Effect of a diazafluoranthen derivative on phospholipases. A study at the air-water interface.

AC-3579 (2-N-methylpiperazinomethyl-1,3-diazafluoranthen 1-oxide) produces in rat hepatocytes a hypertrophy of the endoplasmic reticulum. Two possibilities that can explain this phenomenon are (1) that AC-3579 inactivates the phospholipases, and (2) that an AC-3579-lipid interaction hinders the enzymic activity. To demonstrate these hypotheses, a physicochemical model of biological membrane, the lipid-water interface, has been used. Dipalmitoyl DL-alpha-phosphatidyl-choline was spread at the air-water interface, the enzymes (phospholipase A or phospholipase C) dissolved in the aqueous phase. The enzymic reaction was first studied with and without AC-3579 dissolved in the aqueous phase; no enzymic inactivation was observed. However an AC-3579-lipid complex completely inhibited the enzymic reaction in the case of phospholipase A. An explanation is given in terms of steric hindrance to the enzyme-substrate.     

On the question of an electrokinetic requirement for phospholipase C action

Summary The phospholipase C ofclostridium welchii ( toxin) has an absolute requirement for trace quantities of Ca²⁺. It attacks pure phosphatidylcholine particles (smectic mesophases) having a close-packed bilayer structure only when their surface zeta potential is made positive by the addition of certain divalent ions (e.g., Ca²⁺) to the aqueous phase or by the presence of low concentrations of long chain cations to the lipid. Alternatively, if the rotational freedom of individual phospholipid molecules is increased by the insertion of shortn-alkanols (e.g., hexanol) into the bilayer or when a monolayer of the substrate at an air/water interface is expanded, enzymic hydrolysis can occur without any requirement for a net positive charge on the surface.     

Modelling and experimental studies on lipase-catalyzed isoamyl acetate synthesis in a microreactor

A lipase-catalyzed synthesis of isoamyl acetate was studied in a continuously operated pressure-driven microreactor. The esterification of isoamyl alcohol and acetic acid occurred at the interface between n-hexane and an aqueous phase with dissolved lipase B from Candida antarctica. By adjusting flow rates of both phases, a parallel laminar flow with liquid–liquid boundary in the middle of the microchannel could be reestablished and a separation of phases was achieved at the y-shaped exit of the microreactor. Since product remained in the organic phase, this also enabled its continuous separation from the aqueous phase with the enzyme. A three-dimensional mathematical model was developed, considering the velocity profile developed for steady-state conditions between two immiscible fluids. The model contained convection, diffusion, and enzyme reaction terms, where esterification rate was described with a Ping-Pong Bi-Bi mechanism and inhibition by both substrates. Experimental data, which were in good agreement with model simulations, have demonstrated 35% conversion at residence time 36.5 s at 45 °C and at 0.5 M acetic acid and isoamyl alcohol inlet concentrations, which is much faster as in any literature reported so far. According to model simulations, obtained by non-equidistant finite differences numerical solutions of complex non-linear equations system, further microreactor design and process optimization are feasible.     

Mode of Action of the Phospholipase from Sclerotinia Fungus

By using the purified phospholipase A and B of Sclerotinia Libertiana Fcl., enzymic degradation of soy-lecithin was investigated. From the paperchromatography experiments, it was concluded that the phospholipase A specifically hydrolyzes the ester linkage of unsaturated fatty acid of soy-lecithin whereas the phospholipase B hydrolyzes the linkage of saturated acid of soy-lysolecithin. Phospholipase B also could hydrolyze the two fatty acids from the soy-lecithin, however, the hydrolysis rate was rather inhibited by combination with the phospholipase A. Moreover, the phospholipase B activity on soy-lecithin and soy-lysolecithin was found to be increased by the presence of soy-lecithin and soy-lysolecithin in the reaction mixture, and to be inhibited by the addition of Tween 20.     

