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.     

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.     

Octacyanomolybdate-doped-poly(4-vinylpyridine) ionomer film electrode for the electrocatalytic oxidation of <Emphasis Type="SmallCaps">l</Emphasis>-ascorbic acid

Poly(4-vinylpyridine) (PVP)-based anion exchange polymers are not studied as much as cation exchange polymers Nafion and Eastman Kodak AQ for electroanalytical applications. Similarly, octacyanomolybdate [Mo(CN)₈ ⁴⁻] has not been studied much as a redox mediator. This communication presents results from examinations of the behaviour of Mo(CN)₈ ⁴⁻-doped PVP ionomer film electrode to highlight the opportunities for realization of the application of this composite electrode for l-ascorbic acid (AH₂) estimation via electrocatalytic mediation in acidic medium. The modified electrodes were characterized by cyclic voltammetry and rotating disc electrode voltammetry. PVP coatings possess strong anion-binding capacity for Mo(CN)₈ ⁴⁻ mediator with an extraction coefficient of 990, and electrostatically cross-linked PVP films offer insignificant resistance to permeation of AH₂, facilitating a cross-exchange reaction between the substrate and the mediator in the entire film volume. They show effective electrocatalytic oxidation of AH₂, with the oxidation potential of AH₂ decreased by ∼200 mV in overpotential compared to that at bare electrode. Mo(CN)₈ ⁴⁻/PVP composite electrode does not respond to the more common interferents of l-ascorbic acid estimation even at high positive potentials. These and several other attractive potentialities of the modified electrode are demonstrated by direct determination of AH₂ in a commercial vitamin C tablet without any special treatment, with the value closely agreeing (±0.75%) with the reference method.     

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.     

Kinetics and mechanism of the reaction of hydroxopentaaquarhodium(III) ion with <Emphasis Type="SmallCaps">l</Emphasis>-Arginine in aqueous solution

The kinetics of the aqua ligand substitution from hydroxopentaaquarhodium(III) ion, [Rh(H₂O)₅(OH)]²⁺, by l-Arginine has been studied spectrophotometrically as a function of Arginine concentration, and temperature, at pH 4.3. The reaction proceeds via a rapid outer sphere association complex formation step followed by two consecutive steps. The first of these involves ligand-assisted anation, while the second involves chelation as the second aqua ligand is displaced. The association equilibrium constant for the outer sphere complex formation has been evaluated together with the rate constants for the two subsequent steps. The activation parameters for both steps have been evaluated using Eyring’s equation. Thermodynamic parameters calculated from the temperature dependence of the outer sphere association equilibrium constants are also consistent with an associative mode of activation. The product of the reaction has been characterized by conductivity measurement and IR spectroscopic analysis.     

Studies on electron transfer reactions: reduction of heteropoly 10-tungstodivanadophosphate by <Emphasis Type="SmallCaps">l</Emphasis>-cysteine in aqueous acid medium

l-cysteine undergoes facile electron transfer with heteropoly 10-tungstodivanadophosphate, [ \textPV^\textV \textV^\textV \textW _{1 0} \textO _{4 0} ]^{5 -} , \left[ {{\text{PV}}^{\text{V}} {\text{V}}^{\text{V}} {\text{W}}_{ 1 0} {\text{O}}_{ 4 0} } \right]^{5 - } , at ambient temperature in aqueous acid medium. The stoichiometric ratio of [cysteine]/[oxidant] is 2.0. The products of the reaction are cystine and two electron-reduced heteropoly blue, [PV^IVV^IVW₁₀O₄₀]⁷⁻. The rates of the electron transfer reaction were measured spectrophotometrically in acetate–acetic acid buffers at 25 °C. The orders of the reaction with respect to both [cysteine] and [oxidant] are unity, and the reaction exhibits simple second-order kinetics at constant pH. The pH-rate profile indicates the participation of deprotonated cysteine in the reaction. The reaction proceeds through an outer-sphere mechanism. For the dianion ⁻SCH₂CH(NH₃ ⁺)COO⁻, the rate constant for the cross electron transfer reaction is 96 M⁻¹s⁻¹ at 25 °C. The self-exchange rate constant for the ^- \textSCH₂ \textCH( \textNH₃ ⁺ )\textCOO ^- \mathord

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.     

Tyrosine alkyl esters as prodrug: the structure and intermolecular interactions of <Emphasis Type="SmallCaps">l</Emphasis>-tyrosine methyl ester compared to <Emphasis Type="SmallCaps">l</Emphasis>-tyrosine and its ethyl and <Emphasis Type="Italic">n</Emphasis>-butyl esters

l-Tyrosine alkyl esters are used as prodrugs for l-tyrosine. Although prodrugs are often designed for their behavior in solution, understanding their solid-state properties is the first step in mastering drug delivery. The crystal structure of l-tyrosine methyl ester has been determined and compared to published structures of l-tyrosine and its ethyl and n-butyl esters. It is almost isostructural with the other esters: it crystallizes in the orthorhombic chiral space group P2₁2₁2₁, a = 5.7634(15) Å, b = 12.111(2) Å, c = 14.3713(19) Å, V = 1003.1(4) Å³ with Z′ = 1. Their main packing motif is a C(9) infinite hydrogen-bond chain, but the conformation of l-tyrosine methyl ester is different from the other two: eclipsed versus U-shaped, respectively. The published structure of the ethyl ester, which was incomplete, has been confirmed by X-ray powder diffraction data. Because l-tyrosine methyl ester is very stable (28 years stored at room temperature), and its hydrolysis rate is relatively low, it should be one of the better prodrugs among the alkyl esters of tyrosine.     

