Purification and characterization of a methylene urea-hydrolyzing enzyme from Rhizobium radiobacter (Agrobacterium tumefaciens)

Slow-release fertilizers are gaining acceptance to increase fertilizer use efficiency and reduce environmental impact. The release of nitrogen from methylene urea, a common slow release N fertilizer, is controlled by microbial decomposition. An enzyme hydrolyzing slow-release nitrogen fertilizer, methylene urea, was purified from Rhizobium radiobacter (Agrobacterium tumefaciens) to homogeneity using a four-step purification procedure with an overall yield of 3%. The active enzyme has a molecular mass of approximately 180 kDa determined by size exclusion chromatography, and the SDS page of the purified protein indicated three subunits of different sizes (62, 34 and 32 kDa). The N-terminal amino acid sequence of the 62 kDa fragment indicates identity with urease subunits from Mycobacterium tuberculosis (73%) and Helicobacter pylori (71%). However, for the internal amino acid sequences of the 62 kDa fragment no matches with known proteins were found. Some internal peptides in the smaller subunits (32 and 34 kDa) are homologous to urease subunits and unknown proteins in Agrobacterium tumefaciens. Based on the kinetic properties, substrate selectivity, and inhibition characteristics, the novel enzyme (MUase) is an intracellular enzyme complex with urease activity. The enzymatic mechanism of methylene urea breakdown was studied using a novel LC-MS method for MU analysis, which indicates that all cold-water soluble nitrogen forms of methylene urea are subjected to hydrolysis, and the hydrolysis proceeds via methylurea, urea and other yet unidentified hydrolysis-products, suggesting that the isolated enzyme complex performs a multistep hydrolysis. The microbiological and molecular data is useful in determining the soil factors affecting the efficacy of methylene urea as a slow release fertilizer in agricultural production systems.     

Enhanced bioremediation of soil containing 2,4-dinitrotoluene by a genetically modified Sinorhizobium meliloti

The symbiotic nitrogen-fixing soil bacterium, Sinorhizobium meliloti, is well known for its ability to interact with the leguminous plant Medicago sativa L. It has, however, not been reported that this species possesses the capability to degrade toxic nitroaromatic compounds, such as 2,4-dinitrotoluene (DNT) which is commonly associated with the degradation of the explosive trinitrotoluene (TNT). In this study, the pJS1 DNT-biodegradative plasmid was genetically transferred to S. meliloti strain USDA 1936, which was confirmed by plasmid profile analysis. Several standard analytical and chemical tests including high performance liquid chromatography (HPLC), nitrite (NO₂) release assays, rhizosphere population and plant greenhouse studies were conducted to test the ability of S. meliloti to degrade 2,4-DNT. The possible presence of 2,4-DNT remaining in the treated soil was tested, and no 2,4-DNT had been absorbed by the soil. The pJS1-carrying recombinant strain DHK1 produced ‘ARC’ alfalfa plants that were almost 2-fold higher in shoot dry weight than that produced by the parent strain on soil containing 0.14 mM 2,4-DNT. The transconjugant strain DHK1 reduced significantly one-third more 2,4-DNT in both 0.14 and 0.28 mM contaminated soil, and in 0.55 mM contaminated soil it degraded 94% of the 2,4-DNT present. In liquid cultures, however, only about 4% reduction in 2,4-DNT concentrations was obtained in 10 days. We interpret the results as clearly establishing that genetic modification was successfully used, for the first time, to improve the capability of the symbiotic nitrogen-fixing soil bacterium S. meliloti DHK1 to bioremediate in situ 2,4-DNT-contaminated soil in the presence of alfalfa plants.     

Multiple forms of xylose reductase in Candida intermedia: comparison of their functional properties using quantitative structure-activity relationships, steady-state kinetic analysis, and pH studies