Impedance analysis of phosphatidylcholine membranes modified with valinomycin

The effect of the ion carrier valinomycin on the electrochemical features of the phosphatidylcholine membrane was investigated by electrochemical impedance spectroscopy. Phosphatidylcholine and valinomycin were chosen for the study because they fulfil essential functions in lively organisms. The experimental impedance values obtained in the presence of different amounts of carrier, studied with several potassium ion concentrations, were used for the research ability of valinomycin to form a 1:1 potassium ion complex on the lipid bilayer/electrolyte solution interface. Based on derived mathematical equations, the heterogeneous equilibrium constant (K _h), association rate constant of the complex (k _R) and dissociation rate constant of the complex (k _D) were calculated. The result of the investigation is the proposal of a new method for the determination of the parameters used to describe the chemical reaction at the interface between a carrier molecule from the membrane and a monovalent ion from the aqueous phase.     

New insights on O<Subscript>2</Subscript> uptake mechanisms in two-phase partitioning bioreactors

Numerous reports show that both transfer and uptake of poorly-water soluble substrates are significantly enhanced in two-phase partitioning bioreactors (TPPBs). A number of hypotheses have been put forward to explain these enhancements and among them, the occurrence of direct substrate or oxygen uptake from the vector/water interface has been suggested. The objective of this paper was to quantify the direct oxygen uptake from the vector/water interface in a culture of Pseudomonas putida, performed in a stirred tank reactor, using glucose as substrate and silicone oil as vector. Despite of a sufficient dissolved O₂ concentration in the vector phase (17 mg l⁻¹) and a significant vector surface area (4,000 m⁻¹) no significant direct O₂ uptake from the vector/water interface was observed, compared to O₂ uptake from the aqueous phase. From these results it was concluded that, direct O₂ or substrate uptake from the vector/water interface might not be significant in TPPBs.     

Selection and micropropagation of nodulating and non-nodulating clones ofAlnus crispa (Ait.) Pursh

Summary 600,000 seedlings ofAlnus crispa were inoculated with a 111 mixture of theFrankia strains ACN1 ^AG , AGN1 _exo ^AG and MGP10i. After 3 successive inoculations and screenings, one individual, AC-4, was selected as non-nodulating (Nod^–) with Frankiae. This selected individual AC-4 (Nod^–) and two other clones ofA. crispa, AC-2 and AC-5, known for their ability to nodulate (Nod⁺) and two other clones ofA. crispa, AC-2 and AC-5, known for their ability to nodulate (Nod⁺) withFrankia werein vitro propagated. The different clones ofA. crispa in culture required different kinds and concentrations of sugar during the in vitro multiplication and rooting stages. Nodulation tests using 7Frankia strains indicated that the clone AC-4 (Nod^–) was non-nodulating with 6 of the 7Frankia strains tested. One strain,Frankia ANNI, isolated from one unique nodule produced on the mother-plant AC-4, induced 38% of the AC-4 plantlets to nodulate but with a number of nodules 10 to 20 times less than the clones AC-2 (Nod⁺) and AC-5 (Nod⁺). Morphological observations of the roots of AC-4 (Nod^–) indicated that this clone had few and abnormally short root hairs.     

In situ analysis of oxygen consumption and diffusive transport in high‐temperature acidic iron‐oxide microbial mats

