Extracellular oxidation of D-glucose by some members of the Enterobacteriaceae

Extracellular D-glucose oxidation by 5 enterobacterial species was studied with the purpose of selecting conditions useful for taxonomic studies. Extracellular production of gluconate from ¹⁴C-glucose by bacterial cells was evidenced by DEAE-cellulose paper chromatography. Escherichia coli oxidized glucose only when pyrroloquinoline quinone (PQQ) was added, whereas Serratia marcescens, Yersinia frederiksenii, Erwinia cypripedii and Cedecea lapagei oxidized D-glucose without added PQQ. 2-Deoxyglucose was found to be an excellent non-metabolized analogue of D-glucose in oxidation experiments. D-glucose oxidation was inhibited by KCN, p-chloromercuribenzoic acid and carbonyl cyanide m-chlorophenylhydrazone; and activated by p-benzoquinone. Iodoacetate had no action. Comparative cellulose thin-layer chromatography including 2-ketogluconate and 2,5-diketogluconate (produced by Janthinobacterium lividum) as standards, showed that gluconate was oxidized to 2-ketogluconate by S. marcescens and E. cypripedii, and 2-ketogluconate was oxidized to 2,5-diketogluconate by E. cypripedii. The diversity of D-glucose oxidation products in the Enterobacteriaceae could have some taxonomic applications.     

Stereoselective synthesis and absolute configuration of the C33-C42 fragment of symbiodinolide

Cross-metathesis of methyl ester which was prepared from symbiodinolide with ethylene was performed to give the C33-C42 degraded fragment. This fragment was estimated to be (36S,40S)-diol by the modified Mosher method. Stereoselective synthesis of the (36S,40S)-diol and its diastereomer (36R,40S)-diol was achieved from l-aspartic acid. Synthetic bis-(S)- and (R)-MTPA esters which were derivatized from the (36S,40S)-diol exhibited spectroscopic data identical with those of bis-(S)- and (R)-MTPA esters derived from the degraded product. Thus, the absolute stereochemistry of the C33-C42 fragment was elucidated to be (36S,40S).     

Synthesis and characterization of novel vinyl copolymers containing N-vinylphthalimide: Comonomers reactivity ratios and thermal stability

N-Vinylphthalimide (NVPh) was copolymerized with p-methylstyrene (PMS), p-methoxystyrene (PMOS) and p-chlorostyrene (PClS) at 60 °C, with 2,2′-azo-bis-isobutyronitrile as an initiator. Copolymer composition was determined by elemental analysis in case of the N-vinylphthalimide and p-methylstyrene comonomer pair, whereas proton nuclear magnetic resonance was used for the analysis of the two other copolymers.The reactivity ratios for each comonomer pair were estimated by the classical Fineman-Ross and Kelen-Tüdõs linear techniques. These data showed that N-vinylphthalimide was less reactive in all the cases and that the comonomer distribution, that was basically random in the poly(N-vinylphthalimide-co-p-methylstyrene) and poly(N-vinylphthalimide-co-p-chlorostyrene) copolymers, was rather alternate in the third poly(N-vinylphthalimide-co-p-methoxystyrene) copolymer. The difference observed in the reactivity ratios was discussed in reference to the structure of the comonomer units and the parent radicals. The thermal properties of the copolymers and model homopolymers were investigated by differential scanning calorimetry and thermogravimetric analysis.     

Thermodynamic, IR spectral and X-ray diffraction studies of the ����-cyclodextrin-para-aminobenzoic acid�� inclusion complex

The inclusion complex with stoichiometric composition 1:1 was formed as a result of intermolecular interactions between para-aminobenzoic acid and ??-cyclodextrin. The stability constant of ????-cyclodextrin-para-aminobenzoic acid?? inclusion complex at 289, 292 and 313 K was calculated by the Ketelar equation. The influence of temperature on the stability of ????-cyclodextrin-para-aminobenzoic acid?? inclusion complex was also examined, and thermodynamic parameters involved in the complex formation (??G, ??H, ??S) were calculated. Supramolecular complex formation between para-aminobenzoic acid and ??-cyclodextrin was confirmed by X-ray diffraction and IR spectroscopy studies.     