The xylose-fermenting yeast Candida intermedia produces two isoforms of xylose reductase: one is NADPH-dependent (monospecific xylose reductase; msXR), and another is shown here to prefer NADH approximately 4-fold over NADPH (dual specific xylose reductase; dsXR). To compare the functional properties of the isozymes, a steady-state kinetic analysis for the reaction d-xylose + NAD(P)H + H(+) <--> xylitol + NAD(P)(+) was carried out and specificity constants (k(cat)/K(aldehyde)) were measured for the reduction of a series of aldehydes differing in side-chain size as well as hydrogen-bonding capabilities with the substrate binding pocket of the enzyme. dsXR binds NAD(P)(+) (K(iNAD+) = 70 microM; K(iNADP+) = 55 microM) weakly and NADH (K(i) = 8 microM) about as tightly as NADPH (K(i) = 14 microM). msXR shows uniform binding of NADPH and NADP(+) (K(iNADP+) approximately K(iNADPH) = 20 microM). A quantitative structure-activity relationship analysis was carried out by correlating logarithmic k(cat)/K(aldehyde) values for dsXR with corresponding logarithmic k(cat)/K(aldehyde) values for msXR. This correlation is linear with a slope of approximately 1 (r (2) = 0.912), indicating that no isozyme-related pattern of substrate specificity prevails and aldehyde-binding modes are identical in both XR forms. Binary complexes of dsXR-NADH and msXR-NADPH show the same macroscopic pK of approximately 9.0-9.5, above which the activity is lost in both enzymes. A lower pK of 7.4 is seen for dsXR-NADPH. Specificity for NADH and greater binding affinity for NAD(P)H than NAD(P)(+) are thus the main features of enzymic function that distinguish dsXR from msXR.     

Thermodynamic parameters of enzymes in grassland soils from Galicia, NW Spain

The thermodynamic parameters of the enzymes catalase, dehydrogenase, casein-protease, α-N-benzoyl-l-argininamide (BAA)-protease, urease, Carboxymethyl (CM)-cellulase, invertase, β-glucosidase and arylsulphatase, were investigated in grassland soils from a European temperate-humid zone (Galicia, NW Spain). The effect of temperature on enzyme activity was determined at 5, 18, 27, 37, 57 and 70 °C. The temperature-dependence of the rate of substrate hydrolysis varied depending on the enzyme and soil. In general, the soil containing the least amount of organic matter (OM) showed the lowest enzyme activity for all temperatures and enzymes, whereas soils with similar OM contents showed similar levels of activity for the entire temperature range. Temperature had a noteworthy effect on the activity of oxidoreductases. Product formation in the reaction catalyzed by dehydrogenase increased with increasing temperature until 70 °C, which was attributed to chemical reduction of iodonitrotetrazolium violet (INT) at high temperatures. Catalase activity was not affected above 37 °C, which may be explained either by non-enzymatic decomposition of hydrogen peroxide or by the fact that catalase has reached kinetic perfection, and is therefore not saturated with substrate.The Arrhenius equation was used to determine the activation energy (E_a) and the temperature coefficient (Q₁₀) for all enzymes. The values of E_a and Q₁₀ for each enzyme differed among soils, although in general the differences were small, especially for those enzymes that act on substrates of low molecular weight. In terms of the values of E_a and Q₁₀ and the differences established among soils, the results obtained for those enzymes that act on substrates of high molecular weight differed most from those corresponding to the other enzymes. Thus the lowest E_a and Q₁₀ values corresponded to BAA-protease, and the highest values to CM-cellulase and casein-protease. Except for catalase in one of the soils, the values of E_a and Q₁₀ for the oxidoreductases were similar to those of most of the hydrolases. In general, the effect of temperature appeared to be more dependent on the type of enzyme than on the characteristics of the soil.     

Purification and characterization of a hexanol-degrading enzyme extracted from apple

An enzyme having activity toward n-hexanol was purified from apple, and its biochemical characteristics were analyzed. The purification steps consisted of sedimentation with ammonium sulfate, DEAE Sepharose Fast Flow ion exchange chromatography, and Sephadex G-100 column. The obtained enzyme had a yield of 16.00% with a specific activity of 18879.20 U/mg protein and overall purification of 142.77-fold. The enzyme showed activity to isoamylol, 1-propanol, n-hexanol, and isobutanol but not toward methanol and ethanol. With n-hexanol as a substrate, the optimum conditions were pH 4.0 and 30 °C for enzyme activity and pH 3.0-4.0 and temperatures below 40 °C for enzyme stability. The enzyme activity was increased significantly by adding l-cysteine and Fe(2+) at all tested concentrations and slightly by Zn(2+) at a high concentration but decreased by additions of EDTA, Ga(2+), K(+), Mg(2+), sodium dodecyl sulfate (SDS), sodium aluminum sulfate (SAS), dithiothreitol (DTT), and glutathione (GSH). The enzyme activities toward n-hexanol and n-hexanal were increased by NADH but decreased by NAD(+), in contrast to a decrease toward n-hexane by addition of both NAD(+) and NADH.     

