Biotransformation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by a rabbit liver cytochrome P450: insight into the mechanism of RDX biodegradation by Rhodococcus sp. strain DN22

A unique metabolite with a molecular mass of 119 Da (C(2)H(5)N(3)O(3)) accumulated during biotransformation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by Rhodococcus sp. strain DN22 (D. Fournier, A. Halasz, J. C. Spain, P. Fiurasek, and J. Hawari, Appl. Environ. Microbiol. 68:166-172, 2002). The structure of the molecule and the reactions that led to its synthesis were not known. In the present study, we produced and purified the unknown metabolite by biotransformation of RDX with Rhodococcus sp. strain DN22 and identified the molecule as 4-nitro-2,4-diazabutanal using nuclear magnetic resonance and elemental analyses. Furthermore, we tested the hypothesis that a cytochrome P450 enzyme was responsible for RDX biotransformation by strain DN22. A cytochrome P450 2B4 from rabbit liver catalyzed a very similar biotransformation of RDX to 4-nitro-2,4-diazabutanal. Both the cytochrome P450 2B4 and intact cells of Rhodococcus sp. strain DN22 catalyzed the release of two nitrite ions from each reacted RDX molecule. A comparative study of cytochrome P450 2B4 and Rhodococcus sp. strain DN22 revealed substantial similarities in the product distribution and inhibition by cytochrome P450 inhibitors. The experimental evidence led us to propose that cytochrome P450 2B4 can catalyze two single electron transfers to RDX, thereby causing double denitration, which leads to spontaneous hydrolytic ring cleavage and decomposition to produce 4-nitro-2,4-diazabutanal. Our results provide strong evidence that a cytochrome P450 enzyme is the key enzyme responsible for RDX biotransformation by Rhodococcus sp. strain DN22.     

Biotransformation of 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane (CL-20) by denitrifying Pseudomonas sp. strain FA1

The microbial and enzymatic degradation of a new energetic compound, 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane (CL-20), is not well understood. Fundamental knowledge about the mechanism of microbial degradation of CL-20 is essential to allow the prediction of its fate in the environment. In the present study, a CL-20-degrading denitrifying strain capable of utilizing CL-20 as the sole nitrogen source, Pseudomonas sp. strain FA1, was isolated from a garden soil. Studies with intact cells showed that aerobic conditions were required for bacterial growth and that anaerobic conditions enhanced CL-20 biotransformation. An enzyme(s) involved in the initial biotransformation of CL-20 was shown to be membrane associated and NADH dependent, and its expression was up-regulated about 2.2-fold in CL-20-induced cells. The rates of CL-20 biotransformation by the resting cells and the membrane-enzyme preparation were 3.2 +/- 0.1 nmol h(-1) mg of cell biomass(-1) and 11.5 +/- 0.4 nmol h(-1) mg of protein(-1), respectively, under anaerobic conditions. In the membrane-enzyme-catalyzed reactions, 2.3 nitrite ions (NO(2)(-)), 1.5 molecules of nitrous oxide (N(2)O), and 1.7 molecules of formic acid (HCOOH) were produced per reacted CL-20 molecule. The membrane-enzyme preparation reduced nitrite to nitrous oxide under anaerobic conditions. A comparative study of native enzymes, deflavoenzymes, and a reconstituted enzyme(s) and their subsequent inhibition by diphenyliodonium revealed that biotransformation of CL-20 is catalyzed by a membrane-associated flavoenzyme. The latter catalyzed an oxygen-sensitive one-electron transfer reaction that caused initial N denitration of CL-20.     

Effect of amino acids on production of xylanase and pectinase from Streptomyces sp. QG-11-3

Xylanase and pectinase production by Streptomyces sp. QG-11-3 was stimulated by DL-norleucine, L-leucine, DL-isoleucine, L-lysine monohydrochloride and DL--phenylalanine by up to 3.72- and 2.78-fold, respectively, whereas the combination of DL-norleucine, L-leucine and DL-isoleucine synergistically stimulated the xylanase and pectinase production by up to 6.72- and 5.62-fold, respectively. Glycine, DL-norvaline, DL-methionine, and DL-aspartic acid showed no significant stimulatory effect on enzyme production.     

A mechanistic study of thermal C-H reductive elimination from a six-coordinate d iridium complex

The thermolysis of trans-IrL₂(CO)Cl(H)(C₆H₅) (1abd; L=P(i-Pr)₃; H trans to CO) produces benzene and the Vaska-type complex IrL₂(CO)Cl. A mechanistic study of the reaction has shown that 1a reversibly loses CO at 120 °C (as evidenced by the incorporation of ¹³CO) and isomerizes to the previously unreported 1b (H trans to Cl). It was found that 1b is the complex primarily responsible for the formation of benzene upon thermolysis under CO atmosphere; direct loss of benzene from 1a was determined to be, at most, a minor pathway. Benzaldehyde was also formed as a product of thermolysis of 1a under CO atmosphere. The first-order rate constant for benzene elimination in the absence of CO was found to be 8.5 × 10⁻⁵ s⁻¹. The presence of only 5 Torr CO results in a decrease to 2.0 × 10⁻⁵ s⁻¹, but little further inhibition is observed above 5 Torr CO. Added dihydrogen (100 Torr) was found to effect a novel catalysis of benzene elimination from 1a in the absence of CO atmosphere; it is suggested that trace amounts of dihydrogen, present in solutions of 1a, are responsible for the enhanced rate of elimination in the absence of CO. The thermolysis of 1-d₆ in toluene was found to proceed without any toluene incorporation, implying that arene loss is irreversible.     

