Ring-cleaving cyanuric acid amidohydrolase activity in the atrazine-mineralizing Ralstonia basilensis M91-3

The purpose of this study was to characterize the cyanuric acid amidohydrolase reaction in Ralstonia basilensis M91-3, an atrazine-mineralizing soil bacterium. This ring fission reaction is the last aromatic step in the degradative pathway of atrazine and other s-triazines. The products and molar stoichiometry of the cyanuric acid amidohydrolase reaction were one mol biuret (H₂N·CO·NH·CO·NH₂) and one mol CO₂ per mol cyanuric acid hydrolyzed, as confirmed by ¹³C-NMR and gas chromatography. The optimum pH and temperature, substrate specificity, and kinetic parameters were also characterized for the purified enzyme. The native enzyme had two forms of different sizes, 204 kDa and 160 kDa. Each was a tetramer or pentamer of 44 kDa and 33 kDa, respectively.     

Gene Sequence and Properties of an s-Triazine Ring-Cleavage Enzyme from Pseudomonas sp. Strain NRRLB-12227

Pesticides based on the s-triazine ring structure are widely used in cultivation of food crops. Cleavage of the s-triazine ring is an important step in the mineralization of s-triazine compounds and hence in their complete removal from the environment. Cyanuric acid amidohydrolase cleaves cyanuric acid (2,4,6-trihydroxy-s-triazine), which yields carbon dioxide and biuret; the biuret is subject to further metabolism, which yields CO₂ and ammonia. The trzD gene encoding cyanuric acid amidohydrolase was cloned into pMMB277 from Pseudomonas sp. strain NRRLB-12227, a strain that is capable of utilizing s-triazines as nitrogen sources. Hydrolysis of cyanuric acid was detected in crude extracts of Escherichia coli containing the cloned gene by monitoring the disappearance of cyanuric acid and the appearance of biuret by high-performance liquid chromatography (HPLC). DEAE and hydrophobic interaction HPLC were used to purify cyanuric acid amidohydrolase to homogeneity, and a spectrophotometric assay for the purified enzyme was developed. The purified enzyme had an apparent K_m of 0.05 mM for cyanuric acid at pH 8.0. The enzyme did not cleave any other s-triazine or hydroxypyrimidine compound, although barbituric acid (2,4,6-trihydroxypyrimidine) was found to be a strong competitive inhibitor. Neither the nucleotide sequence of trzD nor the amino acid sequence of the gene product exhibited a significant level of similarity to any known gene or protein.     

Interaction of poly-5-bromouridylic acid. II. Thermodynamic analysis of its interaction with adenosine and 9-methyladenine

The interaction of poly-5-bromouridylic acid [poly(BU)] with adenosine and 9-methyladenine was studied by equilibrium dialysis, optical melting, and microcalorimetry. The stacking free energy, ω, was estimated as ?17.6 kJ/mol for adenosine·2poly(BU) and ?18.8 kJ/mol for 9-methyladenine·2poly(BU) from the binding isotherms constructed from equilibrium dialysis results. The binding isotherms constructed from a series of melting curves also gave ω values for adenosine·2poly(BU). The thermal stability of the complex depends on monomer concentration, and the partial molar enthalpies of the complex formation at the midpoint of the transition were evaluated from the T_m coefficients as a function of free monomer concentration. The values of ?92.0 and ?90.4 kJ/mol were obtained for adenosine·2poly(BU) and 9-methyladenine·2poly(BU) in 0.4M NaCl–0.02M Na-cacodylate–5 × 10^?4M EDTA (pH 7.0), respectively. Microcalorimetric measurements provided lower integral heats of reaction values for these complexes, i.e., ?73.2 kJ/mol for adenosine·2poly(BU) and ?71.5 kJ/mol for 9-methyladenine·2poly(BU). A comparison with a polyribouridylic acid system provided a quantitative understanding of a stabilization by bromination in terms of thermodynamic parameters.     

