Microbial oxidation of 2-bromostyrene by Pseudomonas Putida 39/D. Isolation and identification of metabolites

2-Bromostyrene was subjected to microbial oxidation by two microorganisms. Oxidation by Pseudomonas putida 39/D yielded a mixture of ring dihydroxylated (1S,2R)-4-bromo-3-ethenylcyclohexa-3,5-diene-1,2-diol (4a) (optical purity o.p.>92%) and side chain dihydroxylated (1R)-1-(2-bromophenyl)ethan-1,2-diol (5a) (o.p. 91%) (ratio 4a:5a = 1:4) in a yield of 100 mg/L. Pseudomonas sp. NCIB 9816-11 afforded only 5a.     

Biotransformation of substituted pyridines with dioxygenase-containing microorganisms

A series of 2-, 3- and 4-substituted pyridines was metabolised using the mutant soil bacterium Pseudomonas putida UV4 which contains a toluene dioxygenase (TDO) enzyme. The regioselectivity of the biotransformation in each case was determined by the position of the substituent. 4-Alkylpyridines were hydroxylated exclusively on the ring to give the corresponding 4-substituted 3-hydroxypyridines, while 3-alkylpyridines were hydroxylated stereoselectively on C-1 of the alkyl group with no evidence of ring hydroxylation. 2-Alkylpyridines gave both ring and side-chain hydroxylation products. Choro- and bromo-substituted pyridines, and pyridine itself, while being poor substrates for P. putida UV4, were converted to some extent to the corresponding 3-hydroxypyridines. These unoptimised biotransformations are rare examples of the direct enzyme-catalysed oxidation of pyridine rings and provide a novel synthetic method for the preparation of substituted pyridinols. Evidence for the involvement of the same TDO enzyme in both ring and side-chain hydroxylation pathways was obtained using a recombinant strain of Escherichia coli (pKST11) containing a cloned gene for TDO. The observed stereoselectivity of the side-chain hydroxylation process in P. putida UV4 was complicated by the action of an alcohol dehydrogenase enzyme in the organism which slowly leads to epimerisation of the initial (R)-alcohol bioproducts by dehydrogenation to the corresponding ketones followed by stereoselective reduction to the (S)-alcohols.     

Surface physicochemical properties of Pseudomonas fluorescens and impact on adhesion and transport through porous media

The physiological state of an examined Pseudomonas fluorescens strain had a significant impact on its adhesion to glass surfaces and transport through glass-bead columns. In both batch and column studies collision efficiencies, , for exponential phase cells were much larger (≈2–3) than for stationary or decay phase cells (≈0.5–0.7). Centrifugation of exponential phase cells substantially reduced collision efficiencies (≈0.8). Over the examined range (0.02–0.2 M), ionic strength had no impact on cell attachment. The Lewis acid/base (A/B) character of the cell surface varied with physiological states: exponential phase cells exhibited larger values of the electron–donor parameter of the polar surface tension component, γ_S⁻, than stationary or decay phase cells, resulting in larger calculated cell hydrophilicities. A reduction in exponential phase cell ζ-potential was observed upon centrifugation. Traditional Derjaguin–Landau–Verwey–Overbeek (DLVO) interaction energy profiles (between cells and glass surfaces) indicated energy maxima of the order of 90–130kT, and secondary energy minima of less than 10kT. Extended DLVO modeling predicted infinite energy barriers attributable to repulsive A/B interactions, and similar magnitude secondary energy minima. A pseudo-chemical kinetic approach was used to calculate activation energies of adhesion from experimental collision efficiencies. Collision efficiencies were also predicted from a diffusion-governed mass transport model incorporating interacting force fields. Predicted energy barriers underestimated cell collision efficiencies, suggesting that secondary energy minimum interactions governed initial attachment of cells. The partial reversibility of adhesion upon ionic strength reduction supported the secondary minimum interaction hypothesis.     

