Synthesis of <Emphasis Type="Italic">o</Emphasis>(<Emphasis Type="Italic">m</Emphasis>)-carborane-containing azomethines

A method of preparative synthesis of o(m)-carborane-containing azomethines via the condensation of o(m)-carboranyl-C-methylene-4-formylbenzoates with aliphatic, cycloaliphatic, and aromatic amines was developed.     

Excess enthalpies of binary mixtures of <Emphasis Type="Italic">ortho</Emphasis>-, <Emphasis Type="Italic">meta</Emphasis>-, <Emphasis Type="Italic">para</Emphasis>-structural isomers containing aliphatic group

To obtain further systematic information for the isomer systems, the excess molar enthalpies for binary (o + m), (o + p), (m + p)-isomers of methoxymethylbenzene, ethylmethylbenzene, diethylbenzene, chloromethylbenzene, tolunitrile and fluorobenzonitrile, tolylacetonitrile were measured at 298.15 K. In this article, the results are discussed and compared with those of previous works. The excess enthalpies of binary systems in different solid and liquid states were measured when the pure component of o-/m-tolunitrile, fluorobenzonitrile was titrated into the prior (o + p) or (m + p) mixtures. A series calculation for the interaction energies (IE) between the isomers was carried out for the pair molecules by ab initio MO of Gaussian 09. Correlations between the excess enthalpies at a molar fraction of x = 0.5 and the intermolecular energy are discussed.     

The electrochemical polymerization of <Emphasis Type="Italic">o</Emphasis>-phenylenediamine on <Emphasis Type="SmallCaps">l</Emphasis>-tyrosine functionalized glassy carbon electrode and its application

The polymerization of o-phenylenediamine (OPD) on l-tyrosine (Tyr) functionalized glassy carbon electrode (GCE) and its electro-catalytic oxidation towards ascorbic acid (AA) had been studied in this report. l-Tyrosine was first covalently grafted on GCE surface via electrochemical oxidation, which was followed by the electrochemical polymerization of OPD on the l-tyrosine functionalized GCE. Then, the poly(o-phenylenediamine)/l-tyrosine composite film modified GCE (POPD-Tyr/GCE) was obtained. X-ray photo-electron spectroscopy (XPS), field emission scanning electron microscope (SEM), and electrochemical techniques have been used to characterize the grafting of l-tyrosine and the polymerization and morphology of OPD film on GCE surface. Due to the doping of the carboxylic functionalities in l-tyrosine molecules, the POPD film showed good redox activity in neutral medium, and thus, the POPD-Tyr/GCE exhibited excellent electrocatalytic response to AA in 0.1 mol l⁻¹ phosphate buffer solution (PBS, pH 6.8). The anode peak potential of AA shifted from 0.58 V at GCE to 0.35 V at POPD-Tyr/GCE with a greatly enhanced current response. A linear calibration graph was obtained over the AA concentration range of 2.5 × 10⁻⁴–1.5 × 10^–3 mol l⁻¹ with a correlation coefficient of 0.9998. The detection limit (3δ) for AA was 9.2 × 10⁻⁵ mol l⁻¹. The modified electrode showed good stability and reproducibility and had been used for the determination of AA content in vitamin C tablet with satisfactory results.     

Bioconversion of <Emphasis Type="SmallCaps">d</Emphasis>-glucose into <Emphasis Type="SmallCaps">d</Emphasis>-glucosone by Glucose 2-oxidase from <Emphasis Type="Italic">Coriolus versicolor</Emphasis> at Moderate Pressures