Development of an Amperometric Biosensor Platform for the Combined Determination of <Emphasis Type="SmallCaps">l</Emphasis>-Malic,Fumaric, and <Emphasis Type="SmallCaps">l</Emphasis>-Aspartic Acid

Three amperometric biosensors have been developed for the detection of l-malic acid, fumaric acid, and l -aspartic acid, all based on the combination of a malate-specific dehydrogenase (MDH, EC 1.1.1.37) and diaphorase (DIA, EC 1.8.1.4). The stepwise expansion of the malate platform with the enzymes fumarate hydratase (FH, EC 4.2.1.2) and aspartate ammonia-lyase (ASPA, EC 4.3.1.1) resulted in multi-enzyme reaction cascades and, thus, augmentation of the substrate spectrum of the sensors. Electrochemical measurements were carried out in presence of the cofactor β-nicotinamide adenine dinucleotide (NAD⁺) and the redox mediator hexacyanoferrate (III) (HCFIII). The amperometric detection is mediated by oxidation of hexacyanoferrate (II) (HCFII) at an applied potential of + 0.3 V vs. Ag/AgCl. For each biosensor, optimum working conditions were defined by adjustment of cofactor concentrations, buffer pH, and immobilization procedure. Under these improved conditions, amperometric responses were linear up to 3.0 mM for l-malate and fumarate, respectively, with a corresponding sensitivity of 0.7 μA mM^?1 (l-malate biosensor) and 0.4 μA mM^?1 (fumarate biosensor). The l-aspartate detection system displayed a linear range of 1.0–10.0 mM with a sensitivity of 0.09 μA mM^?1. The sensor characteristics suggest that the developed platform provides a promising method for the detection and differentiation of the three substrates.     

Mechanistic investigations on the oxidation of <Emphasis Type="SmallCaps">l</Emphasis>-valine by diperiodatocuprate(III) in aqueous alkaline medium: a kinetic model

The oxidation of l-valine (l-val) by diperiodatocuprate(III) (DPC) in aqueous alkaline medium at a constant ionic strength of 3.0 × 10⁻³ mol dm⁻³ was studied spectrophotometrically at 298 K and follows the rate law;

Rapid Determination of <Emphasis Type="SmallCaps">l</Emphasis>-Glutamine using Engineered <Emphasis Type="Italic">Escherichia coli</Emphasis> Overexpressing Glutamine Synthetase

A genetically engineered Escherichia coli was developed as the source of enzyme for rapidly quantifying glutamine. E. coli BL21 (DE3) cells overexpressing a glutamine synthetase from Bacillus subtilis were prepared as tube aliquots and used in a small volume of nontoxic mixture. The current method was compared to high performance liquid chromatography analysis, Sigma kit (GLN-1) and Mecke method. The method is applicable to a wide range of glutamine concentrations (0.05–2.5 mM) and correlates well to the detection results obtained from high performance liquid chromatography (Pearson correlation is 0.978 at the 0.01 level). Moreover, the whole assay procedure takes less than 15 min and uses nontoxic reagents, so it can be applied to monitor glutamine production and utilization conveniently.     

A Comprehensive Review on <Emphasis Type="SmallCaps">l</Emphasis>-Asparaginase and Its Applications

l-asparaginase (LA) catalyzes the degradation of asparagine, an essential amino acid for leukemic cells, into ammonia and aspartate. Owing to its ability to inhibit protein biosynthesis in lymphoblasts, LA is used to treat acute lymphoblastic leukemia (ALL). Different isozymes of this enzyme have been isolated from a wide range of organisms, including plants and terrestrial and marine microorganisms. Pieces of information about the three-dimensional structure of l-asparaginase from Escherichia coli and Erwinia sp. have identified residues that are essential for catalytic activity. This review catalogues the major sources of l-asparaginase, the methods of its production through the solid state (SSF) and submerged (SmF) fermentation, purification, and characterization as well as its biological roles. In the same breath, this article explores both the past and present applications of this important enzyme and discusses its future prospects.     

