Purification and Characterization of a Dimethoate-Degrading Enzyme of Aspergillus niger ZHY256, Isolated from Sewage

A dimethoate-degrading enzyme from Aspergillus niger ZHY256 was purified to homogeneity with a specific activity of 227.6 U/mg of protein. The molecular mass of the purified enzyme was estimated to be 66 kDa by gel filtration and 67 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point was found to be 5.4, and the enzyme activity was optimal at 50°C and pH 7.0. The activity was inhibited by most of the metal ions and reagents, while it was induced by Cu²⁺. The Michaelis constant (K_m) and V_max for dimethoate were 1.25 mM and 292 μmol min⁻¹ mg of protein⁻¹, respectively.     

Purification and partial characterization of a membrane-associated lipoxygenase in tomato fruit

Membrane-associated lipoxygenase from green tomato (Lycopersicon esculentum L. cv Caruso) fruit has been purified 49-fold to a specific activity of 8.3 μmol·min⁻¹·mg⁻¹ of protein by solubilization of microsomal membranes with Triton X-100, followed by anion- exchange and size-exclusion chromatography. The apparent molecular mass of the enzyme was estimated to be 97 and 102 kD by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography, respectively. The purified membrane lipoxygenase preparation consisted of a single major band following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which cross-reacts with immunoserum raised against soluble soybean lipoxygenase 1. It has a pH optimum of 6.5, an apparent K_m of 6.2 μm, and V_max of 103. μmol·min⁻¹·mg⁻¹ of protein with linoleic acid as substrate. Corresponding values for the partially purified soluble lipoxygenase from tomato are 3.8 μm and 1.3 μmol·min⁻¹·mg⁻¹ of protein, respectively. Thus, the membrane-associated enzyme is kinetically distinguishable from its soluble counterpart. Sucrose density gradient fractionation of the isolated membranes indicated that the membrane-associated lipoxygenase sediments with thylakoids. A lipoxygenase band with a corresponding apparent mol wt of 97,000 was identified immunologically in sodium dodecyl sulfate-polyacrylamide gel electrophoresis-resolved proteins of purified thylakoids prepared from intact chloroplasts isolated from tomato leaves and fruit.     

Purification and Characterization of an Aminopeptidase from Lactococcus lactis subsp. cremoris Wg2

An aminopeptidase was purified to homogeneity from a crude cell extract of Lactococcus lactis subsp. cremoris Wg2 by a procedure that included diethyl-aminoethane-Sephacel chromatography, phenyl-Sepharose chromatography, gel filtration, and high-performance liquid chromatography over an anion-exchange column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band with a molecular weight of 95,000. The aminopeptidase was capable of degrading several peptides by hydrolysis of the N-terminal amino acid. The peptidase had no endopeptidase or carboxypeptidase activity. The aminopeptidase activity was optimal at pH 7 and 40°C. The enzyme was completely inactivated by the p-chloromecuribenzoate mersalyl, chelating agents, and the divalent cations Cu²⁺ and Cd²⁺. The activity that was lost by treatment with the sulfhydryl-blocking reagents was restored with dithiothreitol or β-mercapto-ethanol, while Zn²⁺ or Co²⁺ restored the activity of the 1,10-phenantroline-treated enzyme. Kinetic studies indicated that the enzyme has a relatively low affinity for lysyl-p-nitroanilide (K_m, 0.55 mM) but that it can hydrolyze this substrate at a high rate (V_max, 30 μmol/min per mg of protein).     

Partial purification and properties of phleinase induced in stem base of orchardgrass after defoliation

Phleinase induced in stem base of orchardgrass (Dactylis glomerata L.) after defoliation was partially purified with ammonium sulfate precipitation, DEAE-Sephadex chromatography, gel filtration, and preparative polyacrylamide gel electrophoresis. The molecular weight of phleinase was 57,000 as determined by gel chromatography. The enzyme showed normal Michaelis-Menten kinetics and its K_m value was 91 millimolar for phlein of mean degree of polymerization 60 as substrate. Reaction velocity of the enzyme was proportional to molarity of phlein irrespective of its chain length (mean degree of polymerization, 30 to 314). Phleinase attacked terminal fructosyl linkage of phlein by multi-chain mechanism. Phleinase cleaved β-2,6 linkage, β-2,6 linkage branched with β-2,1 linkage, and β-2,1 linkage of fructan in order of affinity, but not sucrose. Phleinase exhibited an optimum activity at pH 5.5 at 40°C. Its complete inactivation occurred at 60 and 70°C without and with phlein, respectively. Heat inactivation of the enzyme was enhanced by p-chloromercuribenzoate and protected partially by l-cysteine. The enzyme was inhibited by sulfhydryl reagents such as p-chloromercuribenzoate and Hg²⁺. The modes of action of phleinase were compared with those of the related enzymes.     