Glucose 2-oxidase (pyranose oxidase, pyranose:oxygen-2-oxidoreductase, EC 1.1.3.10) from Coriolus versicolor catalyses the oxidation of d-glucose at carbon 2 in the presence of molecular O₂ producing d-glucosone (2-keto-glucose and d-arabino-2-hexosulose) and H₂O₂. It was used to convert d-glucose into d-glucosone at moderate pressures (i.e. up to 150 bar) with compressed air in a modified commercial batch reactor. Several parameters affecting biocatalysis at moderate pressures were investigated as follows: pressure, [enzyme], [glucose], pH, temperature, nature of fluid and the presence of catalase. Glucose 2-oxidase was purified by immobilized metal affinity chromatography on epoxy-activated Sepharose 6B-IDA-Cu(II) column at pH 6.0. The rate of bioconversion of d-glucose increased with the pressure since an increase in the pressure with compressed air resulted in higher rates of conversion. On the other hand, the presence of catalase increased the rate of reaction which strongly suggests that H₂O₂ acted as inhibitor for this reaction. The rate of bioconversion of d-glucose by glucose 2-oxidase in the presence of either nitrogen or supercritical CO₂ at 110 bar was very low compared with the use of compressed air at the same pressure. The optimum temperature (55°C) and pH (5.0) of d-glucose bioconversion as well as kinetic parameters for this enzyme were determined under moderate pressure. The activation energy (E _a) was 32.08 kJ mol⁻¹ and kinetic parameters (V _max, K _m, K _cat and K _cat/K _m) for this bioconversion were 8.8 U mg⁻¹ protein, 2.95 mM, 30.81 s⁻¹ and 10,444.06 s⁻¹ M⁻¹, respectively. The biomass of C. versicolor as well as the cell-free extract containing glucose 2-oxidase activity were also useful for bioconversion of d-glucose at moderate pressures. The enzyme was apparently stable at moderate pressures since such pressures did not affect significantly the enzyme activity.     

Optimization of Fermentation Conditions for the Biosynthesis of <Emphasis Type="SmallCaps">l</Emphasis>-Threonine by <Emphasis Type="Italic">Escherichia coli</Emphasis>

In this study, the fed-batch fermentation technique was applied to improve the yield of l-threonine produced by Escherichia coli TRFC. Various fermentation substrates and conditions were investigated to identify the optimal carbon source, its concentration and C/N ratio in the production of l-threonine. Sucrose was found to be the optimal initial carbon source and its optimal concentration was determined to be 70 g/L based on the results of fermentations conducted in a 5-L jar fermentor using a series of fed-batch cultures of E. coli TRFC. The effects of glucose concentration and three different feeding methods on the production of l-threonine were also investigated in this work. Our results showed that the production of l-threonine by E. coli was enhanced when glucose concentration varied between 5 and 20 g/L with DO-control pulse fed-batch method. Furthermore, the C/N ratio was a more predominant factor than nitrogen concentration for l-threonine overproduction and the optimal ratio of ammonium sulfate to sucrose (g/g) was 30. Under the optimal conditions, a final l-threonine concentration of 118 g/L was achieved after 38 h with the productivity of 3.1 g/L/h (46% conversion ratio from glucose to threonine).     

Molecular structure of <Emphasis Type="Italic">N</Emphasis>-(<Emphasis Type="Italic">o</Emphasis>-and <Emphasis Type="Italic">p</Emphasis>-hydroxybenzyl)cytisine

The molecular structures of N-(o-and p-hydroxybenzyl)cytisine were investigated by NMR spectroscopy, x-ray structure analysis, and molecular modeling. It was found that NMR resonances of the OH and aromatic protons in N-(o-hydroxybenzyl)cytisine were doubled because of the presence of two conformers in solution. __________ Translated from Khimiya Prirodnykh Soedinenii, No. 2, pp. 165–168, March–April, 2008.     