Phosphotriesterases of flavobacterium sp

An enzyme system having phosphotriesterase activity was partially purified from Flavobacterium sp. by means of gel filtration and preparative gel isotachophoresis. Flavobacterium phosphotriesterase showed maximum activity between pH 8–10 and was unaffected by the presence of metal ions. Non-ionic detergents were potent and irreversible inhibitors of activity. Inhibition was also observed with mercurial thiol reagents and cysteine, although in the latter case inhibition could be reversed by oxidation in air or with K₃Fe(CN)₆. Activity was restricted towards substrates having electron withdrawing aromatic or heterocyclic leaving group such as parathion, paraoxon, diazinon and their analogues. Substrate analogues having the weakly electrophilic 4-aminophenyl group were not hydrolysed and in some cases acted as competitive inhibitors. Product inhibition by 4-nitrophenol (but not by the phosphorus containing moiety) was observed.     

Extraction,purification and properties of soil hydrolases

Invertase, cellulase, phosphatases, protease and β-glucosidase were extracted from permanent pasture soil with 0.2 M phosphate buffer (pH 8) in the presence of 0.2 M EDTA. This extract was further treated with ammonium and salmine sulphates. Attempts were made to fractionate these enzyme activities by gel and anion-exchange chromatography. Specific activities were estimated in all fractions and some characteristics of the purified enzymes (optimum pH, temperature and substrate concentration, and K_m and V_max) were investigated. The results indicated that extracted enzyme activities occurred partly in soil as a carbohydrate-enzyme complex and partly as a humo-carbohydrate complex.     

Partial purification of polyphenol oxidase from Chinese cabbage Brassica rapa L

Polyphenol oxidase (PPO) was purified and characterized from Chinese cabbage by ammonium sulfate precipitation and DEAE-Toyopearl 650M column chromatography. Substrate staining of the crude protein extract showed the presence of three isozymic forms of this enzyme. The molecular weight of the purified enzyme was estimated to be approximately 65 kDa by gel filtration on Toyopearl HW-55F. On SDS-PAGE analysis, this enzyme was composed of a subunit molecular weight of 65 kDa. The optimum pH was 5.0, and this enzyme was stable at pH 6.0 but was unstable below pH 4.0 or above pH 7.0. The optimum temperature was 40 degrees C. Heat inactivation studies showed temperatures >40 degrees C resulted in loss of enzyme activity. PPO showed activity to catechol, pyrogallol, and dopamine (K(m) and V(max) values were 682.5 mM and 67.6 OD/min for catechol, 15.4 mM and 14.1 OD/min for pyrogallol, and 62.0 mM and 14.9 OD/min for dopamine, respectively). The most effective inhibitor was 2-mercaptoethanol, followed in decreasing order by ascorbic acid, glutathione, and L-cysteine. The enzyme activity of the preparation was maintained for 2 days at 4 degrees C but showed a sudden decreased after 3 days.     

藏系绵羊乳酸脱氢酶A的分离纯化和酶学性质

采用HiTrapTM Blue HP亲和层析和DEAE-Sephadex离子交换层析分离方法，对1只雄性高原藏系绵羊(Ovis aries) 大腿骨骼肌乳酸脱氢酶-A(LDH-A)进行了分离纯化，并对其酶学参数进行了测定。纯化的藏系绵羊乳酸脱氢酶-A比活力为100.94 U/mg蛋白，纯化倍数为 12.0。经PAGE和SDS-PAGE分析均显示1条带。酶动力学参数测定显示：Km(米氏常数)NADH(reduced nicotinamide adenine denucleotide)=0.022，Km丙酮酸钠=0.444，均比家兔的高；丙酮酸还原反应的最适pH约为5.8；草酸和二价汞能抑制LDH-A的活力；二价汞是LDH-A的可逆性抑制剂。     

Purification and biochemical properties of dipeptidyl peptidase I from porcine skeletal muscle

Dipeptidyl peptidase I (DPP I; EC 3.4.14.1) was purified from porcine skeletal muscle after several steps such as heat treatment, ammonium sulfate fractionation, gel filtration chromatography, and HPLC anion exchange chromatography. The purified enzyme showed a native molecular mass of approximately 200 kDa on Sephacryl S-200 column chromatography. Two protein bands of 65 and 42 kDa were obtained by SDS-PAGE, indicating its oligomeric nature. Maximum activity was reached at pH 5.5 and 55 degrees C. DPP I shared some common substrate specificities, both on synthetic derivatives and on real peptides, with porcine muscle DPP III. The enzyme required reducing agents for full activation, although the halide requirement was not proved. DPP I was inhibited by the assayed cysteine peptidase inhibitors except p-CMB. The serine peptidase inhibitor 3, 4-DCI also inhibited the enzyme as did the divalent cations Co(2+), Mn(2+), and Zn(2+). On the basis of its properties, DPP I may contribute to the generation of dipeptides during the processing of meat and/or meat products, including cooked ham.     