Nitrogen regulation of Saccharomyces cerevisiae invertase

The regulation of extracellular enzymes is of great biotechnological interest. We studied the regulatory role of the URE 2 gene on the periplasmic invertase of Saccharomyces cerevisiae, because its periplasmic asparaginase is regulated by the URE2/GLN3 system. Enzymatic activity was measured in the isogenic strains P40-1B, the ure2 mutant P40-3C, and the P40-3C strain transformed with the pIC-CS plasmid carrying the URE2 gene. The assays were performed using midlog and stationary phase cells and nitrogen-starved cells from these growth phases. During exponential growth, the level of invertase in both wild-type and ure 2 mutant cells was comparable. However, the invertase activity in ure2 mutant cells from stationary phase was sixfold lower than in the wild-type cells. When P40-3C cells were transformed with the pIC-CS plasmid, the wild-type phenotype was restored. On nitrogen starvation in the presence of sucrose, the invertase activity in wild-type cells from midlog phase decreased three times, whereas in stationary cells, the activity decreased eight times. However, invertase activity doubled in ure 2 mutant cells from both phases. When these cells were trans-formed with the aforementioned plasmid, the wild-type phenotype was restored, although a significant invertase decrease in stationary cells was not observed. These results suggested that the URE2 protein plays a role in invertase activity.     

Enzyme-catalysed synthesis and absolute configuration assignments of cis-dihydrodiol metabolites from 1,4-disubstituted benzenes

A series of ten cis-dihydrodiol metabolites has been obtained by bacterial biotransformation of the corresponding 1,4-disubstituted benzene substrates using Pseudomonas putida UV4, a source of toluene dioxygenase (TDO). Their enantiomeric excess (ee) values have been established using chiral stationary phase HPLC and 1H NMR spectroscopy. Absolute configurations of the majority of cis-dihydrodiols have been established using stereochemical correlation and X-ray crystallography and the remainder have been tentatively assigned using NMR spectroscopic methods but finally confirmed by circular dichroism (CD) spectroscopy. These configurational assignments support and extend the validity of an empirical model, previously used to predict the preferred stereochemistry of TDO-catalysed cis-dihydroxylation of ten 1,4-disubstituted benzene substrates, to more than twenty-five examples.     

A novel magnetite-immobilized cell process for heavy metal removal from industrial effluent

The sorption and desorption of copper (II) (Cu[II]) ions from the wastewater by magnetite-immobilized cells of Pseudomonas putida 5-x with acidic pretreatment were studied. Pretreating cells with 0.6 N HCl was found to enhance greatly the adsorption capacity of biomass up to 85.6 mg/g and had no significant effect on the loss of P. putida 5-x cells during biosorbent pretreatment. The biosorption capacity to Cu²⁺ of magnetite-immobilized cells of P. putida 5-x harvested during various growth phases was also investigated. The experimental results illustrated that the adsorption capacity to Cu²⁺ of P. putida 5-x cultured in sulfate-limiting medium reached maximum during the late stationary growth phase or early death phase, and reached minimum during the log growth phase. The mechanism of copper sequestering by this type of biomass was studied via transmission electron microscopy. A degradation of the peptidoglycan layer of the cell wall was observed in the acidic pretreatment, but no further degradation appeared after the adsorption-desorption cycle. Cu(II) accumulated mostly on the surface of the cell walls and was effectively desorbed by the acidic treatment during the desorption process.

     

3,4-二(3-苯氧基-4-氟苯基)-2,5-二苯基苯基接枝聚硅氧烷气相色谱固定相的合成与表征（英文）

合成了一种耐高温的3,4-二(3-苯氧基-4-氟苯基)-2,5-二苯基苯基接枝聚硅氧烷(DPFP)固定相,使用静态涂渍法将其涂渍到毛细管柱内壁上,制成气相色谱柱。分离裂解乙烯的色谱图显示DPFP固定相在360℃时仍具有良好的分离能力。DPFP固定相的柱效为3 324块/米(保留因子(k)4.24,萘,0.25 mm i.d.)。麦克雷诺常数计算结果显示DPFP固定相属中等极性。溶剂化参数模型结果显示DPFP固定相与溶质之间的主要作用力为偶极-诱导偶极作用力、氢键碱性作用力。Grob试剂分离结果显示DPFP色谱柱具有良好的选择性与惰性。另外,芳香族同分异构体、苯取代物、多环芳烃、脂肪酸酯及脂肪醇都得到了良好的分离,表明DPFP固定相在应用方面有巨大的潜力。     

Production of Anti-Methicillin-Resistant <Emphasis Type="Italic">Staphylococcus</Emphasis> Activity from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> sp. Strain B38 Newly Isolated from Soil

B38 bacterial strain, isolated from Tunisian soil showed a strong antimicrobial activity. Based on biochemical characterization and 16S rDNA sequence analysis, B38 strain was identified as Bacillus subtilis. Cell culture supernatant showed antibacterial activity against clinical isolates of methicillin-resistant Staphylococcus species and several Gram-positive and Gram-negative bacteria. Antifungal activity against phytopathogenic fungi was also observed. Antibacterial activity production started at early exponential growth phase, and maximum activity was reached at the stationary phase. This antibacterial activity was neither affected by proteases, lipase, and organic solvents, nor by surfactants. It was stable over a wide pH range and still active after autoclaving at 121 °C during 20 min. Thin layer chromatography followed by bioautography assay allowed the detection of four active spots with R _f values of 0.30, 0.47, 0.70, and 0.82. The single spot with R _f 0.30 showed antifungal activity, whereas the spots with R _f values of 0.47, 0.70, and 0.82 exhibited antibacterial activity.     