Manganese application alleviates the water deficit-induced decline of N2 fixation

Water deficit is a very serious constraint on N₂ fixation rates and grain yield of soybean (Glycine max Merr.). Ureides are transported from the nodules and they accumulate in the leaves during soil drying. This accumulation appears responsible for a feedback mechanism on nitrogen fixation, and it is hypothesized to result from a decreased ureide degradation in the leaf. One enzyme involved in the ureide degradation, allantoate amidohydrolase, is manganese (Mn) dependent. As Mn deficiency can occur in soils where soybean is grown, this deficiency may aggravate soybean sensitivity to water deficit. In situ ureide breakdown was measured by incubating soybean leaves in a 5 mol m ^{? 3} allantoic acid solution for 9 h before sampling leaf discs in which remnant ureide was measured over time. In situ ureide breakdown was dramatically decreased in leaves from plants grown without Mn. At the plant level, allantoic acid application in the nutrient solution of hydroponically grown soybean resulted in a higher accumulation of ureide in leaves and lower acetylene reduction activity (ARA) by plants grown with 0 mol m ^{? 3} Mn than those grown with 6·6 mol m ^{? 3} Mn. Those plants grown with 6·6 mol m ^{? 3} Mn in comparison with those grown with 52·8 mol m ^{? 3} Mn had, in turn, higher accumulated ureide and lower ARA. To determine if Mn level also influenced N₂ fixation sensitivity to water deficit, a dry‐down experiment was carried out by slowly dehydrating plants that were grown in soil under four different Mn nutritions. Plants receiving no Mn had the lowest leaf Mn concentration, 11·9 mg kg ^{? 1}, and had N₂ fixation more sensitive to water deficit than plants treated with Mn in which leaf Mn concentration was in the range of 21–33 mg kg ^{? 1}. The highest Mn treatments increased leaf Mn concentration to 37·5 mg kg ^{? 1} and above but did not delay the decline of ARA with soil drying, although these plants showed a significant increase in ARA under well‐watered conditions.     

Microbial production, purification, and characterization of (S)-specific N-acetyl-2-amino-1-phenyl-4-pentene amidohydrolase from Rhodococcus globerulus K1/1

Rhodococcus globerulus K1/1 was found to express an inducible (S)-specific N-acetyl-2-amino-1-phenyl-4-pentene amidohydrolase. Optimal bacterial growth and amidohydrolase expression were both observed at about pH 6.5. Purification of the enzyme to a single band in a Coomassie blue-stained SDS-PAGE gel was achieved by nucleic acid and ammonium sulfate precipitation of Rhodococcus globerulus K1/1 crude extract and column chromatography on TSK Butyl-650(S) Fractogel and Superose 12HR. The amidohydrolase was purified to a homogeneity leading to a tenfold increase of the specific activity with a recovery rate of 65%. At pH 7.0 and 23 °C the enzyme showed no loss of activity after 30 days incubation. The amidohydrolase was stable up to 55 °C. The enzyme was inhibited strongly only by 10 mM Zn²⁺ among the tested metal cations and was inhibited 100% by 0.01 mM phenylmethanesulfonyl fluoride. The molecular weight of the native enzyme was estimated to be 92 kDa by gel filtration and 55 kDa by SDS-PAGE, suggesting a homodimeric structure. Received: 8 February 1999 / Received revision: 3 May 1999 / Accepted: 7 May 1999     

Coenzyme F420 dependent N5, N10-methylenetetrahydromethanopterin dehydrogenase in methanol grown Methanosarcina barkeri