Purification and partial characterization of Lactobacillus species SK007 lactate dehydrogenase (LDH) catalyzing phenylpyruvic acid (PPA) conversion into phenyllactic acid (PLA)

Phenyllactic acid (PLA) is a novel antimicrobial compound synthesized by lactic acid bacteria (LAB), and its production from phenylpyruvic acid (PPA) is an effective approach. In this work, a lactate dehydrogenase (LDH), which catalyzes the reduction of PPA to PLA, has been purified to homogeneity from a cell-free extract of Lactobacillus sp. SK007 by precipitation with ammonium sulfate, ion exchange, and gel filtration chromatography. The purified enzyme had a dimeric form with a molecular mass of 78 kDa (size exclusion chromatography) or 39 kDa (SDS-PAGE). The ratio of enzyme activity with PPA to that with pyruvate being almost invariable at every purification step indicated that, in Lactobacillus sp. SK007, LDH is responsible for the conversion of PPA into PLA. HPLC profiles of PPA transformation into PLA by growing cells, cell-free extract, and purified LDH of Lactobacillus sp. SK007 were also investigated. Results showed that the presence of NADH was found to be necessary for the enzymatic production of PLA from PPA. The purified LDH displayed optimal activity for PPA at pH 6.0 and 40 degrees C. The Km values of the enzyme for PPA and pyruvate were 1.69 and 0.32 mM, respectively. Moreover, because other screened LAB strains exhibiting relatively high LDH activity toward PPA produced also considerable amounts of PLA, LDH activity for PPA could be therefore used as a screening marker for PLA-producing LAB.     

Purification and characterization of a peroxidase from corn steep water

Three cationic peroxidases have been detected in early, middle, and late corn steep water, with pI values of approximately 8.9, approximately 9.5, and >10.0. The major cationic corn steep water peroxidase (CSWP), with a pI >10, was purified 36400-fold with a 12% recovery from late steep water by a combination of acetone and ammonium sulfate precipitation and sequential chromatography on CM-cellulose, phenyl-Sepharose, and Sephadex G-75. The UV-vis spectrum of purified CSWP is typical of other plant class III peroxidases. The RZ (A(403)/A(280)) of CSWP was between 2.6 and 2.9. It is not glycosylated and exhibited an M(r) of 30662 +/- 7 by MALDI-TOF MS. The pH optimum of CSWP depends on the substrate, and it is active on 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid), guaiacol, ferulic acid, o-dianisidine, o-phenylenediamine, and pyrogallol but is not active on either syringaldazine or ascorbate. At 75 degrees C and pH 4.5, the enzyme has half-lives of 22.7 min (0 mM Ca(2+)) and 248 min (1 mM Ca(2+)). The enzyme is stable at room temperature (22-25 degrees C), losing <3% of the activity at pH 4.5 and <10% at pH 6.2 over 400 h in the presence of 1 mM Ca(2+).     

Dinoseb as a specific inhibitor of nitrogen fixation in soil

N₂ fixation, estimated by reduction of C₂H₂, by a loamy soil amended with glucose was highly sensitive to the pesticide, 4,6-dinitro-o-sec-butylphenol (DNBP).At a concentration of 6 parts/10⁶ DNBP inhibited reduction of C₂H₂ by 90% in an anaerobic artificial soil system. Studies of the nitrogen-fixing enzyme system isolated from Azotobacter suggested that DNBP may compete with ATP for the same site on the enzyme.     

Purification and characterization of phytase induced in tomato roots under phosphorus-deficient conditions

Phytase (myo-inositol hexakisphosphate phosphohydrolase; EC 3.1.3.8) was purified from roots of tomato plants grown under phosphorus-deficient conditions using five purification schemes. The phytase was successfully separated from the major acid phosphatase to an electrophoretic homogeneity. The native molecular weight of this enzyme was estimated to be about 164 kD by Bio-Gel P-200 gel filtration. The molecular weight of the subunit on SDS-PAGE was approximately 82 kD, indicating that the native form of the enzyme was a homodimer. The isoelectric point of tomato phytase was about 5.5. The enzyme exhibited a high affinity for phytic acid (K _m = 38 μM), and was strongly inhibited by phosphate, molybdate and fluoride. Among other characteristics of tomato phytase, the pH and temperature optima were 4.3 and 45°C, respectively. Tomato phytase contained a fairly high concentration of aspartic, glutamic acid and glycine residues.     