Temperature- and pressure-responsive properties of <Emphasis Type="SmallCaps">l</Emphasis>- and<Emphasis Type="SmallCaps"> dl</Emphasis>-forms of poly(<Emphasis Type="Italic">N</Emphasis>-(1-hydroxymethyl)propylmethacrylamide) in aqueous solutions

The structural transition of the l- and dl forms of poly(N-(1- hydroxymethyl)propylmethacrylamide (PHMPMA) in aqueous solution was studied by measuring the pressure dependence of the apparent scattering intensity, differential scanning calorimetry (DSC), and circular dichroism (CD). The thermodynamic implications of the results are discussed in relation to the chiral structure of the side chain, and differences in the thermal and barometric transitions. T-P diagrams of the transition showed characteristic ellipsoid features. Antagonism of the temperature and pressure effects was observed only for P(dl-HMPMA). For P(l-HMPMA), the transition temperature (T _tr) decreased with increasing pressure, and the highest T _tr was observed at atmospheric pressure (0.1 MPa). For both polymers, the highest P _trs were observed at the lowest temperatures. The l polymer showed a specific negative peak in its CD spectrum at around 220 nm in the lower temperature region and the temperature dependence was reproduced by a single-step transition, with the midpoint corresponding to the T _tr obtained from the scattering measurements. Coupled with the results from the DSC, the different behavior between the P(l-HMPMA) and P(dl-HMPMA) could be explained in terms of the chain states before and after the transition. The cooperative factors derived from the DSC measurement revealed that about 4 to 5 polymers of the present size were necessary to perform a thermal transition for P(l-HMPMA), and that P(dl-HMPMA) underwent its transition as an almost single molecular event.This revised version was published online in June 2005 with correction to the article category.     

Enhanced Production of <Emphasis Type="SmallCaps">l</Emphasis>-Arginine by Expression of <Emphasis Type="Italic">Vitreoscilla</Emphasis> Hemoglobin Using a Novel Expression System in <Emphasis Type="Italic">Corynebacterium crenatum</Emphasis>

Corynebacterium crenatum SYPA 5-5 is an aerobic and industrial l-arginine producer. It was proved that the Corynebacterium glutamicum/Escherichia coli shuttle vector pJC1 could be extended in C. crenatum efficiently when using the chloramphenicol acetyltransferase gene (cat) as a reporter under the control of promoter tac. The expression system was applied to over-express the gene vgb coding Vitreoscilla hemoglobin (VHb) to further increase the dissolved oxygen in C. crenatum. As a result, the recombinant C. crenatum containing the pJC-tac-vgb plasmid expressed VHb at a level of 3.4 nmol g⁻¹, and the oxygen uptake rates reached 0.25 mg A₅₆₂⁻¹ h⁻¹ which enhanced 38.8% compared to the wild-type strain. Thus, the final l-arginine concentration of the batch fermentation reached a high level of 35.9 g L⁻¹, and the biomass was largely increased to 6.45 g L⁻¹, which were 17.3% and 10.5% higher than those obtained by the wild-type strain, respectively. To our knowledge, this is the first report that the efficient expression system was constructed to introduce vgb gene increasing the oxygen and energy supply for l-arginine production in C. crenatum, which supplies a good strategy for the improvement of amino acid products.     

Extracellular Production and Characterization of Two <Emphasis Type="Italic">Streptomyces</Emphasis><Emphasis Type="SmallCaps">l</Emphasis>-Asparaginases

l-Asparaginase (ASNase) has proved its use in medical and food industries. Sequence-based screening showed the thermophilic Streptomyces strain Streptomyces thermoluteus subsp. fuscus NBRC 14270 (14270 ASNase) to positive against predicted ASNase primary sequences. The 14270 ASNase gene and four l-asparaginase genes from Streptomyces coelicolor, Streptomyces avermitilis, and Streptomyces griseus (SGR ASNase) were expressed in Streptomyces lividans using a hyperexpression vector: pTONA5a. Among those genes, only 14270 ASNase and SGR ASNase were successful for overexpression and detected in culture supernatants without an artificial signal peptide. Comparison of the two Streptomyces enzymes described above demonstrated that 14270 ASNase was superior to SGR ASNase in terms of optimum temperature, thermal stability, and pH stability.     