The dehydrogenation of N ⁵,N ¹⁰-methylenetetrahydromethanopterin (CH₂=H₄MPT) to N ⁵,N ¹⁰-methenyltetrahydromethanopterin (CH≡H₄MPT⁺) is an intermediate step in the oxidation of methanol to CO₂ in Methanosarcina barkeri. The reaction is catalyzed by CH₂=H₄MPT dehydrogenase, which was found to be specific for coenzyme F₄₂₀ as electron acceptor; neither NAD, NADP nor viologen dyes could substitute for the 5-deazaflavin. The dehydrogenase was anaerobically purified almost 90-fold to apparent homogeneity in a 32% yield by anion exchange chromatography on DEAE Sepharose and Mono Q HR, and by affinity chromatography on Blue Sepharose. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed only one protein band with an apparent mass of 31 kDa. The apparent molecular mass of the native enzyme determined by polyacrylamide gradient gel electrophoresis was 240 kDa. The ultraviolet/visible spectrum of the purified enzyme was almost identical to that of albumin suggesting the absence of a chromophoric prosthetic group. Reciprocal plots of the enzyme activity versus the substrate concentrations were linear: the apparent K _m for CH₂=H₄MPT and for coenzyme F₄₂₀ were found to be 6 μM and 25 μM, respectively. V_max was 4,000 μmol min^-1·mg^-1 protein (k_cat=2,066 s^-1) at pH 6 (the pH optimum) and 37°C. The Arrhenius activation energy was 40 kJ/mol. The N-terminal amino acid sequence was found to be 50% identical with that of the F₄₂₀-dependent CH₂=H₄MPT dehydrogenase isolated from H₂/CO₂ grown Methanobacterium thermoautotrophicum.     

Barbiturase, a novel zinc-containing amidohydrolase involved in oxidative pyrimidine metabolism.

Barbiturase, which catalyzes the reversible amidohydrolysis of barbituric acid to ureidomalonic acid in the second step of oxidative pyrimidine degradation, was purified to homogeneity from Rhodococcus erythropolis JCM 3132. The characteristics and gene organization of barbiturase suggested that it is a novel zinc-containing amidohydrolase that should be grouped into a new family of the amidohydrolases superfamily. The amino acid sequence of barbiturase exhibited 48% identity with that of herbicide atrazine-decomposing cyanuric acid amidohydrolase but exhibited no significant homology to other proteins, indicating that cyanuric acid amidohydrolase may have evolved from barbiturase. A putative uracil phosphoribosyltransferase gene was found upstream of the barbiturase gene, suggesting mutual interaction between pyrimidine biosynthesis and oxidative degradation. Metal analysis with an inductively coupled radiofrequency plasma spectrophotometer revealed that barbiturase contains approximately 4.4 mol of zinc per mol of enzyme. The homotetrameric enzyme had K(m) and V(max) values of 1.0 mm and 2.5 micromol/min/mg of protein, respectively, for barbituric acid. The enzyme specifically acted on barbituric acid, and dihydro-l-orotate, alloxan, and cyanuric acid competitively inhibited its activity. The full-length gene encoding the barbiturase (bar) was cloned and overexpressed in Escherichia coli. The kinetic parameters and physicochemical properties of the cloned enzyme were apparently similar to those of the wild-type.     

Ureide Catabolism in Soybeans : III. Ureidoglycolate Amidohydrolase and Allantoate Amidohydrolase Are Activities of an Allantoate Degrading Enzyme Complex

We demonstrate that allantoate is catabolized in soybean seedcoat extracts by an enzyme complex that has allantoate amidohydrolase and ureidoglycolate amidohydrolase activities. Soybean seedcoat extracts released ¹⁴CO₂ from [ureido-¹⁴C]ureidoglycolate under conditions in which urease is not detectable. CO₂ and glyoxylate are enzymically released in a one to one ratio indicating that ureidoglycolate amidohydrolase is the responsible activity. Ureidoglycolate amidohydrolase has a K_m of 85 micromolar for ureidoglycolate. Glyoxylate and CO₂ are enzymically released from allantoate at linear rates in a one to 2.3 ratio from 5 to 30 min. This ratio is consistent with the degradation of allantoate to two CO₂ and one glyoxylate with approximately 23% of the allantoate degraded reacting with 2-mercaptoethanol to yield 2-hydroxyethylthio, 2′-ureido, acetate (RG Winkler, JC Polacco, DG Blevins, DD Randall 1985 Plant Physiol 79: 787-793). That [¹⁴C]urea production from [2,7-¹⁴C]allantoate is not detectable indicates that allantoate-dependent glyoxylate production is enzymic and not a result of nonenzymic hydrolysis of a ureido intermediate (nonenzymic hydrolysis releases urea). These results and those from intact tissue studies (RG Winkler DG Blevins, JC Polacco, DD Randall 1987 Plant Physiol 83: 585-591) suggest that soybeans have a second amidohydrolase reaction (ureidoglycolate amidohydrolase) that follows allantoate amidohydrolase in allantoate catabolism. The rate of ¹⁴CO₂ release from [2,7-¹⁴C]allantoate is not reduced when the volume of the reaction mixture is increased, suggesting that the release of ¹⁴CO₂ is not dependent on the accumulation of free intermediates. That [2,7-¹⁴C]allantoate dependent ¹⁴CO₂ release is not proportionally diluted by unlabeled ureidoglycolate indicates that the reaction is carried out by an enzyme complex. This is the first report of ureidoglycolate amidohydrolase activity in any organism and the first in vitro demonstration in plants that the ureido-carbons of allantoate can be completely degraded to CO₂ without a urea intermediate.     

