Efficient Synthesis Of Nicotinic/Isonicotinic Acid By Pseudomonas Putida And Functional Cloning Of Nitrilase

Nitrilase, one of the most important enzyme in nitrilase superfamily, is an potentialbiocatalyst for directly conversion of toxic nitriles into non-toxic acids, which has beensuccessfully adopted in the chemical industry for production of pharmaceuticals,agrochemicals, fine chemicals and so on. Compared with corresponding chemical approaches,enzymatic hydration of nitriles to acids by nitrilase can be conducted under mild reactionconditions with relatively higher yield, greater selectivity, more environment-friendly natureand lower wastes generation. In this paper, a high nitrilase-producing strain was isolated.Then nitrilase production conditions, whole-cell reaction conditions and its application,purification and characterization of the enzyme, cloning and expression of the enzyme weresystematically studied.Utilizing3-cyanopyridine as the sole nitrogen source, this work isolated a bacteriumwith the high nitrilase activity towards3-cyanopyridine. The strain was identified asPseudomonas putida based on morphology, physiological and biochemical characteristics, aswell as16S rRNA gene sequence. The strain was named as P. putida CGMCC3830.3-Cyanopyridine could be fully converted to nicotinic acid by the strain, while no amide wasdetected in the reaction mixture. In order to explore nitrilase production potential, theconditions for production of the enzyme in P. putida CGMCC3830were optimized. Byone-factor-at-a-time method and response surface methodology, glycerol13.54, tryptone11.59, yeast extract5.21, KH2PO41, NaCl1and urea1were proved to be the optimalmedium component (g·L-1); the satisfactory culture conditions were obtained as follows:medium initial pH6.0, culture temperature30?, medium volume55mL/250mL and culturetime36h. It resulted in the maximal nitrilase production of36.12U·mL-1from2.02U·mL-1.The influences of reaction conditions on nitrilase activity using P. putida CGMCC3830as whole cell biocatalyst were investigated. The optimum reaction pH was in pH7.2-7.8, andoptimum reaction temperature was at45?. The half-life(t1/2) of P. putida nitrilase activity at30?,35?,40?and45?was78.9h,45.2h,29.6h and15.0h, respectively. Enzymeactivity was strongly suppressed by Hg2+, Ag+and Al3+. The surfactant showed no significantinfluence on the reaction. The substrate specificity of P. putida was investigated and it showedhigh activity toward aromatic nitriles3-cyanopyridine and4-cyanopridine.3-and4-cyanopyridine (100mmol·L-1) was fully converted into nicotinic acid and isonicotinic acid,respectively, within20min using3mg cdw·mL-1resting cells. Fed-batch reaction was carriedout for bioconversion of3-and4-cyanopyridine.147g·L-1of nicotinic acid was obtained in330min, and172g·L-1of isonicotinic acid was accumulated in380min. This study laid thefoundation for industrial application.The steps of nitrilase purification have been determined. Nitrilase from P. putidaCGMCC3830was purified from cell free extract by ammonium sulfate fractionation,Butyl-Sepharose chromatography, Q-Sepharose chromatography, and SuperdexTM200gelfiltration chromatography, with a final yield of17.1%. An approximately18.1-foldpurification was obtained with a specific activity of54.4U·mg-1for purified nitrilase. According to gel filtration and SDS-PAGE, the enzyme was an approximately307kDahomooligomer, which consisted of8subunits with relatively molecular41kDa. Isoelectricpoint of purified enzyme was measured by two-dimensionalelectrophoresis to be5.2.N-terminal amino acid residues of the enzyme were determined to be MVTYTNKFXAA.The characterization of P. putida CGMCC3830purified nitrilase was investigated. Theoptimal pH and temperature of the enzyme was7.5and40?, respectively. Enzyme activitywas suppressed by Ag+and Hg2+as well as N-ethylmaleimide and iodoacetamide. Metalchelating agent EDTA and8-hydroxyquinoline were proved to have no significant effects onnitrilase activity. Reductant DTT, L-cysteine and L-glutathione showed no obvious influence,but2-mercaptoethanol and ascorbic acid exhibited inhibitory effects on the enzyme. Theenzyme showed high specific activities toward aromatic nitriles such as3-cyanopyridine,4-cyanopyridine, and2-chloro-4-cyanopyridine. According to the classification method?theenzyme could be classified as an aromatic nitrilase. The kinetic parameters Kmand Vmaxof theenzyme for3-cyanopyridine were27.9mmol·L-1and84.0U·mg-1, respectively.Cloning and sequence analysis of P. putida CGMCC3830nitrilase gene was carried out.Gene was cloned through CODEHOP PCR, Degenerate PCR and TAIL-PCR. The openreading frame (ORF) was identified which consisted of1113bp encoding a protein of370amino acids, with a theoretical molecular mass of40.9kDa and a theoretical pI of5.25. Theidentities of gene and amino sequence of P. putida CGMCC3830nitrilase to the otherreported microorganism nitrilase were all lower than64%. A neighbor-joining phylogenetictree was constructed using amino acid sequences of nitrilase?the enzyme was located in thesame clade with R. rhodochrous J1and R. rhodochrous NCIMB11216nitrilase, which werearomatic nitrilases.Heterologous expression of P. putida CGMCC3830nitrilase gene and purification ofrecombinant enzyme was carried out. This nitrilase gene was ligated with pET-28a(+)expression plasmid and used to transform E. coli Rosetta-gami (DE3) for its functionalidentification. Biotransformation experiment demonstrated that this recombinant strain alsoharbored good nitrilase activity. The activity of the recombinant strain could reach up to10U·mL-1with3-cyanopyridine as the substrate.3-and4-cyanopyridine (100mmol·L-1) wasfully converted into nicotinic acid and isonicotinic acid, respectively, within10min using3mg cdw·mL-1cells. Subsequently, the recombinant nitrilase was purified by Ni-NTA affinitychromatography. Under pH7.5and temperature30?, the purified recombinant enzymespecific activity reached48.8U·mg-1with3-cyanopyridine as the substrate.