Biochemical and Genetic Characterization of an FK506-Sensitive Peptidyl Prolyl cis-trans Isomerase from a Thermophilic Archaeon, Methanococcus thermolithotrophicus

A peptidyl prolyl cis-trans isomerase (PPIase) was purified from a thermophilic methanogen, Methanococcus thermolithotrophicus. The PPIase activity was inhibited by FK506 but not by cyclosporine. The molecular mass of the purified enzyme was estimated to be 16 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 42 kDa by gel filtration. The enzyme was thermostable, with the half-lives of its activity at 90 and 100°C being 90 and 30 min, respectively. The catalytic efficiencies (k_cat/K_m) measured at 15°C for the peptidyl substrates, N-succinyl-Ala-Leu-Pro-Phe-p-nitroanilide and N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, were 0.35 and 0.20 μM⁻¹ s⁻¹, respectively, in chymotrypsin-coupled assays. The purified enzyme was sensitive to FK506 and therefore was called MTFK (M. thermolithotrophicus FK506-binding protein). The MTFK gene (462 bp) was cloned from an M. thermolithotrophicus genomic library. The comparison of the amino acid sequence of MTFK with those of other FK506-binding PPIases revealed that MTFK has a 13-amino-acid insertion in the N-terminal region that is unique to thermophilic archaea. The relationship between the thermostable nature of MTFK and its structure is discussed.     

Purification and Characterization of a Dipeptidase from Lactobacillus helveticus SBT 2171

A metal-dependent dipeptidase was purified to homogeneity from a cell extract of Lactobacillus helveticus SBT 2171 by fast protein liquid chromatography. The enzyme was purified 237-fold from the extract, with a yield of 1.8%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band with a molecular weight of 50,000. The dipeptidase hydrolyzes a range of only dipeptides. Dipeptides containing proline, glutamic acid, and aspartic acid are not hydrolyzed. The enzyme was shown to be a metalloenzyme with a pH optimum of 8.0 and a temperature optimum of 55(deg)C. Dithiol-reducing reagents exert strong inhibition on enzyme activity. Kinetic studies indicated that the enzyme has a relative average affinity for leucyl-leucine (K(infm), 0.5 mM). The negative immunoresponse of the purified enzyme with monoclonal antibodies raised against a dipeptidase from Lactococcus lactis subsp. cremoris Wg2 shows that both enzymes can be immunologically distinguished.     

Production and Purification of Extracellular D-Xylose Isomerase from an Alkaliphilic, Thermophilic Bacillus sp.

An alkaliphilic, thermophilic Bacillus sp. (NCIM 59) produced extracellular xylose isomerase at pH 10 and 50°C by using xylose or wheat bran as the carbon source. The distribution of xylose isomerase as a function of growth in comparison with distributions of extra- and intracellular marker enzymes such as xylanase and β-galactosidase revealed that xylose isomerase was truly secreted as an extracellular enzyme and was not released because of sporulation or lysis. The enzyme was purified to homogeneity by ammonium sulfate precipitation followed by gel filtration, preparative polyacrylamide gel electrophoresis, and ion-exchange chromatography. The molecular weight of xylose isomerase was estimated to be 160,000 by gel filtration and 50,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating the presence of three subunits. The enzyme is most active at pH 8.0 and with incubation at 85°C for 20 min. Divalent metal ions Mg²⁺, Co²⁺, and Mn²⁺ were required for maximum activity of the enzyme. The K_m values for D-xylose and D-glucose at 80°C and pH 7.5 were 6.66 and 142 mM, respectively, while K_cat values were 2.3 × 10² s^-1 and 0.5 × 10² s^-1, respectively.     