Bacterial dioxygenase- and monooxygenase-catalysed sulfoxidation of benzo[b]thiophenes

Asymmetric heteroatom oxidation of benzo[b]thiophenes to yield the corresponding sulfoxides was catalysed by toluene dioxygenase (TDO), naphthalene dioxygenase (NDO) and styrene monooxygenase (SMO) enzymes present in P. putida mutant and E. coli recombinant whole cells. TDO-catalysed oxidation yielded the relatively unstable benzo[b]thiophene sulfoxide; its dimerization, followed by dehydrogenation, resulted in the isolation of stable tetracyclic sulfoxides as minor products with cis-dihydrodiols being the dominant metabolites. SMO mainly catalysed the formation of enantioenriched benzo[b]thiophene sulfoxide and 2-methyl benzo[b]thiophene sulfoxides which racemized at ambient temperature. The barriers to pyramidal sulfur inversion of 2- and 3-methyl benzo[b]thiophene sulfoxide metabolites, obtained using TDO and NDO as biocatalysts, were found to be ca.: 25-27 kcal mol(-1). The absolute configurations of the benzo[b]thiophene sulfoxides were determined by ECD spectroscopy, X-ray crystallography and stereochemical correlation. A site-directed mutant E. coli strain containing an engineered form of NDO, was found to change the regioselectivity toward preferential oxidation of the thiophene ring rather than the benzene ring.     

Use of Chemically Bonded β‐Cyclodextrin Silica Stationary Phase for Liquid Chromatographic Separation of Structural Isomers

The retention behavior of five disubstituted benzene derivatives and two naphthalene derivatives is examined by using a chemically bonded β‐cyclodextrin silica stationary phase with the moiety containing the s‐triazine. The chromatographic results of five disubstituted benzene derivatives and two naphthalene derivatives show that effective separation is achieved on this stationary phase by high‐performance liquid chromatography. The results of the present investigation indicate that the formation of inclusion complexes plays a dominant role in the separation mechanism. However, the selectivity can be significantly enhanced by the n‐n interactions between the s‐triazine ring of the chemically bonded β‐cyclodextrin silica stationary phase and the aromatic ring of solutes. For example, the effective separation of the o‐, m‐, and p‐toluidine isomers on this stationary phase with the moiety containing the s‐triazine ring was better than on that of some β‐cyclodextrin bonded stationary phases without the moiety containing s‐triazine ring.     

Naphthalene sulphonic acids--new test compounds for characterization of the columns for reversed-phase chromatography

Non-substituted naphthalene sulphonic acids are strong acids, which are completely ionised in aqueous and aqueous-organic solutions. Because of repulsive electrostatic interactions, they are more or less excluded from the pores of the column packing materials commonly used in reversed-phase chromatography. The ionic exclusion can be suppressed by increasing the ionic strength of the mobile phase. In aqueous sodium sulphate solutions, very good selectivity was observed for isomeric naphthalene di- and tri-sulphonic acids, allowing reversed-phase separations of these strongly ionic compounds without addition of ion-pairing reagents to the mobile phase. The retention of the isomeric acids increases proportionally to the dipole moment, which can be explained by its effect on increasing exposure of the naphthalene ring to hydrophobic interactions with the non-polar stationary phases. Chromatographic behaviour of isomeric naphthalene di- and trisulphonic acids was investigated on 25 different columns for reversed-phase chromatography. The elution order of the isomers is the same on all the columns, but very strong stationary phase effects were observed on the retention and on the band asymmetry, depending on polar interactions with residual silanol groups and other polar adsorption centres in the stationary phases. These effects are independent of the organic solvents, as the tests are performed in purely aqueous mobile phases and allow classification of the columns into several groups.     