Quantification of benzene,toluene, ethylbenzene and o-xylene in internal combustion engine exhaust with time-weighted average solid phase microextraction and gas chromatography mass spectrometry

A new and simple method for benzene, toluene, ethylbenzene and o-xylene (BTEX) quantification in vehicle exhaust was developed based on diffusion-controlled extraction onto a retracted solid-phase microextraction (SPME) fiber coating. The rationale was to develop a method based on existing and proven SPME technology that is feasible for field adaptation in developing countries. Passive sampling with SPME fiber retracted into the needle extracted nearly two orders of magnitude less mass (n) compared with exposed fiber (outside of needle) and sampling was in a time weighted-averaging (TWA) mode. Both the sampling time (t) and fiber retraction depth (Z) were adjusted to quantify a wider range of C_gas. Extraction and quantification is conducted in a non-equilibrium mode. Effects of C_gas, t, Z and T were tested. In addition, contribution of n extracted by metallic surfaces of needle assembly without SPME coating was studied. Effects of sample storage time on n loss was studied. Retracted TWA–SPME extractions followed the theoretical model. Extracted n of BTEX was proportional to C_gas, t, D_g, T and inversely proportional to Z. Method detection limits were 1.8, 2.7, 2.1 and 5.2 mg m⁻³ (0.51, 0.83, 0.66 and 1.62 ppm) for BTEX, respectively. The contribution of extraction onto metallic surfaces was reproducible and influenced by C_gas and t and less so by T and by the Z. The new method was applied to measure BTEX in the exhaust gas of a Ford Crown Victoria 1995 and compared with a whole gas and direct injection method.     

The use of redox reactions in the analysis of dyes and dye industry intermediates: IV. Oxidation of benzidine,o-tolidine,and o-dianisidine with chloramine T and N-bromosuccinimide

The oxidation of benzidine, o-tolidine, and o-dianisidine with chloramine T and N-bromosuccinimide was studied, the stoichiometry was established, and a mechanism was proposed. An extraction-photometric determination of these diamines was developed, based on the reaction with excess chloramine T or N-bromosuccinimide and the measurement of the absorbance of the N,N′-dihalogeno-diphenoquinonediimines formed, after their extraction into chloroform.     

Viscosity of heavy n-alkanes and diffusion of gases therein based on molecular dynamics simulations and empirical correlations

The viscosity of pure n-alkanes and n-alkane mixtures was studied by molecular dynamics (MD) simulations using the Green–Kubo method. n-Alkane molecules were modeled based on the Transferable Potential for Phase Equilibria (TraPPE) united atom force field. MD simulations at constant number of molecules or particles, volume and temperature (NVT) were performed for n-C₈ up to n-C₉₆ at different temperatures as well as for binary and six-component n-alkane mixtures which are considered as prototypes for the hydrocarbon wax produced during the Gas-To-Liquid (GTL) Fischer–Tropsch process. For the pure n-alkanes, good agreement between our simulated viscosities and existing experimental data was observed. In the case of the n-alkane mixtures, the composition dependence of viscosity was examined. The simulated viscosity results were compared with literature empirical correlations. Moreover, a new macroscopic empirical correlation for the calculation of self-diffusion coefficients of hydrogen, carbon monoxide, and water in n-alkanes and mixtures of n-alkanes was developed by combining viscosity and self-diffusion coefficient values in n-alkanes. The correlation was compared with the simulation data and an average absolute deviation (AAD) of 11.3% for pure n-alkanes and 14.3% for n-alkane mixtures was obtained.     

Yeasts and Lactic Acid Bacteria Mixed-Specie Biofilm Formation is a Promising Cell Immobilization Technology for Ethanol Fermentation

We previously found that some Saccharomyces cerevisiae and Lactobacillus plantarum remarkably formed mixed-specie biofilm in a static co-culture and deduced that this biofilm had potential as immobilized cells. We investigated the application of mixed-specie biofilm formed by S. cerevisiae BY4741 and L. plantarum HM23 for ethanol fermentation in repeated batch cultures. This mixed-specie biofilm was far abundantly formed and far resistant to washing compared with S. cerevisiae single biofilm. Adopting mixed-specie biofilm formed on cellulose beads as immobilized cells, we could produce enough ethanol from 10 or 20 % glucose during ten times repeated batch cultures for a duration of 10 days. Cell numbers of S. cerevisiae and L. plantarum during this period were stable. In mixed-specie biofilm system, though ethanol production was slightly lower compared to S. cerevisiae single-culture system due to by-production of lactate, pH was stably maintained under pH 4 without artificial control suggesting high resistance to contamination. Inoculated model contaminants, Escherichia coli and Bacillus subtilis, were excluded from the system in a short time. From the above results, it was indicated that the mixed-specie biofilm of S. cerevisiae and L. plantarum was a promising immobilized cell for ethanol fermentation for its ethanol productivity and robustness due to high resistance to contamination.     