Acid phosphatase synthesis inAspergillus flavus

Proteins with phosphatase activity were produced during the growth ofAspergillus flavus in a phosphate-supplemented liquid synthetic medium. The best carbon and nitrogen sources for the synthesis of phosphatase were glucose and ammonium sulfate, respectively. The proteins were separated by molecular exclusion and ion exclusion chromatography (IEC) into three components one of which showed phosphatase activity. The molar mass of the enzyme was approximately 62 kDa. The purified enzyme exhibited an optimum activity at pH 4.0 and at 45°C. The activity of the enzyme was stimulated by Ca²⁺ and Mg²⁺ but inhibited by fluoride, iodoacetic acid, ethylenediaminetetraacetic acid and 2,4-dinitrophenol, and exhibited an apparentK _M of approximately 420 μmol/L.     

Calorimetric Glucose Determination by Glucose Oxidase —Methylene Blue

The reaction between glucose and methylene blue, catalyzed by glucose oxidase (GOD)was analysed calorimetrically. The amount of heat produced under saturating methylene blue concentrations ( > 10^?2 mol/1)was measured with glucose concentration and time as parameters (kinetic procedure) Kinetic constants (pseudo one substrate kinetics) were derived from the experimental data: K_M(glucose)= 1.18 × 10^?3 mol/l and V_max = 0.085 J/mg GOD min (3.89 · 10^?6 mol/mg GOD min) Comparison of caloric with optical measurements gave an enthalpy of reaction of 22.52 kJ/mol. Considering the observed substrate inhibition, glucose determinations are possible up to glucose concentrations of 0.1 mol/l.     

Characterization of an ethanol‐tolerant 1,4‐β‐xylosidase produced by Pichia membranifaciens

Aims: The purification and biochemical properties of the 1,4‐β‐xylosidase of an oenological yeast were investigated. Methods and Results: An ethanol‐tolerant 1,4‐β‐xylosidase was purified from cultures of a strain of Pichia membranifaciens grown on xylan at 28°C. The enzyme was purified by sequential chromatography on DEAE cellulose and Sephadex G‐100. The relative molecular mass of the enzyme was determined to be 50 kDa by SDS‐PAGE. The activity of 1,4‐β‐xylosidase was optimum at pH 6·0 and at 35°C. The activity had a K_m of 0·48 ± 0·06 mmol l^?1 and a V_max of 7·4 ± 0·1 μmol min^?1 mg^?1 protein for p‐nitrophenyl‐β‐d ‐xylopyranoside. Conclusions: The enzyme characteristics (pH and thermal stability, low inhibition rate by glucose and ethanol tolerance) make this enzyme a good candidate to be used in enzymatic production of xylose and improvement of hemicellulose saccharification for production of bioethanol. Significance and Impact of the Study: This study may be useful for assessing the ability of the 1,4‐β‐xylosidase from P. membranifaciens to be used in the bioethanol production process.     