Enhanced <Emphasis Type="SmallCaps">l</Emphasis><Emphasis Type="Italic">-</Emphasis>(+)-Lactic Acid Production by an Adapted Strain of <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> using Corncob Hydrolysate

Corncob is an economic feedstock and more than 20 million tons of corncobs are produced annually in China. Abundant xylose can be potentially converted from the large amount of hemicellulosic materials in corncobs, which makes the crop residue an attractive alternative substrate for a value-added production of a variety of bioproducts. Lactic acid can be used as a precursor for poly-lactic acid production. Although current industrial lactic acid is produced by lactic acid bacteria using enriched medium, production by Rhizopus oryzae is preferred due to its exclusive formation of the l-isomer and a simple nutrition requirement by the fungus. Production of l-(+)-lactic acid by R. oryzae using xylose has been reported; however, its yield and conversion rate are poor compared with that of using glucose. In this study, we report an adapted R. oryzae strain HZS6 that significantly improved efficiency of substrate utilization and enhanced production of l-(+)-lactic acid from corncob hydrolysate. It increased l-(+)-lactic acid final concentration, yield, and volumetric productivity more than twofold compared with its parental strain. The optimized growth and fermentation conditions for Strain HZS6 were defined.     

Thermal properties of amphiphilic biodegradable triblock copolymer of <Emphasis Type="SmallCaps">l</Emphasis>,<Emphasis Type="SmallCaps">l</Emphasis>-lactide and ethylene glycol

Samples of poly(l,l-lactide)-block-poly(ethylene glycol)-block-poly(l,l-lactide) (PLLA-PEG-PLLA) were synthesized from l,l-lactide polymerization using stannous 2-ethylhexanoate, Sn(Oct)₂ as initiator and di-hydroxy-terminated poly(ethylene glycol) (PEG) (M _n  = 4000 g mol⁻¹) as co-initiator. The chemical linkage between the PEG segment and the PLA segments was characterized by Fourier transform infrared spectroscopy (FTIR). Thermogravimetry analysis (TG) revealed the copolymers composition and was capable to show the deleterious effect of an excess of Sn(Oct)₂ in the polymer thermal stability, while Differential Scanning Calorimetry (DSC) allowed the observation of the miscibility between the PLLA and PEG segments in the different copolymers.     

Cloning,Expression, and Characterization of a Novel (<Emphasis Type="Italic">S</Emphasis>)-Specific Alcohol Dehydrogenase from <Emphasis Type="Italic">Lactobacillus kefir</Emphasis>

A gene encoding a novel (S)-specific NADH-dependent alcohol dehydrogenase (LK-ADH) was isolated from the genomic DNA of Lactobacillus kefir DSM 20587 by thermal asymmetric interlaced-polymerase chain reaction. The nucleotide sequence of (S)-LK-ADH gene (adhS) was determined, which consists of an open reading frame of 1,044 bp, coding for 347 amino acids with a molecular mass of 37.065 kDa. After a BLAST similarity search in GenBank database, the amino acid sequence of (S)-LK-ADH showed some homologies to several zinc containing medium-chain alcohol dehydrogenases. This novel gene was deposited into GenBank with the accession number of EU877965. adhS gene was subcloned into plasmid pET-28a(+), and recombinant (S)-LK-ADH was successfully expressed in E. coli BL21(DE3) by isopropyl-β-d-1-thiogalactopyranoside induction. Purified enzyme showed a high enantioselectivity in the reduction of acetophenone to (S)-phenylethanol with an ee value of 99.4%. The substrate specificity and cofactor preference of recombinant (S)-LK-ADH were also tested.     

<Emphasis Type="Italic">o</Emphasis>-Semiquinone metal complexes as derivatives of sterically hindered di-<Emphasis Type="Italic">o</Emphasis>-quinone

ESR spectroscopy was used to study a series of mono- and binuclear o-semiquinone metal complexes, viz., derivatives of sterically hindered di-o-quinone 1,2-bis(2,5-di-tert-butylcyclohexadiene-1,5-dion-3,4-yl)ethane (1). Hindered rotation was found in the o-semiquinone moiety of the generated complexes. Magnetochemical measurements for polymeric bis(o-semiquinolate)copper(II) based on di-o-quinone 1 showed two antiferromagnetic channels of interaction between the paramagnetic centers. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 7, pp. 1580–1584, July, 2005.     