The role of dissolved oxygen as a principal electron acceptor for microbial metabolism was investigated within Fe(III)‐oxide microbial mats that form in acidic geothermal springs of Yellowstone National Park (USA). Specific goals of the study were to measure and model dissolved oxygen profiles within high‐temperature (65–75°C) acidic (pH = 2.7–3.8) Fe(III)‐oxide microbial mats, and correlate the abundance of aerobic, iron‐oxidizing Metallosphaera yellowstonensis organisms and mRNA gene expression levels to Fe(II)‐oxidizing habitats shown to consume oxygen. In situ oxygen microprofiles were obtained perpendicular to the direction of convective flow across the aqueous phase/Fe(III)‐oxide microbial mat interface using oxygen microsensors. Dissolved oxygen concentrations dropped from ～ 50–60 μM in the bulk‐fluid/mat surface to below detection (< 0.3 μM) at a depth of ～ 700 μm (～ 10% of the total mat depth). Net areal oxygen fluxes into the microbial mats were estimated to range from 1.4–1.6 × 10^?4 μmol cm^?2 s^?1. Dimensionless parameters were used to model dissolved oxygen profiles and establish that mass transfer rates limit the oxygen consumption. A zone of higher dissolved oxygen at the mat surface promotes Fe(III)‐oxide biomineralization, which was supported using molecular analysis of Metallosphaera yellowstonensis 16S rRNA gene copy numbers and mRNA expression of haem Cu oxidases (FoxA) associated with Fe(II)‐oxidation.     

Biodegradation by an Arthrobacter Species of Hydrocarbons Partitioned into an Organic Solvent

An Arthrobacter strain mineralized naphthalene and n-hexadecane dissolved in 2,2,4,4,6,8,8-heptamethylnonane. The extent of mineralization increased with greater volumes of solvent. Measurements under aseptic conditions of the partitioning of naphthalene into the aqueous phase from the solid phase or from heptamethylnonane showed that the rates were rapid and did not limit mineralization. The rate of mineralization of hexadecane was rapid, although partitioning of the compound into aqueous solution was not detected. The Arthrobacter sp. grown in media with or without heptamethylnonane did not excrete products that increased the aqueous solubility of naphthalene and hexadecane. Measurements of the number of cells in the aqueous phase showed that the Arthrobacter sp. attached to the heptamethylnonane-water interface, but attachment was evident even without a substrate in the heptamethylnonane. Tests with small inocula of the Arthrobacter sp. demonstrated that at least a portion of naphthalene or hexadecane dissolved in heptamethylnonane was degraded by cells attached to the solvent-water interface. The cells did not adhere in the presence of 0.1% Triton X-100. The surfactant prevented mineralization of the hexadecane initially dissolved in heptamethylnonane, but it increased the rate and extent of mineralization of naphthalene initially dissolved in heptamethylnonane. The data show that organic solvents into which hydrophobic compounds partition affect the biodegradation of those compounds and that attachment of microorganisms to the organic solvent-water interface may be important in the transformation.     

Enhanced biodegradation of phenanthrene in a biphasic culture system

Degradation of phenanthrene byPseudomonas aeruginosa AK1 was examined in (i) an aqueous mineral salts medium to which phenanthrene particles of varying size (i.e. diameter) were added, and (ii) an aqueous/organic biphasic culture system consisting of mineral salts medium supplemented with 2,2,4,4,6,8,8-heptamethylnonane (HMN) as the phenanthrene-carrying organic phase. In both systems, the rate of phenanthrene biodegradation could be significantly enhanced by manipulations leading to improved phenanthrene mass transfer into the aqueous phase. With crystalline phenanthrene, the rate of biodegradation was found to be directly correlated to the particle surface area, whereas in the biphasic system the rate of biodegradation of the dissolved phenanthrene was mainly governed by the HMN/water interface area. In the latter system, exponential growth with a doubling time t _d of 6–8 hours has been achieved under conditions of intensive agitation of the medium indicating that phenanthrene degradation by strain AK1 is limited mainly by physicochemical parameters. Addition of selected surfactants to the culture medium was found to accelerate phenanthrene degradation by strain AK1 only under conditions of low agitation (in the presence of HMN) and after pretreatment of phenanthrene crystals by ultrasonication (in the absence of HMN). Evidence is presented that the stimulating effect of the surfactants was primarily due to improved dispersion of phenanthrene particle agglomerates (in the aqueous mineral salts medium supplemented with phenanthrene crystals) or of the phenanthrene-carrying lipophilic solvent drops (in the aqueous/organic biphasic culture system) whereas the solubilizing activity towards phenanthrene was neglectible. Under conditions of intensive mixing of the culture medium (i.e. if a high particle surface area or HMN/water interface area, respectively, is provided), the addition of surfactants did not enhance phenanthrene biodegradation.     