The occurrence of N-acetylaspartate amidohydrolase (aminoacylase II) in the developing rat

Of The amino acids and derivatives, N-acetyl-L-aspartic acid is present in a uniquely high level (5–6 μmol/g) in the brain of mammals after myelination has occurred. Much lower levels (0·06–0·17 μmol/g) are found prior to this stage of brain development (Tallan , 1957). In non-nervous tissues, on the other hand, only trace amounts of this acetyl amino acid are present (Tallan , Moore and Stein , 1956). N-acetyl aspartic acid serves as an excellent source of acetyl groups for lipogenesis in the developing rat brain (D'Adamo and Yatsu , 1966; Dadamo , Gidez and Yatsu , 1968). Non-nervous tissues such as kidney and mammary gland also rapidly metabolize the acetyl amino acid, the former tissue converting the acetyl group primarily to CO₂ and the latter to fatty acids (Benuck and D'Adamo , 1968). An enzyme with a high specificity for N-acetyl-L-aspartic acid initially termed aminoacylase II, was originally isolated from hog kidney by Birnbaum et al. (1952). Since the physiological role of the substrate is not known, it was of interest to study the occurrence of this enzyme, N-acetyl-L-aspartate amidohydrolase (EC 3.5.1.15), in developing tissues of the rat.     

Hydrolysis of 3′,4′-dichloropropionanilide by an aryl acylamidase from Taraxacum officinale

An aryl acylamidase (aryl-acylamine amidohydrolase, E.C. 3.5. 1.a) which hydrolyses the herbicide propanil (3′,4′-dichloropropionanilide), was isolated from dandelion roots and partially purified and characterized. Specificity tests on the enzyme revealed that it could hydrolyse various chlorine ring-substituted propionanilides and 3,4-dichloroanilide alkyl compounds. The partially purified enzyme was inhibited by several sulfhydryl reagents and metal ions. The pH optimum was broad, between 7·4 and 7·8. The apparent activation energy, determined from an Arrhenius plot, was 9·0 kcal/mol (37 700 J/mol) for the hydrolysis of 3′,4′-dichloropropionanilide. The apparent K_m was 1·7 × 10⁻⁴ M with propanil as substrate.     

Discrimination between rival laccase inhibition models from data sets with one inhibitor concentration using a penalized likelihood analysis and Akaike weights

Laccase from the white-rot fungus Fomes fomentarius was used for the biodegradation of ferulic acid (FA) in the presence of chloride anions. The initial reaction rates of substrate depletion were obtained by reverse-phase HPLC determination of remaining FA since substrate and reaction products have absorption peaks at similar wavelengths. Modelling of time-course data was accomplished by discrimination of the best enzyme inhibition equation from an initial set of seven different models based on Michaelis–Menten kinetics: competitive; uncompetitive; non-competitive; mixed; mixed hyperbolic; mixed parabolic; mixed hyperbolic and parabolic. Corrected Akaike information criterion was used to evaluate the relative merit of each kinetic model in order to rank them and find the more likely one. The discrimination results showed that the models with higher probabilities were the competitive and mixed inhibition types, but Akaike weights supported the selection of competitive inhibition (CI). After optimization by nonlinear regression, laccase kinetic parameters of FA biodegradation in the presence of chloride anions were: V_max?=?0.11?μmol?min^?1?mg^?1, K_m?=?44?μmol?L^?1 and a CI constant K_ic?=?14?mmol?L^?1.     

Optimization of culture conditions and bench-scale production of anticancer enzyme L-asparaginase by submerged fermentation from Aspergillus terreus CCT 7693