GTP Cyclohydrolase I: Purification, Characterization, and Effects of Inhibition on Nitric Oxide Synthase in Nocardia Species

GTP cyclohydrolase I (GTPCH) catalyzes the first step in pteridine biosynthesis in Nocardia sp. strain NRRL 5646. This enzyme is important in the biosynthesis of tetrahydrobiopterin (BH₄), a reducing cofactor required for nitric oxide synthase (NOS) and other enzyme systems in this organism. GTPCH was purified more than 5,000-fold to apparent homogeneity by a combination of ammonium sulfate fractionation, GTP-agarose, DEAE Sepharose, and Ultragel AcA 34 chromatography. The purified enzyme gave a single band for a protein estimated to be 32 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular mass of the native enzyme was estimated to be 253 kDa by gel filtration, indicating that the active enzyme is a homo-octamer. The enzyme follows Michaelis-Menten kinetics, with a K_m for GTP of 6.5 μM. Nocardia GTPCH possessed a unique N-terminal amino acid sequence. The pH and temperature optima for the enzyme were 7.8 and 56°C, respectively. The enzyme was heat stable and slightly activated by potassium ion but was inhibited by calcium, copper, zinc, and mercury, but not magnesium. BH₄ inhibited enzyme activity by 25% at a concentration of 100 μM. 2,4-Diamino-6-hydroxypyrimidine (DAHP) appeared to competitively inhibit the enzyme, with a K_i of 0.23 mM. With Nocardia cultures, DAHP decreased medium levels of NO₂⁻ plus NO₃⁻. Results suggest that in Nocardia cells, NOS synthesis of nitric oxide is indirectly decreased by reducing the biosynthesis of an essential reducing cofactor, BH₄.     

Purification and characterization of novel cutinase from nasturtium (Tropaeolum majus) pollen.

Cutinase from pollen grains of Tropaeolum majus was purified by Sephadex G-100 gel filtration, QAE-Sephadex chromatography, and isoelectric focusing. The purified enzyme was homogeneous as judged by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. The molecular weight of the enzyme was estimated to be 40,000 by both Sephadex G-100 gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This cutinase was found to be a glycoprotein containing about 7% carbohydrate and the isoelectric point of this enzyme was 5.45. It catalyzed hydrolysis of p-nitrophenyl esters of C₂ to C₁₈ fatty acids with similar K_m and V. The purified cutinase showed an optimum pH of 6.8 with cutin as the substrate, whereas with p-nitrophenyl esters of fatty acids the optimum pH was 8.0. This enzyme did not show any metal ion requirement. Unlike the previously studied fungal cutinases, the present pollen enzyme was strongly inhibited by thiol-directed reagents such as N-ethylmaleimide and p-hydroxymercuribenzoate whereas it was totally insensitive to the active serine-directed reagent, diisopropylfluorophosphate. The purified pollen cutinase showed preference for primary alcohol esters, but it did not catalyze hydrolysis of tripalmitoyl or trioleyl glycerol at significant rates. The properties of the pollen enzyme are, in general, in sharp contrast to those of the fungal cutinase, and the present results strongly suggest that the pollen enzyme belongs to a new class of cutinases. Another esterase which preferentially hydrolyzed p-nitrophenyl acetate was also found in the extracellular fluid. This enzyme, separated from cutinase, showed a pI of 5.6 and it was sensitive to diisopropylfluorophosphate, but not to SH-directed reagents.     

Purification and Characterization of a Low-Molecular-Weight Phospholipase A2 from Developing Seeds of Elm

Phospholipase A₂ (PLA₂) was purified about 180,000 times compared with the starting soluble-protein extract from developing elm (Ulmus glabra) seeds. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified fraction showed a single protein band with a mobility that corresponded to 15 kD, from which activity could be recovered. When analyzed by matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry, the enzyme had a deduced mass of 13,900 D. A 53-amino acid-long N-terminal sequence was determined and aligned with other sequences, giving 62% identity to the deduced amino acid sequence of some rice (Oryza sativa) expressed sequence tag clones. The purified enzyme had an alkaline pH optimum and required Ca²⁺ for activity. It was unusually stable with regard to heat, acidity, and organic solvents but was sensitive to disulfide bond-reducing agents. The enzyme is a true PLA₂, neither hydrolyzing the sn-1 position of phosphatidylcholine nor having any activity toward lysophosphatidylcholine or diacylglycerol. The biochemical data and amino acid sequence alignments indicate that the enzyme is related to the well-characterized family of animal secretory PLA₂s and, to our knowledge, is the first plant enzyme of this type to be described.     