Optical enzyme sensor for determining <Emphasis Type="SmallCaps">l</Emphasis>-lysine content using <Emphasis Type="SmallCaps">l</Emphasis>-lysine oxidase from the rockfish <Emphasis Type="BoldItalic">Sebastes schlegeli</Emphasis>

l-Lysine (l-Lys) in living bodies is critical for metabolism; therefore, determination of its levels in food is important. Most enzymatic methods for l-Lys analysis are performed using l-lysine oxidase (LyOx), but commercially manufactured LyOx is generally not highly selective for l-Lys among amino acids. We previously isolated LyOx as an antibacterial protein secreted from the skin of the rockfish Sebastes schlegeli. In the present study, we developed an optical enzyme sensor system for rapid and continuous determination of l-Lys using this LyOx. The system comprised an immobilized LyOx membrane, an optical oxygen probe, a flow system, and a personal computer. The amount of l-Lys was detected as a decrease in the oxygen concentration due to the LyOx reaction. The specificity of the sensor was examined against various amino acids. The sensor response was specific for l-Lys. Good reproducibility was obtained in 58 assays. The response of the sensor using commercially prepared LyOx was unstable compared with the response using LyOx isolated in our laboratory. Our sensor system could be used for 5 weeks without our having to change the enzyme membrane. The calibration curve for a standard l-Lys solution was linear from 0.1 to 3.0 mmol L⁻¹. One assay could be completed within 2 min. The sensor was applied to determine the l-Lys content in food samples such as bonito cooking water and scallop hepatopancreas. The values obtained using the sensor and conventional high-performance liquid chromatography methods were well correlated.     

Enthalpy characteristics of dissolution of <Emphasis Type="SmallCaps">l</Emphasis>-cysteine and <Emphasis Type="SmallCaps">l</Emphasis>-asparagine in aqueous solutions of acetonitrile and dimethyl sulfoxide at 298.15 K

The integral enthalpies of dissolution Δ_sol H ^m of l-cysteine and l-asparagine in mixtures of water with acetonitrile and dimethyl sulfoxide at the concentration of organic solvent up to 0.32 molar fractions were measured by means of dissolution calorimetry. The standard enthalpies of dissolution (Δ_sol H°) and transfer (Δ_trans H°) of the amino acids from water to a mixed solvent were calculated. The enthalpy coefficients of pair interactions for L-cysteine and L-asparagine with cosolvent molecules are positive, except for the L-asparagine-water-acetonitrile system. The concepts on the prevailing effect of specific interactions in solutions and the influence of the nature of the cosolvents and lateral substituents of the amino acids on the thermochemical characteristics of dissolution were used to explain the data obtained.     

High Soluble Expression of <Emphasis Type="SmallCaps">d</Emphasis>-Amino Acid Oxidase in <Emphasis Type="Italic">Escherichia coli</Emphasis> Regulated by a Native Promoter

To express high-active soluble d-amino acid oxidase (DAAO), a constitutive plasmid that is regulated by a native hydantoinase promoter (P_Hase), was constructed. A d-amino acid oxidase gene (dao) was ligated with the P_Hase and cloned into pGEMKT to constitutively express protein of DAAO without the use of any inducer such as isopropyl β-d-1-thiogalactopyranoside which is poisonous to the cells and environment. The ribosome binding site region, host strain, and fermentation conditions were optimized to increase the expression level. When cultivated in a 5-m³ fermenter, the enzyme activity of JM105/pGEMKT-R-DAAO grown at 37 °C was found to be 32 U/mL and increase 16-fold over cells of BL21(DE3)/pET-DAAO grown at 28 °C. These results indicate the success of our approaches to overproducing DAAO in soluble form in Escherichia coli.     

