Streptomyces-catalyzed Nitroaromatic Reduction And The Purification, Cloning And Expression Of Its Nitroreductase

Nitroaromatic compounds are widely used in the synthesis of pharmaceuticals, pesticide and dyes, and they are recoginized as the major environmental pollutants for their structural stability, low solubility, high toxic and hard to degrade. One of the most important degradative pathways of nitroaromatic compounds is nitroreduction for the electron-withdrawing substituents in the aromatic rings. Nitroreduction is also the major pathway of preparing arylamines in fine chemical production. Therefore, the reduction of nitroaromatic compounds showed dual significances on environmental protection and resource utilization. At present, chemical reduction of nitroaromatic compounds is the general methods for nitroreduction in chemistry and industry, but rigorous conditions and environmental contaminations are two major limitations/disadvantages. Comparing to chemical reduction, biocatalysis is one of the sustainably developmental technologies with mild conditions, simple reaction methods and environmental friendliness. However, the bioavailability of biocatalysts are largely limited by the high toxicity and low solubility of nitroaromatic compounds. One of effective solutions is to digout mcrobials and/or enzymes with high efficiency. In this study, two new Streptomyces with highly efficient-nitroreductive activity were isolated and both native and recombinant nitroreductases were obtained, which may provide new biocatalyst sources for nitroreduction and the application of chemical industry and bioremediation.The main findings of this work include:(1) Two Streptomyces sp. strains with highly efficient-nitroreducing activity were isolated from soil. Based on the morphologic observation, physical and biochemical characteristics and phylogenetic analysis of 16S rDNA, they were classfied into Actinomycetate Streptomyces, and were designed as Streptomyces mirabilis DUT001 and Streptomyces resistomycificus DUT002, respectively.The 16S rDNA sequences were deposited into GenBank, and the access number were EU071006 and EU216596. the strains were conserved in the China General Microbiological Culture Collection Center as No.2517 and 2518, respectively. (2) The characteristics of reduction of nitroaromatic compounds by strains DUT001 and DUT002 were determined. The initial culture temperature and pH of these two strains were 30℃and 6.5-7.5, respectively. The optimal carbon and nitrogen sources of these strains for nitro-reduction were glucose and yeast extract. 1%(w/v) of the non-ion surfactant Triton X-100, calcium alginate immobilization or 0.1 mM of redox mediator AQDS improved nitro-conversion. However, organic solvents inhibited the reaction by the order: isopropanol>acetonitrile>acetone>methanol≈alcohol>DMSO. The seventh generation of Streptomyces mirabilis DUT001 and Streptomyces resistomycificus DUT002 by domestication in the presence of nitroaromatic compounds could increase the capacity of nitroreduction with 1.8 and 1.5 folds, respectively. The substrate spectrum of these two Streptomyces included polycyclic nitroaromatic compounds such as 4-nitro-1,8-naphthalic anhydride,3-nitro-1,8-naphthalic anhydride,3-nitrophthalimide and 4-nitrophthalimide, and dinitro compounds such as 1,3-dinitrobenzene and 1,4-dinitrobenzene. The conversion rate varied between 70% and 95%. Moreover, some azo dyes such as acid red B and K-2BP and substituted fluorenones could also be reduced.(3) Purification and characterization of nitroreductase from Streptomyces mirabilis DUT001. The NADH-dependent nitroreductase from Streptomyces mirabilis DUT001 was purified through ammonium sulphate precipitation, Sepharose Q anion exchange chromatography, Gel-200 filtration chromatography and polyacrylamide gel electrophoresis (PAGE). The purification fold and yield was 33.79 and 32%, respectively. The enzyme was identified as a single band of 37 kDa by SDS-PAGE. The N-terminal tweenty amino acids were DINGGGATLPQLYLTPDVLT. The alignment with blast data showed very low similarity to the known nitroreductases. The optimal reaction temperature and pH of the purified nitroreductase were 40℃and 7.5, respectively. The reaction mechanism of the enzyme was line with the sequential mechanism in the double-substrate reactions, according to the results of analysis of the kinetics data and the concentration of the cofactor and the nitro substrate were not interfered with each other. The specific nitroreductase inhibitors inhibited the activity of the purified enzyme by the following order:menadione> dicoumarol> sodium benzoate> antimycin A and they were competitive inhibitors for this enzyme. The nitroreductase activity increased by 1%(w/v) of Triton X-100. The metal ions increasing the nitroreductase activity followed this sequence, Mg2+> Ca2+> Sr2+> Mn2+; and the activity was significantly inhibited by EDTA and EGTA.(4) Nitroreductase coded gene, snr was cloned and expressed in both a cell-free expression system and E. coli cell. The snr gene from DUT002 was extracted and was successfully expressed in vivo and in vitro. Modelling and docking results indicated that this nitroreductase functioned as a dimmer and snr showed very low similarity with other known nitroreductases by sequence allignment. Based on phylogenetic analysis, it was classfied into the NfsB family. The recombinant NADH-dependent nitroreductase was purified by immobilized metal affinity chromatography and gel filtration and was identified as a single band of 25 kDa by SDS-PAGE. The optimal temperature and pH of nitroreduction activity were 30℃and 7.5, respectively. Polycyclic nitroaromatic compounds such as 4-nitro-1,8-naphthalic anhydride, 3-nitro-1,8-naphthalic anhydride,3-nitrophthalimide and 4-nitrophthalimide and dinitro compounds such as 1,3-dinitrobenzene and 1,4-dinitrobenzene could be reduced by the recombinant nitroreductase.In summary, two soil Streptomyces sp. strains were isolated. Both native and recombinant nitroreductases were obtained and showed highly efficient nitroreduction capacity. They reduced the nitroaromatic compounds with high efficiency. The optimal conditions and factors of nitroreduction by strains and putative nitroreductase were analyzed in detail. The catalysis mechanism by nitroreductase was tentatively explored. This study will provide new biocatalysts and/or enzyme resources for nitroreduction, and facilatate application of biocatalysts in chemical industry and enviromental bioremediation.