Isolation, purification and properties of a thermostable chitinase from an alkalophilic Bacillus sp. BG-11

An alkalophilic, chitinase-producing Bacillus sp. BG-11 was isolated which produced an extracellular chitinase and which was purified 16.5-fold, using standard purification techniques. The purified chitinase exhibited a broad pH and temperature optima of 7.5-9.0 and 45 deg C-55 deg C, respectively. The chitinase was stable between pH 6.0-9.0 and 50°C for more than 2 h. Half lives of enzyme at 60 deg C, 70 deg C and 80 deg C were 90 min, 30 min and 20 min respectively. Km value was 12 mg chitin per ml. Shelf life was 60 days at 4°C. Ca2+, Ni2+ and Triton-X-100 stimulated the activity up to 20% whereas Ag+, Hg2+, dithiothreitol, -mercaptoethanol, glutathione, iodoacetic acid and iodoacetamide inhibited the activity up to 50%.     

Complete Amino Acid Sequence of a Subunit from Rapeseed Protein

A subunit of molecular weight 18300 has been separated and isolated from seeds of Brassica campestris L. This subunit was cleaved by using cyanogen bromide, trypsin, Staphylococcus aureus V8 protease and chymotrypsin; the fragments obtained from enzymatlc and chemical cleavages were separated and isolated by polyacrylamide gel electrophoresis and gel filtration. The amino acid analyses were carried out. The complete amino acid sequence of the subunit containing 172 amino acid residues has been established by manual Edman method.     

Direct enantioseparation of some β-adrenergic blocking agents using impregnated thin-layer chromatography

The resolution of (±)-atenolol, (±)-propranolol and (±)-metoprolol into their enantiomers was achieved by TLC on silica-gel plates impregnated with optically pure

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-lysine (0.5%) and

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-arginine (0.5%) as the chiral selectors. In all cases, different combinations of acetonitrile–methanol solvent systems were found to be successful in resolving these compounds. Spots were detected using iodine vapour. The detection limit for both (±)-atenolol and (±)-propranolol was 2.6 μg and for (±)-metoprolol, it was 0.26 μg.     

Measurement and comparison of head scatter factor for 7 MV unflattened (FFF) and 6 MV flattened photon beam using indigenously designed columnar mini phantom

Aim

To measure and compare the head scatter factor for 7 MV unflattened and 6 MV flattened photon beam using a home-made designed mini phantom.

Background

The head scatter factor (Sc) is one of the important parameters for MU calculation. There are multiple factors that influence the Sc values, like accelerator head, flattening filter, primary and secondary collimators.

Materials and methods

A columnar mini phantom was designed as recommended by AAPM Task Group 74 with high and low atomic number material for measurement of head scatter factors at 10 cm and d_max dose water equivalent thickness.

Results

The Sc values measured with high-Z are higher than the low-Z mini phantoms observed for both 6MV-FB and 7MV-UFB photon energies. Sc values of 7MV-UFB photon beams were smaller than those of the 6MV-FB photon beams (0.6–2.2% (Primus), 0.2–1.4% (Artiste) and 0.6–3.7% (Clinac iX (2300CD))) for field sizes ranging from 10 cm × 10 cm to 40 cm × 40 cm. The SSD had no influence on head scatter for both flattened and unflattened beams. The presence of wedge filters influences the Sc values. The collimator exchange effects showed that the opening of the upper jaw increases Sc irrespective of FF and FFF.

Conclusions

There were significant differences in Sc values measured for 6MV-FB and unflattened 7MV-UFB photon beams over the range of field sizes from 10 cm × 10 cm to 40 cm × 04 cm. Different results were obtained for measurements performed with low-Z and high-Z mini phantoms.     

Soil nitrogen and microbial biomass carbon dynamics in native forests and derived agricultural land uses in a humid tropical climate of India

Sedum alfredii, a cadmium (Cd) and zinc (Zn) hyperaccumulator at a mine located in Qu Zhou City, Zhejiang Province, China, can accumulate Cd and Zn exceeding 1,000 and 10,000 mg kg^?1, respectively in its shoot (dry weight) when growing under metal-contaminated habitats. Several strains of bacteria were isolated from the rhizosphere of S. alfredii thriving in different Pb/Zn mines in Hunan Province and Zhejiang Province, China, which can resist high levels of heavy metals. Among the different strains isolated, Burkholderia cepacia showed the highest ability in mobilizing Cd and Zn as well as resisting high concentrations of soluble Zn (500 mg L^?1). The soluble Zn concentration in the medium increased from 13 to 72 and 99% (p?<?0.001) after bacterial inoculation in the medium supplemented with insoluble zinc oxide and zinc carbonate, respectively, while pH dropped from 7 to 2.93. The soluble Cd concentration was also increased from 8 to 96% (p?<?0.001), and pH decreased from 7 to 2.65. Short-chain organic acids were also analyzed and the results indicated that oxalic acid, tartaric acid, formic acid and acetic acid had a significant correlation (p?<?0.001) with the concentrations of Cd and Zn being mobilized during the assay. The present results implicated that certain bacteria associated with metal hyperaccumulators could contribute significantly in mobilizing heavy metals, which would enhance the phytoextraction process.