent to the β-subunit, SI859A1₀0581, was necessary for the functional expression of the recombinant NHase₀8, and did not affect the form of the expressed product in E. coli cells. Importantly, the NHase exhibited adequate thermal stability, with a half-life of 1.5 h at 50℃. To meet the needs of the large-scale preparation of recombinant NHase₀8, an industrial auto-inductio medium （AIM） was designed and optimized. AIM can efficiently produce the recombinant NHase as the traditional IPTG induction method, In a 10-L fermenter, a glycerol-limited feeding method was performed, and a final biomass of 39.5 g/L dry cell weight and activity of 2170 U/ml culture was achieved, and the expression of level was higher than 25% of the total cell protein.Nicotinamide is the main form of vitamin B3, and is precursor of some co-enzyme （nicotinamide adenine dinucleotide, NAD+, and nicotinamide adenine dinucleotide phosphate, NHDP+）. Nicotinamide has a wide range of applications and market prospects as food and feed additives. The recombinant NHase_F1 and NHase₀8 all efficiently hydrate 3-cyanopyridine to produce nicotinamide. The specific activity of the purified NHase_F1 was 26.6 U/mg protein, and NHase₀8 was 404.5 U/mg protein. In a 1-L reaction mixture,3.6 mol of 3-cyanopyridine was completely converted to nicotinamide in four feedings by recombinant NHase₀8, and niacin was not detected. Therefore, given the high efficiency and thermal stability of recombinant NHase₀8, it was more suitable for the industrial production of nicotinamide, and the auto-induction strategy in this work was also conducive to produce the recombinant NHase₀8 in industry.