Preparation of Optically Active Allothreonine by Separating from a Diastereoisomeric Mixture with Threonine

A simple procedure is described to obtain D- and L-allothreonine (D- and L-aThr). A mixture of N-acetyl-D-allothreonine (Ac-D-aThr) and N-acetyl-L-threonine (Ac-L-Thr) was converted to a mixture of their ammonium salts and then treated with ethanol to precipitate ammonium N-acetyl-L-threoninate (Ac-L-Thr·NH₃) as the less-soluble diastereoisomeric salt. After separating Ac-L-Thr·NH₃ by filtration, Ac-D-aThr obtained from the filtrate was hydrolyzed in hydrochloric acid to give D-aThr of 80% de, recrystallized from water to give D-aThr of >99% de. L-aThr was obtained from a mixture of the ammonium salts of Ac-L-aThr and Ac-D-Thr in a similar manner.     

Structures and Properties of a Diastereoisomeric Molecular Compound of (2S,3S)- and (2R,3S)-N-Acetyl-2-amino-3-methylpentanoic Acids

An X-ray crystal structural analysis revealed that (2S,3S)-N-acetyl-2-amino-3-methylpentanoic acid (N-acetyl-L-isoleucine; Ac-L-Ile) and (2R,3S)-N-acetyl-2-amino-3-methylpentanoic acid (N-acetyl-D-alloisoleucine; Ac-D-aIle) formed a molecular compound containing one Ac-L-Ile molecule and one Ac-D-aIle molecule as an unsymmetrical unit. This molecular compound is packed with strong hydrogen bonds forming homogeneous chains consisting of Ac-L-Ile molecules or Ac-D-aIle molecules and weak hydrogen bonds connecting these homogeneous chains in a fashion similar to that observed for Ac-L-Ile and Ac-D-aIle. Recrystallization of an approximately 1:1 mixture of Ac-L-Ile and Ac-D-aIle from water gave an equimolar molecular compound due to its lower solubility than that of Ac-D-aIle or especially Ac-L-Ile. The results suggest that the equimolar mixture of Ac-L-Ile and Ac-D-aIle could be obtained from an Ac-L-Ile-excess mixture by recystallization from water.     

<Emphasis Type="Italic">N</Emphasis>-Acyl derivatives of Asn,new bacterial <Emphasis Type="Italic">N</Emphasis>-acyl <Emphasis Type="Italic">D</Emphasis>-amino acids with surfactant activity

Summary. New N-acyl D-amino acids were isolated from Bacillus pumilus IM 1801. Their structures were determined by chemical analysis and mass spectrometry. The lipid part was identified as a mixture of fatty acids with 11, 12, 13, 15, and 16 carbon atoms in the iso, anteiso or n configuration linked by an amide bond with a D-asparagine. They exhibited surfactant properties.     

A Metal Ion as a Cofactor Attenuates Substrate Inhibition in the Enzymatic Production of a High Concentration of <Emphasis Type="SmallCaps">D</Emphasis>-glutamate Using <Emphasis Type="Italic">N</Emphasis>-acyl-<Emphasis Type="SmallCaps">D</Emphasis>-glutamate Amidohydrolase

N-Acyl-D-glutamate amidohydrolase (D-AGase) was inhibited by 94 % when 1 mol/l N-acetyl-DL- glutamate was used as a substrate. The addition of 1 mM Co²⁺ stabilized D-AGase. Moreover, the substrate inhibition was weakened to 88% with the addition of 0.4 mM Co²⁺ to the reaction mixture. Although D-AGase is a zinc-metalloenzyme, the addition of Zn²⁺ from 0.01 to 10 mM did not increase the D-glutamic acid production in the saturated substrate. Under optimal conditions, 0.38 M D-glutamic acid was obtained from N-acyl-DL-glutamate with 100% of the theoretical yield after 48 h.     