Isolation and identification of iron-reducing bacteria from gley soils

Seventy-one facultative anaerobic bacteria, capable of reducing iron oxide in pure culture, were isolated from three differently gleyed subsoils. The bacteria were picked at random from poured plates (10⁻⁵ and 10⁻⁶) inoculated with serially diluted soil samples. An attempt was made to identify these strains by morphological and biochemical tests. Among these 71 iron-reducing bacteria, all except three were capable of reducing nitrate to nitrite and 35 reduced nitrite further into gaseous compounds (denitrification), but only one strain (Bacillus subtilis) produced H₂S. Based upon their physiological and morphological properties, 38 strains were allotted to the genus Pseudomonas, 31 sporeformers to the genus Bacillus and two were regarded to be coryneform (Arthrobacter?) bacteria. Species identified were Ps. denitrificans (23), ps. stutzeri (8) ps. fluorescens-putida (5), Bacillus cereus (6), B. cereus var. mycoides (14) and Bacillus subtilis (9). Two spore-forming bacilli, two non-pigmented pseudomonads and two coryneform type of bacteria could not be identified. The significance of the enzyme nitrate reductase (nitratase) of these bacteria for anaerobic respiration and as a mechanism of iron reduction is discussed.     

Purification and characterization of polyphenol oxidase from garland chrysanthemum (Chrysanthemum coronarium L.)

Polyphenol oxidase (PPO) of garland chrysanthemum (Chrysanthemum coronarium L.) was purified approximately 32-fold with a recovery rate of 16% by ammonium sulfate fractionation, ion exchange chromatography, hydrophobic chromatography, and gel filtration. The purified enzyme appeared as a single band on PAGE and SDS-PAGE. The molecular weight of the enzyme was estimated to be about 47000 and 45000 by gel filtration and SDS-PAGE, respectively. The purified enzyme quickly oxidized chlorogenic acid and (-)-epicatechin. The K(m) value (Michaelis constant) of the enzyme was 2.0 mM for chlorogenic acid (pH 4.0, 30 degrees C) and 10.0 mM for (-)-epicatechin (pH 8.0, 40 degrees C). The optimum pH was 4.0 for chlorogenic acid oxidase (ChO) and 8.0 for (-)-epicatechin oxidase (EpO). In the pH range from 5 to 11, their activities were quite stable at 5 degrees C for 22 h. The optimum temperatures of ChO and EpO activities were 30 and 40 degrees C, respectively. Both activities were stable at up to 50 degrees C after heat treatment for 30 min. The purified enzyme was strongly inhibited by l-ascorbic acid and l-cysteine at 1 mM.     

Kocuria rosea HN01, a newly alkaliphilic humus-reducing bacterium isolated from cassava dreg compost

Purpose

The aims of this study were to isolate an alkaliphilic humus-reducing bacterium, investigate the fastest microbial reduction of humus analog as affected by different cultivation, and examine its ability for iron(III) oxide reduction and organochlorine pollutants (OCPs) degradation.

Materials and methods

A strain of pure culture, designated as HN01, was isolated from cassava dreg compost using anaerobic enrichment procedure with glucose as the electron donor and anthraquinone-2,6-disulphonate (AQDS) as the sole terminal electron acceptor. The isolate strain was identified using phenotypic and phylogenetic analysis. Iron(III) oxides and OCPs were chosen as potential electron acceptors. Strict anaerobic techniques and sterile conditions were applied throughout the incubation experiments, purged with O₂-free N₂ for 15 min. The concentration of reduced AQDS and Fe(II) was then quantified using a UV–vis spectrophotometer. The concentration of OCPs was analyzed by gas chromatography with a micro-electron capture detector. Cell number was determined by direct plate counting on aerobic Luria–Bertani medium agar medium at pH 9.