Acylative kinetic resolution of racemic aromatic β-hydroxy esters catalyzed by chiral nucleophilic N-(1-arylethyl)benzoguanidines

An efficient acylative kinetic resolution of racemic aromatic β-hydroxy esters with cyclohexanecarboxylic anhydride was achieved using newly designed (R)-N-methylbenzoguanidine ((R)-NMBG) derivatives. A series of (R)-NMBG derivatives was synthesized by modifying the original (R)-NMBG catalyst with the introduction of branched N-substituents containing a stereogenic center, and their catalytic performance was evaluated. (R,R)-N-(1-(β-1-Naphthyl)ethyl)benzoguanidine [(R,R)-NβNpEtBG] was found to function as an efficient acyl transfer catalyst for the reaction of a broad variety of substrates, regardless of the substituent type and substitution pattern.     

Agrobacterium-Mediated Transformation in Alpinia galanga (Linn.) Willd. for Enhanced Acetoxychavicol Acetate Production

Agrobacterium-mediated transformations ensure elevated amounts of secondary metabolite accumulation with genetic and biosynthetic stability. In the present study, Alpinia galanga rich in bioactive compounds was genetically transformed using different strains of Agrobacterium rhizogenes viz. LBA 9402, A ₄ , 532, 2364 and PRTGus. Even though a higher growth rate was obtained with the LBA 9402 strain, maximum acetoxychavicol acetate accumulation (ACA) was seen in the PRTGus transformant. PRTGus root line has shown 10.1 fold higher ACA content in comparison to the control roots. The lowest ACA production was shown by the A ₄ transformant (4.9 fold). The quantification of ACA in the transformed roots was carried out by using HPLC, which was found to be in the order of PRTGus > LBA 9402 > 2364 > 532 > A ₄ . The fast growth rate of hairy roots, genetic stability and their ability to synthesize more than one metabolite offer a promising system for the production of valuable secondary metabolites.     

Trihydroxy-2-thiaquinolizidine derivatives as a new class of bicyclic glycosidase inhibitors

Trihydroxy-2-thiaquinolizidines, a new class of bicyclic dideoxy-iminohexitol glycosidase inhibitor derivatives with nominally the d-gluco, l-ido, d-manno and l-gulo configurations were synthesized. X-ray analyses indicated that the preferred conformation for d-gluco and d-manno derivatives was a flat trans-fused system. Unlike deoxynojirimycin, the compound with d-gluco configuration was selective for α-glucosidases (yeast and rice) and showed no inhibitory activity towards β-glucosidase (almond), α-galactosidase (green coffee beans), α-galactosidase (E. coli) and α-mannosidase (jack bean), while the l-ido derivative was specific for β-glucosidase (almond).     

Photoisomerization of octyl methoxycinnamate

The octyl-p-methoxy-trans-cinnamate (E-OMC) was exposed to sunlight to induce the E to Z transformation. Octyl-p-methoxy-cis-cinnamate (Z-OMC) was then purified from the mixture of the E- and the Z-OMC using C-18-semi-preparative HPLC. The UV absorption of the Z configuration at various concentrations in various solvents was measured. Molar absorption coefficient of the compound was then calculated. By using the obtained molar absorption coefficient of Z-OMC and of E-OMC, E to Z photoisomerization of octyl methoxycinnamate (OMC) in various solvents at various concentrations could be monitored by C-18 HPLC using UV detector. The result indicates that equilibrium of photoisomerization depends upon concentration and polarity of the solvent used.     

Copper(II) tetrafluoroborate as a novel and highly efficient catalyst for N-tert-butoxycarbonylation of amines under solvent-free conditions at room temperature

Commercially available copper(II) tetrafluoroborate hydrate was found to be a highly efficient catalyst for chemoselective N-tert-butoxycarbonylation of amines with di-tert-butyl dicarbonate under solvent-free conditions and at room temperature. Various aromatic amines were protected as their N-tert-butyl carbamates in high yields and in short times. No competitive side reactions such as isocyanate, urea, and N,N-di-t-Boc formation was observed. Chemoselective N-tert-butoxycarbonylation was achieved with substrates bearing OH and SH groups. Chiral α-amino acid esters afforded the corresponding N-t-Boc derivatives in excellent yields.     

Para-selectivity of SAPO-5 and mordenite catalysts in the alkylation of cumene with 2-propanol

Alkylation of cumene with 2-propanol was studied on SAPO-5 and mordenite catalysts. The primary products of the alkylation on every catalyst examined here were p- and m-diisopropylbenzene (DIPB) in a ratio of 75:25. The p-DIPB fraction in DIPB isomers (para-selectivity) decreased with increasing yield of DIPB, due to the secondary reaction of p-DIPB to m-DIPB. On SAPO-5, mordenite and silica-alumina, this secondary reaction proceeds through dealkylation of p-DIPB to cumene, followed by re-alkylation of the resultant cumene to m-DIPB. The dealkylation of p-DIPB would occur preferentially over that of m-DIPB. This was due to the higher reactivity of p-DIPB and probably to the reactant molecular shape selectivity of SAPO-5 and mordenite. The para-selectivity was improved by supporting boron oxide on SAPO-5 and mordenite; this improvement was caused by suppression of the secondary dealkylation of p-DIPB.     