Oxygen uptake rate in production of xylitol by Candida guilliermondii with different aeration rates and initial xylose concentrations

The global oxygen uptake rate (OUR) and specific oxygen uptake rates (SOUR) were determined for different values of the volumetric oxygen mass transfer coefficient (15, 43, and 108 h⁻¹), and for varying initial xylose concentrations (50, 100, 150, and 200 g/L) in shaking flasks. The initial cell concentration was 4.0 g/L, and there was only significant growth in the fermentation with the highest oxygen availability. In this condition, OUR increased proportionally to cell growth, reaching maximum values from 2.1 to 2.5 g of O₂/(L·h) in the stationary phase when the initial substrate concentration was raised from 50 to 200 g/L, respectively. SOUR showed different behavior, growing to a maximum value coinciding with the beginning of the exponential growth phase, after which point it decreased. The maximum SOUR values varied from 265 to 370 mg of O₂/(g of cell·h), indicating the interdependence of this parameter and the substrate concentration. Although the volumetric productivity dropped slightly from 1.55 to 1.18 g of xylitol/(L·h), the strain producing capacity (γ _P/X ) rose from 9 to 20.6 g/g when the initial substrate concentration was increased from 50 to 200 g/L. As for the xylitol yield over xylose consumed (γ _P/S ), there was no significant variation, resulting in a mean value of 0.76 g/g. The results are of interest in establishing a strategy for controlling the dynamic oxygen supply to maximize volumetric productivity.     

Synthesis and Applications of a Novel 3,4-<Emphasis Type="Italic">Bis</Emphasis>(2-Fluoro-5-Trifluoromethyl Phenyl)-2,5-Diphenyl Phenyl Grafted Polysiloxane Stationary Phase

A novel polyphenyl-grafted polysiloxane stationary phase named 3,4-bis(2-fluoro-5-(trifluoromethyl)phenyl)-2,5-diphenyl phenyl grafted polysiloxane stationary phase (FFMP) was synthesized through a Diels–Alder reaction with a high column efficiency (average number of plates: 3700 plates/m; achieved by naphthalene at 120 °C) and simultaneously coated on fused silica capillary tubes to prepare a gas chromatographic column with excellent performance. The column performance test results indicated that the FFMP columns could work properly up to 360 °C, as evidenced by the chromatogram of the polyethylene pyrolysis mixture. The thermogravimetric analysis curve showed that the decomposition temperature of the FFMP was up to 380 °C. The FFMP columns were also applied in the separation and analysis of multimixtures, such as Grob test mixtures, benzene mixtures and fatty acid esters, and as well as a medium polar stationary phase (according to the results of McReynolds constants, the sum of ?I was 779.) The FFMF columns exhibited excellent separation selectivity for these substances because of the conjugated system formed by the polyphenyl side chain connected by single bonds. This conjugated system can promote the delocalization of π-electrons as well as enhance the forces of π–π interaction, and the dipole-induced dipole action between the FFMP stationary phase and the analytes.     

MITOCHONDRIAL DNA SYNTHESIS IN NON-GROWTH CONDITION (LIQUID HOLDING) IN Saccharomyces cerevisiae. RELATION WITH GROWTH STAGES and MITOCHONDRIAL DNA REPAIR AFTER UV-IRRADIATION

Abstract— The synthesis of DNA as measured by incorporation of [¹⁴C]adenine in Saccharomyces cerevisiae liquid held in non-growth conditions was followed in controls and UV-irradiated cells. The incorporation into mitochondrial DNA relative to total DNA was higher in these conditions than that observed in growth medium, especially in liquid held stationary phase cells. The absolute amount of mitochondrial DNA synthesized during liquid holding was larger in exponential than in stationary phase cells and increased after UV-irradiation.
The data reported here are discussed in relation to the effects of liquid holding on the UV-induction of rho ⁻ mutants, such effects depending upon the growth stage of the cells at the time of irradiation. A correlation has been found between the initial ability of the cells to synthesize mitochondrial DNA in liquid holding conditions and their capacity to repair UV-induced lesions in this DNA. We propose the hypothesis that the opposite effects of liquid holding on the UV-induction of rho ⁻ mutants observed in exponential versus stationary phase cells are not due to the action of different repair pathways, but result essentially from quantitative differences in mtDNA synthesis.     