Kinetics and mechanism of interaction of hexaaquachromium(III) with <Emphasis Type="SmallCaps">l</Emphasis>-Dopa in aqueous medium: comparative antiparkinsonian studies

The kinetics and mechanism of the substitution reaction between [Cr(H₂O)₆]³⁺ and l-Dopa in aqueous medium has been studied over the range 1.8 ≤ pH ≤ 2.6, 1.68 × 10⁻² mol dm⁻³ ≤ [Dopa] ≤ 5.04 × 10⁻² mol dm⁻³, I = 0.1 mol dm⁻³ (KNO₃) at 50 °C. The reaction takes place via an outer sphere association between Cr³⁺ and l-Dopa followed by chelation. The product was characterized by physicochemical and infrared spectroscopic methods. The antiparkinsonian activity of the product was found to be higher than that of l-Dopa.     

Xymedon conjugate with biogenic acids. Antioxidant properties of a conjugate of Xymedon with <Emphasis Type="SmallCaps">l</Emphasis>-ascorbic acid

Antiradical activity and antioxidant properties of a conjugate of Xymedon with l-ascorbic acid were studied. In contrast to Xymedon, a conjugate of Xymedon with l-ascorbic acid was found to react with free radicals. Pro-oxidant activity of the conjugate of Xymedon with l-ascorbic acid in a chemiluminescence system is weaker as compared to individual l-ascorbic acid. This is the evidence of the increase in the stability of ascorbic acid upon conjugation with a molecule of Xymedon. Conjugate of Xymedon with l-ascorbic acid facilitates the decrease in the concentration of a lipid peroxidation product (malondialdehyde) and the activity of superoxide dismutase in liver and serum of laboratory animals exposed to intoxication with a known hepatotropic toxin CCl₄. This observation shows the contribution of the antioxidant action to the hepatoprotective effect of the studied Xymedon derivative.     

Mechanistic aspects of ligand substitution on <Emphasis Type="Italic">cis</Emphasis>-diaqua(<Emphasis Type="Italic">cis</Emphasis>-1,2-diaminocyclohexane)platinum(II) by Glycine-<Emphasis Type="SmallCaps">l</Emphasis>-Leucine

The kinetics of the interaction of glycine-l-leucine (Glyleu) with cis-[Pt(cis-dach)(OH₂)₂]²⁺ (dach = 1,2-diaminocyclohexane) has been studied spectrophotometrically as a function of [cis-[Pt(cis-dach)(OH₂)₂]²⁺], [Glyleu] and temperature at pH 4.0, where the complex exists predominantly as the diaqua species and Glyleu as a zwitterion. The substitution reaction shows two consecutive steps: the first is the ligand-assisted anation and the second is the chelation step. The activation parameters for both the steps were evaluated using Eyring’s equation. The low ∆H₁^≠ (51.9 ± 2.8 kJmol⁻¹) and large negative value of ∆S₁^≠ (−152 ± 8 JK⁻¹mol⁻¹) as well as ∆H₂^≠ (54.4 ± 1.7 kJmol⁻¹) and ∆S₂^≠ (−162 ± 5 JK⁻¹mol⁻¹) indicate an associative mode of activation for both the aqua ligand substitution processes.     

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).     

Studies on synthesis and in vitro biodegradability of novel optically active nanostructure poly(ester-imide)s containing <Emphasis Type="SmallCaps">l</Emphasis>-phenylalanine and <Emphasis Type="SmallCaps">l</Emphasis>-isoleucine linkages

A series of biodegradable functional amino-acid-based poly(ester-imide)s (PEI)s were designed and synthesized by the direct polycondensation reaction of chiral diacids composed of naturally occurring α-amino acids with 4,4′-thiobis(2-tert-butyl-5-methylphenol) in the presence of tosyl chloride, pyridine, and N,N-dimethylformamide as a condensing agent. These new chiral polymers were characterized with respect to chemical structure and purity using specific rotation experiments, FT-IR, ¹H-NMR, techniques, and elemental analysis. The surface morphology of these polymers was investigated by field emission scanning electron microscopy. The result indicated nanoscale morphology of the obtained polymers. Thermal stability and the weight loss behavior of the resulting PEIs were studied by TGA techniques. All PEIs showed no significant weight loss below 400 °C in a N₂ environment. The monomers and prepared polymers were co-cultivated with airborne fungal spores in culture media to study their biological activity. Soil burial test was also used for evaluation of their biodegradation behavior. The results showed that the synthesized monomers and their derived polymers are biologically active and that their degradation products are probably nontoxic to microbial growth.     

ESI-MS of Peptides in the Presence of <Emphasis Type="SmallCaps">l</Emphasis>-Ascorbic Acid

A mass spectrometric method with electrospray ionisation (ESI-MS) is described for detection and quantification of selected peptides [glutathione, phytochelatin 2, bombesin, salmon luteinising hormone, and peptide fragment Cys(Xx)₁₂]. A reaction coil was used to introduce a solution of sample in mobile phase consisting of acetonitrile (ACN) and an aqueous solution of trifluoroacetic acid (TFA). l-Ascorbic acid (AA), which substantially increased the abundance of the molecular ions of the peptides analysed, was added to aqueous solutions of the peptides or amino acids at a final concentration of 10 mM. This addition probably eliminated the negative effect on the ionisation processes of the strong acid TFA present in the mobile phase, thus enabling analysis of lower concentrations of the peptides.     

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.