The synthesis of (4S,5S)-(−)-isocytoxazone

(4S,5S)-(?)-Isocytoxazone, which is needed for a configurational study, was synthesized from the commercially available (1S,2S)-(+)-2-amino-1-(4-nitrophenyl)-1,3-propanediol in which the p-nitro substituent was replaced by a p-methoxyl group; the thus prepared p-methoxyphenyl amino diol was cyclized via an N-Boc derivative.     

Kinetic Analysis, Expression Pattern, and Production of a Recombinant Fungal Protease Inhibitor of Tasar Silkworm Antheraea mylitta

Antheraea mylitta, a tasar silk-producing insect of Saturniidae family, expresses a fungal protease inhibitor named as A. mylitta fungal protease inhibitor-1 (AmFPI-1). AmFPI-1 inhibits alkaline protease of Aspergillus oryzae but its mechanism of action is not known. To understand the mode of inhibition of AmFPI-1 against the fungal protease, it was purified from the hemolymph of A. mylitta larvae and inhibitory activity against A. oryzae protease was studied. Kinetic analysis of purified AmFPI-1 on alkaline protease of A. oryzae showed that AmFPI-1 acts as a canonical-type competitive inhibitor with equilibrium dissociation constant (K _i ) of 60?nM. Expression of AmFPI-1 in different body tissues of fifth instar A. mylitta larvae was determined by real-time PCR, and the highest expression was observed in fat body followed by integument, silk gland, and gut, indicating that AmFPI-1 has pleiotropic functions including protection from invading fungi. The cDNA of AmFPI-1 was expressed in Escherichia coli, and recombinant His-tagged fusion protein was purified by Ni-NTA chromatography. Recombinant AmFPI-1 showed inhibitory activity against A. oryzae protease and suggested its use in various biological applications to prevent proteolysis.     

An efficient approach to d-threo-3-hydroxyaspartic acid for the synthesis of novel l-threo-oxazolines as selective blockers of glutamate reversed uptake

An efficient, stereoselective synthetic strategy to d-threo-3-hydroxyaspartic acid was developed. Starting from l-(2S,3S)-N-benzoyl-3-hydroxyaspartic acid dimethyl ester by a Deoxo-fluor-catalyzed cyclization reaction, an inversion of configuration at the β-center (erythro isomer), was observed. A base-induced epimerization reaction led to the d-trans-isomer, which was hydrolyzed to give d-threo-3-hydroxyaspartic acid with excellent stereoselectivity and overall yield. Starting from d-threo-3-hydroxyaspartic acid, l-threo-oxazolines can be stereoselectively synthesized.     

Extracellular Enzymes of the White-Rot Fungus Fomes fomentarius and Purification of 1,4-β-Glucosidase

Production of the lignocellulose-degrading enzymes endo-1,4-β-glucanase, 1,4-β-glucosidase, cellobiohydrolase, endo-1,4-β-xylanase, 1,4-β-xylosidase, Mn peroxidase, and laccase was characterized in a common wood-rotting fungus Fomes fomentarius, a species able to efficiently decompose dead wood, and compared to the production in eight other fungal species. The main aim of this study was to characterize the 1,4-β-glucosidase produced by F. fomentarius that was produced in high quantities in liquid stationary culture (25.9 U?ml^?1), at least threefold compared to other saprotrophic basidiomycetes, such as Rhodocollybia butyracea, Hypholoma fasciculare, Irpex lacteus, Fomitopsis pinicola, Pleurotus ostreatus, Piptoporus betulinus, and Gymnopus sp. (between 0.7 and 7.9 U?ml^?1). The 1,4-β-glucosidase enzyme was purified to electrophoretic homogeneity by both anion-exchange and size-exclusion chromatography. A single 1,4-β-glucosidase was found to have an apparent molecular mass of 58 kDa and a pI of 6.7. The enzyme exhibited high thermotolerance with an optimum temperature of 60 °C. Maximal activity was found in the pH range of 4.5–5.0, and K _M and V _max values were 62 μM and 15.8 μmol?min^?1?l^?1, respectively, when p-nitrophenylglucoside was used as a substrate. The enzyme was competitively inhibited by glucose with a K _i of 3.37 mM. The enzyme also acted on p-nitrophenylxyloside, p-nitrophenylcellobioside, p-nitrophenylgalactoside, and p-nitrophenylmannoside with optimal pH values of 6.0, 3.5, 5.0, and 4.0–6.0, respectively. The combination of relatively low molecular mass and low K _M value make the 1,4-β-glucosidase a promising enzyme for biotechnological applications.