Determination of 3-nitrobenzanthrone in surface soil by normal-phase high-performance liquid chromatography with fluorescence detection

A sensitive method for determining 3-nitrobenzanthrone in surface soil was developed. 3-Nitrobenzanthrone was reduced to 3-aminobenzanthrone by refluxing at 60 degrees C with hydrazine and Raney nickel for 20 min, and 3-aminobenzanthrone was determined by normal-phase high-performance liquid chromatography (HPLC) with fluorescence detection. We used a cyanopropyl stationary phase and an n-hexane-ethyl acetate (3:1, v/v) mobile phase, since 3-aminobenzanthrone exhibits fluorescence in a low-polarity solvent such as n-hexane or ethyl acetate, but not in a polar solvent such as water or methanol. The calibration graph showed good linearity (r2>0.9999) in the range of 0.002-2 ng, and the detection limit was 0.002 ng (S/N=3). 3-Nitrobenzanthrone in extracts from surface soil collected in the Chubu area (central area) of Japan was determined after clean-up using silica gel chromatography and high-performance liquid chromatography on a pyrenylethyl stationary phase. The concentration of 3-nitrobenzanthrone in surface soil was determined in the range of 1.2-1020 pg/g soil.     

Optimization of biosurfactant lipopeptide production from Bacillus subtilis S499 by Plackett-Burman design

Bacillus subtilis S499 is well-known for its ability to produce two families of surfactant lipopeptides: Iturin A and Surfactin S1. Fermentation optimization for this strain was performed to amplify the surfactant production. Ten active variables were analyzed by two successive Plackett-Burman designs, consisting respectively of 12 and 16 experiments to give an optimized medium. The amount of biosurfactant lipopeptides in the supernatant of a culture carried out in this optimized medium was about five times higher than that obtained in nonoptimized rich medium. The analysis of the surfactant molecules produced in such optimized conditions has revealed the presence of a third family of lipopeptides: the fengycins. The time-dependent production of these three families of molecules in bioreactors showed that surfactin S1 is produced during the exponential phase and iturin A and fengycins during the stationary phase.     

反相液相色谱法分离2-(4-羟基苯氧基)丙酸酯对映体

合成了三-(4-甲基苯甲酸)纤维素酯(MCTB)手性固定相，用反相高效液相色谱法在该手性固定相上对2-(4-羟基苯氧基)丙酸酯对映体进行拆分．实验结果表明，在三-(4-甲基苯甲酸)纤维素酯手性固定相上，以甲醇与水的体积分数为75：25做流动相能较好的拆分2-(4-羟基苯氧基)丙酸甲酯、乙酯和丁酯对映体，其分离因子分别为1．38、1．49、0．98；同时还发现2-(4-羟基苯氧基)丙酸酯中酯基团的大小对其对映体的分离也有明显的影响，其中以乙酯的拆分效果最佳。     

咪唑侧基功能化聚离子液体修饰的混合模式色谱固定相的制备及其色谱性能

该文合成了咪唑侧基功能化的离子液体单体1-(4-乙烯基苄基)-3-氰甲基溴化咪唑盐,通过表面引发原子转移自由基聚合将该单体接枝到硅胶表面,制备了一种新型混合模式色谱固定相。采用红外光谱、元素分析及热重分析对其结构进行表征。该色谱固定相具有良好的分离能力。通过研究流动相pH对物质保留的影响,验证了物质在该固定相上存在反相-离子交换保留机理。通过与十八烷基硅烷键合硅胶固定相比较,证实了该聚离子液体固定相对物质保留提供了π-π作用。结果表明,对咪唑侧基功能化是制备新型离子液体固定相的可行方法。     

Effect of oxidizing gas pressure on laboratory-scale decontamination of soils polluted by hydrocarbons

Three standard European soils were artificially contaminated with hexachlorobenzene, 4-chlorobiphenyl, and naphthalene. Small piles (ca. 30 mg) of contaminated soil, or neat soil in control runs, were then heated in the crucible of a thermogravimetric analyzer from room temperature to ca. 450°C at 5°C/min. To investigate effects of ambient gas pressure on contaminants removal, soil specimens were subjected to closely similar heating schedules under either 0.1 or 0.01 MPa pressure of air. The lower pressure augmented decontamination, reducing by as much as 20–45°C the temperature necessary for a given extent of pollutant removal, and increasing the maximum rate of decontamination. The precise magnitude and duration of such pressure-induced improvements in decontamination varied with pollutant and soil type. Predictions of a contaminant evaporation-diffusive transport model were in reasonable agreement with experimentally observed pressure trends. Higher diffusion coefficients for pollutant vapor under reduced pressure are believed to be responsible for the observed pressure effects.