Alfalfa Root Nodule Carbon Dioxide Fixation : II. Partial Purification and Characterization of Root Nodule Phosphoenolpyruvate Carboxylase

A nonphotosynthetic phosphoenolpyruvate carboxylase (EC 4.1.1.31) was partially purified from the cytosol of root nodules of alfalfa. The enzyme was purified 86-fold by ammonium sulfate fractionation, DEAE-cellulose, hydroxylapatite chromatography, and reactive agarose with a final yield of 32%. The enzyme exhibited a pH optimum of 7.5 with apparent K_m values for phosphoenolpyruvate and magnesium of 210 and 100 micromolar, respectively. Two isozymes were resolved by nondenaturing polyacrylamide disc gel electrophoresis. Subsequent electrophoresis of these isozymes in a second dimension by sodium dodecyl sulfate slab gel electrophoresis yielded identical protein patterns for the isozymes with one major protein band at molecular weight 97,000. Malate and AMP were slightly inhibitory (about 20%) to the partially purified enzyme. Phosphoenolpyruvate carboxylase comprised approximately 1 to 2% of the total soluble protein in actively N₂-fixing alfalfa nodules.     

Production, Purification, and Characterization of β-(1-4)-Endoxylanase of Streptomyces roseiscleroticus

Twelve species of Streptomyces that formerly belonged to the genus Chainia were screened for the production of xylanase and cellulase. One species, Streptomyces roseiscleroticus (Chainia rosea) NRRL B-11019, produced up to 16.2 IU of xylanase per ml in 48 h. A xylanase from S. roseiscleroticus was purified and characterized. The enzyme was a debranching β-(1-4)-endoxylanase showing high activity on xylan but essentially no activity against acid-swollen (Walseth) cellulose. It had a very low apparent molecular weight of 5,500 by native gel filtration, but its denatured molecular weight was 22,600 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It had an isoelectric point of 9.5. The pH and temperature optima for hydrolysis of arabinoxylan were 6.5 to 7.0 and 60°C, respectively, and more than 75% of the optimum enzyme activity was retained at pH 8.0. The xylanase had a K_m of 7.9 mg/ml and an apparent V_max of 305 μmol · min^-1 · mg of protein^-1. The hydrolysis rate was linear for xylan concentrations of less than 4 mg/ml, but significant inhibition was observed at xylan concentrations of more than 10 mg/ml. The predominant products of arabinoxylan hydrolysis included arabinose, xylobiose, and xylotriose.     

Purification and characterization of α-l-rhamnosidase from Penicillium corylopholum MTCC-2011

An extracellular α-l-rhamnosidase has been purified to electrophoretic homogeneity from the culture filtrate of Penicillium corylopholum MTCC-2011 using a simple procedure consisting of concentration by ultrafiltration and cation exchange column chromatography on carboxymethyl cellulose. The sodium dodesyl sulphate polyacrylamide gel electrophoresis analysis of the purified enzyme gave a single protein band corresponding to the molecular mass of 67.0 kDa. The native – polyacrylamide gel electrophoresis analysis also gave a single protein band confirming the purity of the enzyme and also showing that the enzyme is a monomer in the native state. The K_m and k_cat values of the enzyme were 0.42 mM and 35.7 s^?1, respectively, using p-nitrophenyl α-l-rhamnopyranoside as the substrate. The pH and temperature optima of the enzyme were 6.5 and 57.0 °C, respectively. The purified enzyme preparation successfully hydrolyzed naringin and rutin to prunin and quercetin glucoside, respectively. Thus it can be used for the preparation of these pharmaceutically important compounds.     