Results and discussion

(1) Strain HN01 was identified as Kocuria rosea, and the AQDS reduction by HN01 was observed in NaCl concentrations below 12 % (w/v) (optimum, 10 %) and pH ranges of 6.0–10.0 (optimum, 9.0) with sucrose as electron donor at 30 °C; (2) glucose, sucrose, methanol, ethanol, glycerol, and acetate were the favorable electron donors for AQDS reduction by strain HN01; (3) the strain had the ability of reducing iron(III) oxides in the presence of sucrose at pH 9.0 and its Fe(III)-reducing capacity ranked as goethite (α-FeOOH) > lepidocrocite (γ-FeOOH) > haematite(α-Fe₂O₃); and (4) the strain could effectively dechlorinate p,p′-DDT (1,1,1-trichloro-2,2-bis(4-chlorophenyl) ethane), and the dechlorination rate reached 71.3 %.

Conclusions

This is the first report of a strain of K. rosea capable of reducing AQDS, iron (III) oxides, and p,p′-DDT, which extends the diversity of the alkaliphilic and halotolerant humus/Fe(III)-reducing bacterium associated with dechlorination. The strain may have the potential to be used for bioremediation of an anoxic alkaline wastewater or site contaminated with OCPs.     

Remediation of TNT-Contaminated Soils by Anaerobic Proteinaceous Immobilisation

In this work a practical remediation technique for TNT-contaminated soils is demonstrated at the laboratory scale level. Addition of 2%% proteinaceous material and 20%% compostto a TNT-polluted soil (633 + 210 mg TNT kg^-1) decreased free TNT with 87%% and water leachable TNT with 67%% after 3 weeks anaerobic incubation. Upon subsequent aerobic incubation,there was an increase in soil respiration, in the activity of both groups of nitrifiers and also in the activity of methane-oxidising bacteria compared to untreated TNT-contaminated soil.Italian ryegrass (% Lolium multiflorum var. % Bartolini)was succesfully grown after the treatment and developed an extensive root system within 28 days. After a subsequent anaerobic/aerobic phase and growth of Italian ryegrass comprisinga total treatment time of 10 weeks, earthworms were capable ofsurviving and increasing weight in the remediated soil. The overall procedure of anaerobic immobilisation and subsequent growth of Italian ryegrass offers a low-cost method for practitioners to significantly improve the quality of TNT-contaminated soils.     

Characterization of tyrosinase from the cap flesh of portabella mushrooms

Tyrosinase, purified from the cap flesh tissue of portabella mushrooms, was characterized with regard to its physical and biochemical properties. A native molecular size of 41 kDa for the enzyme was obtained by size exclusion chromatography, whereas SDS-PAGE indicated that the enzyme contained a single subunit with a size of approximately 48 kDa under reduced and nonreduced conditions. The purified enzyme showed a single immunological cross-reacting protein after Western blotting when probed with antibodies against Agaricus bisporus tyrosinase. Isoelectric focusing demonstrated that the enzyme preparation, apparently homogeneous by electrophoresis, still contained three isoforms of pI 5.1, 5.2, and 5.3. The purified enzyme was able to oxidize a variety of mono-, di-, and triphenolic compounds. An apparent K(m) of 5 mM was obtained using catechol as the substrate, and an apparent K(m) of 9 mM was found using L-Dopa as a substrate. Ascorbic acid, kojic acid, tropolone, mercaptobenzothiazole, and salicylhydroxamic acid inhibited the enzyme severely at 100 microM.     

Reduction in the Acute Toxicity of Explosive Wastewater Containing Toxic Nitroaromatic Compounds by a Nanoscale Zerovalent Iron Pretreatment Process

The feasibility of using nanoscale zerovalent iron (nZVI) treatment for reducing the acute toxicity of explosive wastewater, such as 2,4,6-trinitrotoluene (TNT) red water which contains highly toxic nitroaromatic compounds (NACs), has been investigated. The water quality was evaluated before and after nZVI treatment using several different analytical techniques, including UV?CVis spectroscopy, X-ray photoelectron spectroscopy, high-performance liquid chromatography, and gas chromatography/mass spectroscopy. The acute toxicity of the wastewater was tested using a luminescence bacterium bioarray. The results indicated that the most significant toxic NACs, such as dinitrotoluene sulfonates, had been effectively removed from the TNT red water by nZVI together with the small amounts of other NACs. Following 1?h of the nZVI processing treatment, the acute toxicity of the TNT wastewater was reduced by approximately 94?%. This treatment would therefore be useful for the pretreatment of wastewaters prior to the application of a biological process. The reduction in the biotoxicity of the wastewater was based on the reductive conversion processes and adsorption behaviors of nZVI.