Magnetic lipase adsorbed to a magnetic fluid

A magnetic fluid was synthesized by oxidation of ferrous ions (Fe^2&+) in the presence of a synthetic alternating copolymer of polyethylene glycol (PEG) and maleic acid (MA), poly(PEG-MA). The magnetic fluid dispersed stably both in aqueous solution and in organic solvents. Its particle size was approximately 10 nm. The magnetic fluid was mixed with lipase in water, followed by lyophilization. Although the enzyme and the magnetic fluid were dissociated in aqueous solution, they remained associated in organic solvents such as benzene. The magnetic fluid-adsorbed lipase dispersed in benzene and exerted high enzymic activity (2.9 μmol min⁻¹ mg⁻¹ lyophilized powder) for lauryl laurate synthesis from lauric acid and lauryl alcohol, and was readily recovered from the reaction mixture in a magnetic field (6000 Oe) without loss of enzymic activity.     

The kinetic behaviour of a two-enzyme system in biphasic media: coupling hydrolysis and lipoxygenation

Analysis of the kinetic behaviour of a two-enzyme-system carrying out two consecutive reactions was investigated in macroheterogeneous biphasic media (octane/buffer pH 9.6, v/v=1:1). The lipase-catalysed hydrolysis of trilinolein and the subsequent lipoxygenation of the liberated linoleic acid, were coupled in a modified Lewis cell with a well-defined liquid/liquid interfacial area. Trilinolein was dissolved in the organic phase and hydrolysed in the presence of Mucor javanicus lipase at the organic/aqueous interface. Linoleic acid, liberated after hydrolysis was transferred to the aqueous phase and reacted with lipoxygenase. This reaction consumed linoleic acid and produced hydroperoxides, which favoured the transfer of residual linoleic acid, since they possess surface active properties. Catalysis and transfer influenced each other reciprocally. At low substrate concentrations, cooperativity phenomena were observed in the experimental and also the modelled two-enzyme systems. When the initial substrate concentration was high, the kinetic behaviour of the two-enzyme system in a compartmentalised medium, seemed to be independent of the substrate concentration, unlike that observed in homogeneous monophasic enzymology. The numerical integration program used to model the two-enzyme system was based on results obtained in separate studies of the following three phenomena: (1) trilinolein hydrolysis in biphasic medium, (2) linoleic acid transfer across a liquid/liquid interface and (3) lipoxygenation in an aqueous media. Results obtained by modelling were similar to the results observed experimentally.     

Molecular dynamics study of phospholipase A2 on a membrane surface

The desolvation of lipid molecules in a complex of the enzyme human synovial phospholipase A₂ with a lipid membrane is investigated as a mechanism that enhances the overall activity of the enzyme. For this purpose the interaction of the enzyme phospholipase A₂ with a dilauryl-phosphatityl-ethanolamin (DLPE) membrane monolayer surface has been studied by means of molecular dynamics simulations. Two enzyme-membrane complexes, a loose and a tight complex, are considered. For comparison, simulations are also carried out for the enzyme in aqueous solution. The conformation, dynamics, and energetics of the three systems are compared, and the interactions between the protein and lipid molecules are analyzed. Free energies of solvation are calculated for the lipid molecules in the enzyme–membrane interface. Along with the calculated dielectric susceptibility at this interface, the results show the desolvation of lipids in a tightly bound, but not in a loosely bound protein-membrane complex. The desolvated lipids are found to interact mainly with hydrophobic protein residues, Including Leu-2, Val-3, Ala-18, Leu-19, Phe-24, Val-31, and Phe-70. The results also explain why the turnover rate of phospholipase A₂ complexed to a membrane is enhanced after a critical amount of negatively charged reaction product is accumulated. © 1996 Wiley-Liss, Inc.     