Kinetics of Thermostable Alanine Racemase of Bacillus stearothermophilus

Unlabeled D- and L-alanine were racemized in deuterium oxide with an alanine racemase of Bacillus stearothermophilus at saturated concentration of substrate, and various p²H and temperature. Samples of the solution were taken at intervals, and all alanine isomers in the samples were transformed into a mixture of diastereomeric derivatives of methyl N-(–)-camphanylalaninate. Their ratio was measured on a GC-Mass, and the relative rate was calculated at the initial stage of the reaction. There was little difference in the decrease rate of the optical rotation between the enantiomers. Internal proton-transfer to the antipode was almost zero for either substrate. The α-hydrogen was abstracted 1.2–2.3 times faster from D-alanine than from L-alanine. D-Alanine gave an almost even mixture of deuterium labeled D- and L-alanine, while L-alanine gave a mixture of labeled D- and L-alanine at a ratio of 3:1. These results suggest the racemase builds two different bases in the active site. The base for D-alanine may be closer to the enzyme surface, and that for L-alanine inside.     

Molecular simulation of shale gas adsorption and diffusion in inorganic nanopores

We studied the structural and dynamical properties of methane and ethane in montmorillonite (MMT) slit pore of sizes 10, 20 and 30 Å using grand canonical Monte Carlo and classical molecular dynamics (MD) simulations. The isotherm, at 298.15 K, is generated for pressures up to 60 bar. The molecules preferentially adsorb at the surface as indicated by the density profile. In case of methane, we observe only a single layer, at the pore wall, whose density increases with increasing pressure. However, ethane also displays a second layer, though of low density in case of pore widths 20 and 30 Å. In-plane self-diffusion coefficient, D_‖, of methane and ethane is of the order of 10^? 6 m²/s. At low pressure, D_‖ increases significantly with the pore size. However, D_‖ decreases rapidly with increasing pressure. Furthermore, the effect of pore size on D_‖ diminishes at high pressure. Ideal adsorbed solution theory is used to understand the adsorption behaviour of the binary mixture of methane (80%) and ethane (20%) at 298.15 K. Furthermore, we calculate the selectivity of the gases at various pressures of the mixture, and found high selectivity for ethane in MMT pores. However, selectivity of ethane decreases with increase in pressure or pore size.     

Design Efficiency and Estimation for Component Amount Models

Component amount models were introduced by Piepel and Cornell (1985) to describe mixture experiments where the response depends not only on the proportions of the components but also on the total amount. The extended simplex centroid design supported by the barycenters corresponding to the regression functions in the model is D-optimal or fails to be D-optimal depending on the regression model under consideration. For two special component amount models, namely the ν-tic polynomial and the minimum polynomial of degree ν, the efficiency of the simplex centroid design was investigated. This covers the presentation of new results concerning the D-optimality as well as the assessment of the simplex centroid design in non-optimal situations by means of the G-efficiency. Formulae for the least squares estimates as well as their variances are presented.     

Kinetics of Bifidobacterium longum ATCC 15707 fermentations: effect of the dilution rate and carbon source

The effect of the dilution rate on biomass and product synthesis in fermentations of glucose, fructose and a commercial mixture of fructooligosaccharides (FOS) by Bifidobacterium longum ATCC 15707 was studied. Kinetic parameters (maximum specific growth rate, Monod constant, maintenance, and yield coefficients) in the mathematical model of the fermentation were estimated from experimental data. In the FOS mixture fermentations, approximately 12% of the total reducing sugars (mainly fructose) in the feed were not metabolized by the bacterium. In fermentations of fructose and the FOS mixture, biomass concentration increased as the dilution rate increased and, once maximum values were reached [3.90 (D=0.20 h^–1) and 2.54 g l^–1 (D=0.15 h^–1), respectively], decreased rapidly as the culture was washed out. Formic acid was detected at low dilution rates in glucose and fructose fermentations. The main products in fermentations of the three carbon sources were lactic and acetic acids. Average values of the molar ratio between acetic and lactic acids of 1.18, 1.21 and 0.83 mol mol^–1 were obtained in glucose, fructose and FOS mixture fermentations, respectively. In batch fermentations carried out without pH control this molar ratio was lower than 1.5 only when fructose was used as the carbon source.     