L-Asparaginase amidohydrolase (EC 3.5.1.1) has received significant attention owing to its clinical use in acute lymphoblastic leukemia treatment and non-clinical applications in the food industry to reduce acrylamide (toxic compound) formation during the frying of starchy foods. In this study, a sequential optimization strategy was used to determine the best culture conditions for L-asparaginase production from filamentous fungus Aspergillus terreus CCT 7693 by submerged fermentation. The cultural conditions were studied using a 3-level, central composite design of response surface methodology, and biomass and enzyme production were optimized separately. The highest amount of biomass (22.0?g·L^?1) was obtained with modified Czapek–Dox medium containing glucose (14?g·L^?1), L-proline (10?g·L^?1), and ammonium nitrate (2?g·L^?1) fermented at 37.2?°C and pH 8.56; for maximum enzyme production (13.50?U·g^?1), the best condition was modified Czapek–Dox medium containing glucose (2?g·L^?1), L-proline (10?g·L^?1), and inoculum concentration of 4.8?×?10⁸ espore·mL^?1 adjusted to pH 9.49 at 34.6?°C. The L-asparaginase production profile was studied in a 7?L bench-scale bioreactor and a final specific activity of 13.81?U·g^?1 was achieved, which represents an increase of 200% in relation to the initial non-optimized conditions.     

Purification and characterization of a membrane-bound hydrogenase from Sporomusa sphaeroides involved in energy-transducing electron transport

Hydrogenase was solubilized from the cytoplasmic membrane fraction of betaine-grown Sporomusa sphaeroides, and the enzyme was purified under oxic conditions. The oxygen-sensitive enzyme was partially reactivated under reducing conditions, resulting in a maximal activity of 19.8 μmol H₂ oxidized min^–1 (mg protein)^–1 with benzyl viologen as electron acceptor and an apparent K _m value for H₂ of 341 μM. The molecular mass of the native protein estimated by native PAGE and gel filtration was 122 and 130 kDa, respectively. SDS-PAGE revealed two polypeptides with molecular masses of 65 and 37 kDa, present in a 1:1 ratio. The native protein contained 15.6 ± 1.7 mol Fe, 11.4 ± 1.4 mol S^2–, and 0.6 mol Ni per mol enzyme. The hydrogenase coupled with viologen dyes, but not with other various artificial electron carriers, FAD, FMN, or NAD(P)⁺. The amino acid sequence of the N-termini of the subunits showed a high degree of similarity to eubacterial membrane-bound uptake hydrogenases. Washed membranes catalyzed a H₂-dependent cytochrome b reduction at a rate of 0.18 nmol min^–1 (mg protein)^–1. Received: 7 September 1995 / Accepted: 4 December 1995     

Purification and Characterization of Extracellular Phytase from a Marine Yeast <Emphasis Type="Italic">Kodamaea ohmeri</Emphasis> BG3

The extracellular phytase in the supernatant of cell culture of the marine yeast Kodamaea ohmeri BG3 was purified to homogeneity with a 7.2-fold increase in specific phytase activity as compared to that in the supernatant by ammonium sulfate fractionation, gel filtration chromatography (Sephadex™ G-75), and anion-exchange chromatography (DEAE Sepharose Fast Flow Anion-Exchange). According to the data from sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular mass of the purified enzyme was estimated to be 98.2 kDa while the molecular mass of the purified enzyme was estimated to be 92.9 kDa and the enzyme was shown to be a monomer according to the results of gel filtration chromatography. The optimal pH and temperature of the purified enzyme were 5.0 and 65°C, respectively. The enzyme was stimulated by Mn²⁺, Ca²⁺, K⁺, Li⁺, Na⁺, Ba²⁺, Mg²⁺ and Co²⁺ (at a concentrations of 5.0 mM), but it was inhibited by Cu²⁺, Hg²⁺, Fe²⁺, Fe³⁺, Ag⁺, and Zn²⁺ (at a concentration of 5.0 mM). The enzyme was also inhibited by phenylmethylsulfonyl fluoride (PMSF), iodoacetic acid (at a concentration of 1.0 mM), and phenylgloxal hydrate (at a concentration of 5.0 mM), and not inhibited by EDTA and 1,10-phenanthroline (at concentrations of 1.0 mM and 5.0 mM). The K _m, V _max, and K _cat values of the purified enzyme for phytate were 1.45 mM, 0.083 μmol/ml · min, and 0.93 s^-1, respectively.     

Gene sequence and properties of an s-triazine ring-cleavage enzyme from Pseudomonas sp. strain NRRLB-12227.