De novo synthesis of 3'-nucleotidase in germinating wheat embryo

The enzyme 3′-AMP nucleotidase was purified 2,500- to 5,000-fold from extracts of an acetone powder of wheat (Triticum aestivum) embryonic axes germinated for 40 hours. Sodium dodecyl sulfate acrylamide gel electrophoresis and chromatography on Biogel-P100 indicate that the enzyme is monomeric with a molecular weight of 39,000. Extracts of embryos germinated up to 6 hours have only 1% of the 40-hour level of enzyme activity. To see if the increase to 40 hours represents de novo synthesis, extracts were compared for their ability to react with a rabbit antibody prepared against the enzyme. In immunodiffusion tests, 40-hour extracts showed a strong precipitin line coincident with that of the purified enzyme, whereas no precipitation was observed with 1-hour extracts. When the enzyme present in 40-hour extracts was partially inactivated by EDTA, it still blocked the ability of the antibody to inhibit enzyme activity. Extracts of 1-hour embryos, in contrast, were not able to block the inhibitory activity of the antibody. Embryos allowed to take up ³⁵SO₄ between 40 and 46 hours of germination synthesized ³⁵S-labeled 3′-nucleotidase. In contrast, no radioactive protein synthesized by embryos during the first 6 hours of germination coincided on gel electrophoresis with the enzyme. These results indicate that the increase in 3′-nucleotidase activity is a consequence of de novo synthesis of the enzyme.     

Purification and Characterization of an NAD+-Dependent XylB-Like Aryl Alcohol Dehydrogenase Identified in Acinetobacter baylyi ADP1

The gene xylB_ADP1 from Acinetobacter baylyi ADP1 (gene annotation number ACIAD1578), coding for a putative aryl alcohol dehydrogenase, was heterologously expressed in Escherichia coli BL21(DE3). The respective aryl alcohol dehydrogenase was purified by fast protein liquid chromatography to apparent electrophoretic homogeneity. The predicted molecular weight of 39,500 per subunit was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. According to the native M_w as determined by gel filtration, the enzyme forms dimers and therefore seems to be XylB related. The enzyme showed the highest activity at 40°C. For both the reduction and the oxidation reactions, the pH for optimum activity was 6.5. The enzyme was NADH dependent and able to reduce medium- to long-chain n-alkylaldehydes, methyl-branched aldehydes, and aromatic aldehydes, with benzaldehyde yielding the highest activity. The oxidation reaction with the corresponding alcohols showed only 2.2% of the reduction activity, with coniferyl alcohol yielding the highest activity. Maximum activities for the reduction and the oxidation reaction were 104.5 and 2.3 U mg⁻¹ of protein, respectively. The enzyme activity was affected by low concentrations of Ag⁺ and Hg²⁺ and high concentrations of Cu²⁺, Zn²⁺, and Fe²⁺. The gene xylB_ADP1 seems to be expressed constitutively and an involvement in coniferyl alcohol degradation is suggested. However, the enzyme is most probably not involved in the degradation of benzyl alcohol, anisalcohol, salicyl alcohol, vanillyl alcohol, cinnamyl alcohol, or aliphatic and isoprenoid alcohols.     

Biochemical and Genetic Characterization of trans-2′-Carboxybenzalpyruvate Hydratase-Aldolase from a Phenanthrene-Degrading Nocardioides Strain

trans-2′-Carboxybenzalpyruvate hydratase-aldolase was purified from a phenanthrene-degrading bacterium, Nocardioides sp. strain KP7, and characterized. The purified enzyme was found to have molecular masses of 38 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 113 kDa by gel filtration chromatography. Thus, the homotrimer of the 38-kDa subunit constituted an active enzyme. The K_m and kcat values of this enzyme for trans-2′-carboxybenzalpyruvate were 50 μM and 13 s⁻¹, respectively. trans-2′-Carboxybenzalpyruvate was transformed to 2-carboxybenzaldehyde and pyruvate by the action of this enzyme. The structural gene for this enzyme was cloned and sequenced; the length of this gene was 996 bp. The deduced amino acid sequence of this enzyme exhibited homology to those of trans-2′-hydroxybenzalpyruvate hydratase-aldolases from Pseudomonas putida PpG7 and Pseudomonas sp. strain C18.     