The fate of vitamin D3 and indolylbutyric acid applied to cuttings of Populus tremula L. during adventitious root formation

Abstract Treatment of green cuttings of Populus tremula L. with vitamin D₃ enhances adventitious rooting and synergy is observed on simultaneous application with indolylbutyric acid. Translocation experiments with labelled vitamin D₃ and IBA, fed to the base of the cuttings, showed that such synergy was not due to an effect of one substance on the translocation of the other. When indolylbutyric acid was fed to the base of the cuttings, a high proportion was rapidly converted into conjugates which, on saponifaction, released inter alia indolylbutyric acid, but no indolylacetic acid. On the other hand, vitamin D₃ was metabolized to give products similar to those produced in non-sterile aqueous solutions of vitamin D₃ stored under the conditions of culture of the cuttings. Such a mixture is as effective as vitamin D₃ itself in promoting adventitious rooting. A preliminary examination of the products showed that extensive oxidation of vitamin D₃ had occurred and some of the individual components were synthesized and tested for their effect on rhizogenesis in P. tremula.     

Effect of mixed organic compounds extracted from <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> on <Emphasis Type="Italic">Daphnia magna</Emphasis> (water flea)

In this study, the potential for Rhodobacter sphaeroides, a purple nonsulfur photosynthetic strain of bacteria, to act as a physiological activation material for water flea, Daphnia magna, was tested under two different culture conditions: i.e. YTC (a 1:1 mixture of yeast: Tetramin: Ceropyl) as a basal diet with Selenastrum capricormutrum, a green microalga, and YTC with physiologically activating compounds (PACs), which were defined as mixed organic compounds produced in R. sphaeroides. The impact of PACs produced in R. sphaeroides was assessed by the viability, growth and reproduction of D. magna until the third generation. Compared to S. capricormutrum, it was found that the number of baby daphnia increased after addition to D. magna of PACs produced from dark-cultured R. sphaeroides. Also, we demonstrated that PACs produced from dark-cultured R. sphaeroides influenced individual D. magna growth parameters, such as body length, survival and number of broods per female. Here, we also studied the effect of enhanced sensitivity of D. magna to toxic materials, i.e. titanium oxide and methidathion, when D. magna were grown in culture using PACs. The data provide strong evidence that R. sphaeroides has the potential to substantially increase the physiological activity of D. magna.     

Effects of Sucrose and Citric Acid on the Viscometric Behavior of Aqueous Methylcellulose Solution

The flow of the mixture of methylcellulose (MC)—sucrose—citric acid—water was essentially thixotropic. The viscometric behaviors could be expressed by the power law, S = KDⁿ, where S was shearing stress and D rate of shear. The differential viscosity (ηd) of the mixture was strongly dropped by the addition of citric acid. Sucrose had the tendency to increase ηd of the mixtures except in the case of high sucrose concentration. From the results obtained, it has been concluded that citric acid tends to break the intermolecular hydrogen bonds of MC molecules, and that sucrose tends to keep the hydrogen bonds.     

Species of Dickinsonia Sprigg from the Ediacaran of South Australia

An iconic member of the Ediacara Biota, Dickinsonia Sprigg is one of few such taxa with multiple species. Here we use Gaussian finite mixture models to assess the validity of species distinctions for this genus. Our results indicate that the five described species of Dickinsonia from the Ediacara Member, South Australia are better classified as two based on multiple approaches. Two different methods for dimension reduction both provide strong support for two groups, with overlapping but distinct mixture models. The variable selection method produces the most biologically realistic clusters, indicating that the two species can be primarily differentiated based on the greater relative size of the anterior most unit of Dickinsonia costata Sprigg compared with Dickinsonia tenuis Glaessner & Wade. Despite differences in aspect ratio and number of modules, both species regulated growth to maintain overall shape. The greater likelihood of preservation of a midline and an irregular outer margin in D. tenuis highlights differential structural integrity and flexibility. Co-occurrence in the Ediacara Member indicates that both species occupied the same environments and temporal distribution. Smaller maximum and average size for D. costata, combined with higher abundance, may suggest a comparatively shorter lifespan and increased rates of reproduction.     

Purification and Characterization of UDP-Arabinopyranose Mutase from Chlamydomonas reinhardtii

Chlamydomonas reinhardtii cells are surrounded by a mixture of hydroxyprolin-rich glycoproteins consisting of L-arabinose, D-galactose, D-glucose, and D-mannose residues. The L-arabinose residue is thought to be attached by a transfer of UDP-L-arabinofuranose (UDP-Araf), which is produced from UDP-L-arabinopyranose (UDP-Arap) by UDP-arabinopyranose mutase (UAM). UAM was purified from the cytosol to determine the involvement of C. reinhardtii UAM (CrUAM) in glycoprotein synthesis. CrUAM was purified 94-fold to electrophoretic homogeneity by hydrophobic and size-exclusion chromatography. CrUAM catalyzed the reversible conversion between UDP-Arap and UDP-Araf and exhibited autoglycosylation activity when UDP-D-[¹⁴C]glucose was added as substrate. Compared to the properties of native and recombinant CrUAM overexpressed in Escherichia coli, native CrUAM showed a higher affinity for UDP-Arap than recombinant CrUAM did. This increased affinity for UDP-Arap might have been caused by post-translational modifications that occur in eukaryotes but not in prokaryotes.     