<Emphasis Type="Italic">Z</Emphasis>-and <Emphasis Type="Italic">E</Emphasis>-conformers of <Emphasis Type="Italic">N</Emphasis>-chloroacetylcytisine

N-Chloroacetylcytisine was synthesized by acylation of (–)-cytisine. Stable Z- and E-conformers with respect to rotational isomerism around the N-12–CO bond were found in PMR spectra at room temperature. The point at which PMR resonances of the Z- and E-conformers coalesced upon heating was measured. The transition barrier between the conformers was estimated.     

Determination of serum <Emphasis Type="SmallCaps">d</Emphasis>-lactic and <Emphasis Type="SmallCaps">l</Emphasis>-lactic acids in normal subjects and diabetic patients by column-switching HPLC with pre-column fluorescence derivatization

d-Lactic and l-lactic acids were simultaneously determined by means of a column-switching high-performance liquid chromatography (HPLC) with fluorescence detection. As a fluorescence reagent, 4-nitro-7-piperazino-2,1,3-benzoxadiazole (NBD-PZ) was employed for the fluorescence derivatization of lactic acid. The proposed HPLC system adopted both octylsilica (Cadenza CD-C8) and amylose-based chiral columns (CHIRALPAK AD-RH), which proved to give a sufficient enantiomeric separation of the lactic acid derivatives with a separation factor () of 1.32 and a resolution (R_s) of 1.98. Moreover, the features of the first elution of d-lactic acid peak in the proposed HPLC were convenient for the determination of trace amount of serum d-lactic acid, which is known to increase under diabetes. Intra-day and inter-day accuracies were in the range of 90.5–101.2 and 89.0–100.7%, and the intra-day and inter-day precisions were 0.3–1.2 and 0.4–4.8%, respectively. The proposed method was applied to determine d-lactic and l-lactic acids in human serum of normal subjects and diabetic patients, showing that both d-lactic and l-lactic acid concentrations were significantly increased in the serum of diabetic patients (n=31) as compared with normal subjects (n=21). This fact was found for the first time owing to the development of the proposed HPLC method which is able to determine d-lactic and l-lactic acid simultaneously. Finally, serum d-lactic acid concentrations determined by the proposed HPLC method were compared with those from a reported enzymatic assay, and the smaller p value between normal subjects and diabetic patients was shown by the proposed HPLC method.     

Mixtures poly((<Emphasis Type="Italic">R</Emphasis>)-3-hydroxybutyrate) and poly(<Emphasis Type="SmallCaps">l</Emphasis>-lactic acid) subjected to DSC

Blends of bacterial poly((R)-3-hydroxybutyrate) (PHB) and poly(l-lactic acid) (PLLA) synthesized by polycondensation of l-lactic acid or by ring-opening polymerization of l-lactide were studied. Miscibility was investigated through both conventional differential scanning calorimetry (DSC) and temperature-modulated DSC (TMDSC). PHB and low-molar mass PLLA were miscible in a whole concentration range, and a single glass transition temperature was observed. On the other hand, PHB/high-molar mass PLLA mixtures phase separate, giving rise to two glass transition temperatures corresponding to PHB and PLLA. A treatment of blends at 190 °C leads to formation of block/multiblock/random copolymers, and blends become miscible.     

Acid-base and complexation properties of <Emphasis Type="Italic">gem</Emphasis>-diphosphonic and <Emphasis Type="Italic">gem</Emphasis>-diphosphinic acids in aqueous solutions and micellar media of surfactants

Acid-base and complexation properties of gem-disubstituted phosphorus acids, viz., methylenediphosphonic, 1-hydroxyethylidene-1,1-diphosphonic, P,P-diphenylmethylene-diphosphinic, and P, P-diphenyl-1-hydroxyethylidene-1, 1-diphosphinic acids, were studied in aqueous solutions and in the presence of biomimetics (micelles of ionic surfactants). The dissociation constants of the acids and stability constants of complexes with magnesium(II) and copper(II) ions were determined in aqueous solutions and microheterogeneous media containing sodium dodecyl sulfate, cetylpyridinium chloride, or cetylpyridinium nitrate (ionic strength 0.1 mol L^–1, temperature 25°C).Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 9, pp. 1919–1925, September, 2004.