Adhesion to the hydrocarbon phase increases phenanthrene degradation by <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> LP6a

Microbial adhesion is an important factor that can influence biodegradation of poorly water soluble hydrocarbons such as phenanthrene. This study examined how adhesion to an oil–water interface, as mediated by 1-dodecanol, enhanced phenanthrene biodegradation by Pseudomonas fluorescens LP6a. Phenanthrene was dissolved in heptamethylnonane and added to the aerobic aqueous growth medium to form a two phase mixture. 1-Dodecanol was non-toxic and furthermore could be biodegraded slowly by this strain. The alcohol promoted adhesion of the bacterial cells to the oil–water interface without significantly changing the interfacial or surface tension. Introducing 1-dodecanol at concentrations from 217 to 4,100 mg l⁻¹ increased phenanthrene biodegradation by about 30% after 120 h incubation. After 100 h incubation, cultures initially containing 120 or 160 mg l⁻¹ 1-dodecanol had mineralized >10% of the phenanthrene whereas those incubated without 1-dodecanol had mineralized only 4.5%. The production and accumulation of putative phenanthrene metabolites in the aqueous phase of cultures likewise increased in response to the addition of 1-dodecanol. The results suggest that enhanced adhesion of bacterial cells to the oil–water interface was the main factor responsible for enhanced biodegradation of phenanthrene to presumed polar metabolites and to CO₂.     

Genetic architecture of body weight,condition factor and age of sexual maturation in Icelandic Arctic charr (<Emphasis Type="Italic">Salvelinus alpinus</Emphasis>)

The high commercial value from the aquaculture of salmonid fishes has prompted many studies into the genetic architecture of complex traits and the need to identify genomic regions that have repeatable associations with trait variation both within and among species. We searched for quantitative trait loci (QTL) for body weight (BW), condition factor (CF) and age of sexual maturation (MAT) in families of Arctic charr (Salvelinus alpinus) from an Icelandic breeding program. QTL with genome-wide significance were detected for each trait on multiple Arctic charr (AC) linkage groups (BW: AC-4, AC-20; CF: AC-7, AC-20, AC-23, AC-36; MAT: AC-13/34, AC-39). In addition to the genome-wide significant QTL for both BW and CF on AC-20, linkage groups AC-4, AC-7, AC-8, and AC-16 contain QTL for both BW and CF with chromosome-wide significance. These regions had effects (albeit weaker) on MAT with the exception of the region on AC-8. Comparisons with a North American cultured strain of Arctic charr, as well as North American populations of Atlantic salmon (Salmo salar), and rainbow trout (Oncorhynchus mykiss), reveal some conservation in QTL location and structure, particularly with respect to the joint associations of QTL influencing BW and CF. The detection of some differences in genetic architecture between the two aquaculture strains of Arctic charr may be reflective of the differential evolutionary histories experienced by these fishes, and illustrates the importance of including different strains to investigate genetic variation in a species where the intent is to use that variation in selective breeding programs.     

Phospholipase C and the physical states of polar head groups of lipids

Summary Activity of phospholipase C fromClostridium perfringens on liposomes made fromsn-3-phosphatidylcholine, dimyristoyl (DMPC), dipalmitoyl (DPPC) or distearoyl (DSPC) was measured at various temperatures and was correlated with their gel/liquid-crystalline phase transitions (T _c : 23, 41.5, 52°C for DMPC, DPPC, DSPC, respectively). In all cases, the activity of phospholipase C was high in the gel phases of the substrates and was almost zero in their liquid-crystalline phases. Fluorescence depolarization measurements of N-dansyl-sn-3-phosphatidylethanolamine (DPE) and 1,6-diphenyl-1,3,5-hexatriene (DPH) incorporated into the liposomes showed that both the head group and the alkyl chains of the lipids were immobilized in the gel phases but were highly mobile in the liquid-crystalline phase. These results indicate that the rotational mobility of lipids (both of the head groups and the alkyl chains) was not a major factor in the phospholipase C reaction. It is inferred that some electrostatic and/or hydrophobic interactions might play important roles in regulation of the phospholipase C activity.     