Influence of the filamentous cyanobacterium Planktothrix agardhii on population growth and reproductive pattern of the rotifer Brachionus calyciflorus

The population dynamics of B. calyciflorus was investigated using a green alga, Monoraphidium minutum, and a blue-green alga Planktothrix agardhii as food sources, separately and as mixtures. Growth rate (r), egg ratio (ER), and juvenile development time (D _j ) were measured in the laboratory and mortality rate and embryonic development time (D _E ) were calculated. With M. minutum, Brachionus showed a typical growth curve (Monod-kinetics) dependent on food concentration. In contrast B. calyciflorus did not grow well on P. agardhii. With all food concentrations the measured growth rates were about r=0. At low food concentrations r was low with both food types, but the ER of B. calyciflorus was significantly higher with P. agardhii as food source. Furthermore the relative egg volume of females carrying one egg was higher with Planktothrix than with Monoraphidium. An addition of P. agardhii to M. minutum led to increasing growth rates. Highest growth rates were found with complementary food sources. High food concentrations of M. minutum shortened the juvenile development (D _J ) time, but D _j was uneffected by different P. agardhii food concentrations. A mixture of both algae did not shorten D _j compared with M. minutum as single food. The calculated D _E was not effected by different food qualities but the calculated mortality was nearly 3 times higher with P. agardhii as food.     

Structure of the O-polysaccharide from the lipopolysaccharide of Providencia alcalifaciens O60

An O-polysaccharide (O-antigen) was isolated by mild acid degradation of the lipopolysaccharide of Providencia alcalifaciens O60 and studied by sugar and methylation analyses as well as ¹H and ¹³C NMR spectroscopy, including 2D ROESY and ¹H,¹³C HMBC experiments in D₂O and a ROESY experiment in a 9:1 H₂O–D₂O mixture to reveal correlations for NH protons. It was found that the polysaccharide is built up of linear pentasaccharide repeating units containing an amide of d-glucuronic acid with l-serine and has the following structure:The O-antigen studied is structurally and serologically closely related to the O-antigen of Proteus vulgaris O44.     

Sequentially Generated Designs

Procedures for sequential generation of nearly D-optimal designs are described. Two kinds of designs can be obtained: symmetrical block designs and nonsymmetrical ones. It is shown that in a special case when the number of the support points of a continuous D-optimal design equals to the number of regression coefficients the sequential designs can be constructed very easy without use of a computer. A Catalogue containing 135 designs has been developed by use of these procedures. 34 of them can be used for experiments in cuboidal factor space and the remaining for experiments with mixture and process variables. Comparison with other designs is done.     

Microbial Formation of D-Pantothenic Acid-α-glucoside

The formation of D-pantothenic acid-α-glucoside (PaA-α-G) was found from D-pantothenic acid (PaA) and maltose in incubation mixtures of microorganisms, especially Saccharomyces yeasts and Sporobolomyces coralliformis IFO 1032. The reaction conditions were investigated for formation of PaA-α-G by resting cells of Spor. coralliformis. The formation of the compound increased with PaA concentration (3~20 mg/ml). The yield was maximum at 5~10 mg/ml of PaA. Cetyl trimethyl ammonium bromide (0.1 %) promoted the formation of PaA-α-G. Sucrose was the optimal α-glucosyl donor. When 30 mg/ml of sucrose was fed to the reaction mixture (initial sucrose, 100 mg/ml; and PaA, 10 mg/ml) at 12-hr intervals, 5.74 mg/ml (3.30 mg/ml as PaA) of PaA-α-G was formed in 48-hr incubation at 28°C with shaking. PaA-α-G was also formed by yeast α-glucosidase, mold maltase and the cell-free extract of Spor. coralliformis. The compound showed approximately 9~10% and 0.1~0.3% (molar ratio) of activity of PaA for Saccharomyces carlsbergensis ATCC 9080 and Lactobacillus plantarum ATCC 8014, respectively. The compound had the same microbiological activity as authentic 4′-O-(α-D-glucopyranosyl)-D-pantothenic acid.     