Purification and properties of dipeptidyl peptidase IV from Streptococcus mitis ATCC 9811

Dipeptidyl peptidase IV (EC 3.4.14.—) from Streptococcus mitis ATCC 9811 was purified to a specific activity of 56.2 units/mg protein by a series of column chromatographic techniques. The purified enzyme was apparently homogeneous as judged by disc gel electrophoresis. Gel filtration on a calibrated column indicated an apparent molecular weight of 120,000 for the native enzyme. Gel electrophoresis of the denatured enzyme in the presence of sodium dodecyl sulfate in a constant acrylamide concentration resulted in the appearance of a single component for which a molecular weight of 53,000 was calculated. The purified enzyme has an optimum pH between 6.0 and 8.7 and an isoelectric point of 4.0. The K_m value toward glycylprolyl-p-nitroanilide is about 6.0 × 10^?5m. Substrate specificity studies indicated that the purified enzyme hydrolyzes specifically N-terminal X-proline from X-Pro-p-nitroanilides. Inhibition of this enzyme was achieved with Hg²⁺, Pb²⁺, Zn²⁺, EDTA, and diisopropyl phosphorofluoridate, but not with N-ethyl-maleimide and sulfhydryl inhibitors.     

Purification and characterization of an exo-polygalacturonase secreted by Rhizopus oryzae MTCC 1987 and its role in retting of Crotalaria juncea fibre

An acidic polygalacturonase (PG) secreted by Rhizopus oryzae MTCC-1987 in submerged fermentation condition has been purified to electrophoretic homogeneity using ammonium sulphate fractionation and anion exchange chromatography on diethylaminoethyl cellulose. The purified enzyme gave a single protein band in sodium dodecyl sulphatepolyacrylamide gel electrophoresis analysis with a molecular mass corresponding to 75.5 kDa. The K _m and k _cat values of the PG were 2.7 mg/mL and 2.23 × 10³ s^?1, respectively, using citrus polygalacturonic acid as the substrate. The optimum pH of the purified PG was 5.0 and it does not loose activity appreciably if left for 24 hours in the pH range from 5.0 to 12.0. The optimum temperature of purified enzyme was 50°C and the enzyme does not loose activity below 30°C if exposed for two hours. The purified enzyme showed complete inhibition with 1 mM Ag⁺, Hg²⁺ and KMnO₄, while it was stimulated to some extent by Co²⁺. The purified PG exhibited retting of Crotalaria juncea fibre in absence of ethylenediaminetetraacetic acid.     

Purification and Characterization of an Acetyl Xylan Esterase from Bacillus pumilus

Bacillus pumilus PS213 was found to be able to release acetate from acetylated xylan. The enzyme catalyzing this reaction has been purified to homogeneity and characterized. The enzyme was secreted, and its production was induced by corncob powder and xylan. Its molecular mass, as determined by gel filtration, is 190 kDa, while sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single band of 40 kDa. The isoelectric point was found to be 4.8, and the enzyme activity was optimal at 55°C and pH 8.0. The activity was inhibited by most of the metal ions, while no enhancement was observed. The Michaelis constant (K_m) and V_max for α-naphthyl acetate were 1.54 mM and 360 μmol min⁻¹ mg of protein⁻¹, respectively.     

A Novel 2-Aminomuconate Deaminase in the Nitrobenzene Degradation Pathway of Pseudomonas pseudoalcaligenes JS45

2-Aminomuconate, an intermediate in the metabolism of tryptophan in mammals, is also an intermediate in the biodegradation of nitrobenzene by Pseudomonas pseudoalcaligenes JS45. Strain JS45 hydrolyzes 2-aminomuconate to 4-oxalocrotonic acid, with the release of ammonia, which serves as the nitrogen source for growth of the microorganism. As an initial step in studying the novel deamination mechanism, we report here the purification and some properties of 2-aminomuconate deaminase. The purified enzyme migrates as a single band with a molecular mass of 16.6 kDa in 15% polyacrylamide gel electrophoresis under denaturing conditions. The estimated molecular mass of the native enzyme was 100 kDa by gel filtration and 4 to 20% gradient nondenaturing polyacrylamide gel electrophoresis, suggesting that the enzyme consists of six identical subunits. The enzyme was stable at room temperature and exhibited optimal activity at pH 6.6. The K_m for 2-aminomuconate was approximately 67 μM, and the V_max was 125 μmol · min⁻¹ · mg⁻¹. The N-terminal amino acid sequence of the enzyme did not show any significant similarity to any sequence in the databases. The purified enzyme converted 2-aminomuconate directly to 4-oxalocrotonate, rather than 2-hydroxymuconate, which suggests that the deamination was carried out via an imine intermediate.