Reactive extraction of penicillin G in a pilot plant Karr-column

Summary Penicillin G was extracted from a model medium with a secondary amine (Amberlite LA-2) as carrier in n-butylacetate as solvent in a 7.6 m high pilot plant Karr-column at different stroke frequencies, throughput of the phases, concentrations of Penicillin G and carrier and ratios of the throughputs of the aqueous and organic phases. Up to penicillin concentrations of 30 gl^–1, throughputs of the aqueous phase of 100 lh^–1 and throughput ratios of the aqueous phase-to-organic phase of 3, very high degrees of extraction (99%) can be achieved with a penicillin loss below 1%.Symbols a specific interfacial area with regard to the volume of the continuous phase - C partition coefficient - cA, cA, i concentration of carrier (sec. amine) in the bulk at the interface - cAHP, cAHP, i concentration of complex in the bulk at the interface - cH proton concentration - cHP_a, cHP_a,i concentration of free acid in the bulk of the aqueous phase at the interface - cHP_o, cHP_{o, i} concentration of free acid in the bulk of the organic phase, at the interface - cP, cP, i concentration of acid anions in the bulk of the aqueous phase, at the interface - d₃₂ Sauter droplet diameter - E degree of extraction - f stroke frequency - KG reaction equilibrium constant - K_phys distribution coefficient - N number of stages in cascade - t mean residence time of the aqueous phase - _aq throughput of the aqueous phase - _o throughput of the organic phase - Z dimensionless longitudinal coordinate of the column with regard to its active length (4 m) - holdup of the organic phase     

Enzymatic Synthesis of N-formyl-L-aspartyl-L-phenylalanine Methyl Ester (Aspartame Precursor) Utilizing an Extractive Reaction in Aqueous/Organic Biphasic Medium

N-Formyl-L-aspartyl-L-phenylalanine methyl ester (N-formyl aspartame, F-AspPheOMe) was synthesized enzymatically utilizing an extractive reaction in an aqueous/organic biphasic system. The N-formyl aspartame yield in a pure aqueous monophasic system was, in general, ca. 3% , however, it was over 80 % in a water/1-butanol biphasic system using a simultaneously extractive operation of an enzymatic reaction in an aqueous phase and a product separation from an aqueous to an organic phases.     

Factors influencing the extent and development of the oxic zone in sediments

Dissolved oxygen concentrations in river-sediment porewaters are reported and modelled using a zero-order reaction rate and the Monod equation. After mixing the sediments and allowing settling, the dissolved oxygen profile in the bed-sediment was expected to reach a steady-state rapidly (< 1 h). However changes in the vertical profile of oxygen over a period of 38 days revealed that the penetration of oxygen increased and the dissolved oxygen flux at the interface decreased with time, probably as the oxidation kinetics of organic matter and redox reactions in the sediment changed. Experiments with three contrasting silt and sand dominated sediments (organic matter content between 0.9 and 18%) at two water velocities (ca 10 and 20 cm s^–1) showed that the dissolved oxygen profiles were independent of velocity for each of the sediments. The most important controls on the reaction rate were the organic matter content and specific surface area of the sediment. A viscous diffuse-boundary-layer above the sediment was only detected in the experiments with the silt sediment where the sediment oxygen demand was relatively high. In the coarser sediments, the absence of a diffuse layer indicated that slow oxidation processes in the sediment controlled the dissolved oxygen flux at the interface. The problem of determining a surface reference in coarse sediment is highlighted. The results are discussed with reference to other studies including those concerned with estuarine and marine sediments.