Transglycosylation of Glycosyl Residues to Cyclic Tetrasaccharide by Bacillus stearothermophilus Cyclomaltodextrin Glucanotransferase Using Cyclomaltodextrin as the Glycosyl Donor

Cyclomaltodextrin glucanotransferase (EC 2.4.1.19, abbreviated as CGTase) derived from Bacillus stearothermophilus produced a series of transfer products from a mixture of cyclomaltohexaose and cyclic tetrasaccharide (cyclo{→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→}, CTS). Of the transfer products, only two components, saccharides A and D, remained and accumulated after digestion with glucoamylase. The total combined yield of the saccharides reached 63.4% of total sugars, and enzymatic and instrumental analyses revealed the structures of both saccharides. Saccharide A was identified as4-mono-O-α-glucosyl-CTS, {→6)-[α-D-Glcp-(1→4)]-α-D-Glcp-(1→3)-α-D-Glcp-(1→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→}, and sachharide D was 4,4′-di-O-α-glucosyl-CTS, {→6)-[α-D-Glcp-(1→4)]-α-D-Glcp-(1→3)-α-D-Glcp-(1→6)-[α-D-Glcp-(1→4)]-α-D-Glcp-(1→3)-α-D-Glcp-(1→}. These structures led us to conclude that the glycosyltransfer catalyzed by CGTase was specific to the C4-OH of the 6-linked glucopyranosyl residues in CTS.     

A flow cytometry analysis of batch and continuous culture transient diauxic growth in Hansenula polymorpha

A flow cytometry analysis and in vitro enzyme activity study is carried out on the methylotrophic yeast, Hansenula polymorpha, during both (a) batch growth and (b) continuous cultures subjected to single perturbations in either system dilution rate or influent carbon substrate composition. Flow cytometry of yeasts growing diauxically on a glucose: methanol mixture during exponential growth, exhibit DNA and RNA distributions indicative of the S-synthesis-phase of the cell cycle. Cells at the stationary growth stage exhibit DNA and RNA distributions that indicate one portion of the population in the G ₀/G₁ resting phase and another in the M-mitosis-phase.Yeast cells grown at a steady-state of D=0.2 h¹, then shifted to D=0.35 h^–1, at a constant influent substrate mixture, are also examined with both flow cytometry and in vitro enzyme assays. Distributions of DNA, RNA, and total protein at either steady state and during the shift between dilution rates did not resemble any observed in batch culture. Flow cytometry indicates significant changes in cell composition within 20 min of the imposed dilution rate shift. In vitro enzyme assays show a response time in decreasing methanol oxidase activity of 2.5–3 h upon a dilution rate shift-up, while hexokinase activity increases to its steady-state level in less than 3 h. Similar cell compositional changes are reported for shifts in influent substrate methanol: glucose ratio at a constant dilution rate of D=0.35 h ^–1. Results suggest that an unsteady-state regime, oscillating between conditions that promote maximum enzyme activity of either glucose- or methanol-metabolizing enzymes, may allow simultaneous enhanced time-averaged production of both sets of enzymes.     

Species relationships within Diplotaxis (Brassicaceae) and the phylogenetic origin of D. muralis

Nineteen from the ca. 30 Diplotaxis species including all known haploid chromosome numbers have been analysed for isoelectric focusing patterns of Rubisco, allozymes and RAPDs. D. erucoides (n=7) was clearly separated from all other species as were D. harra and D. crassifolia (n=13 each). Taxa with n=8 had different IEF patterns, but allozyme data grouped D. siettiana, D. ibicensis and D. brevisiliqua together. Species with n=9 were characterised by different IEF patterns, and their position was neither resolved in the allozyme nor in the RAPD tree. Only the D. catholica accessions were strongly clustered together. D. viminea and D. siifolia (both n=10) were kept separate, whereas the n=11 taxa D. tenuifolia, D. cretacea and D. simplex grouped together. Data confirm D. viminea as maternal parent of the allotetraploid D. muralis, and D. tenuifolia as the likely parent.