Pesticides based on the s-triazine ring structure are widely used in cultivation of food crops. Cleavage of the s-triazine ring is an important step in the mineralization of s-triazine compounds and hence in their complete removal from the environment. Cyanuric acid amidohydrolase cleaves cyanuric acid (2,4,6-trihydroxy-s-triazine), which yields carbon dioxide and biuret; the biuret is subject to further metabolism, which yields CO(2) and ammonia. The trzD gene encoding cyanuric acid amidohydrolase was cloned into pMMB277 from Pseudomonas sp. strain NRRLB-12227, a strain that is capable of utilizing s-triazines as nitrogen sources. Hydrolysis of cyanuric acid was detected in crude extracts of Escherichia coli containing the cloned gene by monitoring the disappearance of cyanuric acid and the appearance of biuret by high-performance liquid chromatography (HPLC). DEAE and hydrophobic interaction HPLC were used to purify cyanuric acid amidohydrolase to homogeneity, and a spectrophotometric assay for the purified enzyme was developed. The purified enzyme had an apparent K(m) of 0.05 mM for cyanuric acid at pH 8.0. The enzyme did not cleave any other s-triazine or hydroxypyrimidine compound, although barbituric acid (2,4, 6-trihydroxypyrimidine) was found to be a strong competitive inhibitor. Neither the nucleotide sequence of trzD nor the amino acid sequence of the gene product exhibited a significant level of similarity to any known gene or protein.     

Development of a continuous system for the degradation of a cyanuric acid by adsorbed Pseudomonas sp. NRRL B-12228

Cyanuric acid in high concentrations (15.5 mm) was degraded completely by Pseudomonas sp. NRRL B-12228 independently of glucose concentration. In the batch fermentations there was a relation between the glucose concentration, on the one hand, and the liberation of ammonia or production of protein, on the other. The greater the supply of carbon, the more biomass was produced, and fewer NH _inf4 ^sup+ ions were released. Continuous fermentations using adsorbed cells could be performed to degrade cyanuric acid. In spite of different glucose feeding there was only a negligible difference in residues of s-triazine. In a one-step continuous system with dilution rates between 0.021 h^–1 and 0.035 h^–1, even a ratio of 0.65 between glucose and cyanuric acid was not sufficient to degrade the cyanuric acid supplied (320–540 mol l^–1 h^–1) completely. When a continuous two-step system was applied with dilution rates between 0.035 h^–1 and 0.056 h^–1, the consumption of carbon source could be minimized while s-triazine degradation up to 860 mol l^–1 h^–1 was complete. In this way the ratio between glucose and cyanuric acid could be increased to 0.25 (molar C:N ratio = 0.33:1). Thereby the process was made considerably more economic.     

A novel amidohydrolase (DmhA) from Sphingomonas sp. that can hydrolyze the organophosphorus pesticide dimethoate to dimethoate carboxylic acid and methylamine

Objectives

To characterize a novel dimethoate amidohydrolase from Sphingomonas sp. DC-6.

Results

A gene, dmhA, encoding the dimethoate amidohydrolase responsible for transforming dimethoate to dimethoate carboxylic acid and methylamine, was cloned from Sphingomonas sp. DC-6. Sequence analysis and molecular modeling indicate that DmhA shares 31–57 % amino acid sequence identities with other functionally confirmed amidohydrolase. DmhA was expressed in Escherichia coli BL21 (DE3) and purified by Ni–NTA affinity chromatography. The purified DmhA could hydrolyze 4-acetaminophenol, dimethoate and propanil. DmhA activity was optimal at 30 °C and pH 7.5. Hg²⁺, Zn²⁺, Cu²⁺, Cd²⁺, Tween 80, Triton X-100 or SDS strongly inhibited its activity. The K _m and k _cat values of DmhA for dimethoate are 0.02 mM and 1.2 s⁻¹, respectively.

Conclusions

DmhA was confirmed to be a novel dimethoate amidohydrolase which could eliminate the toxicity of dimethoate, providing a novel gene resource for the development of pesticide-degrading enzyme preparation and mechanistic study of dimethoate hydrolysis.