β-fructofuranosidase production by Aspergillus japonicus in shaking batch cultures as affected by initial sucrose concentration

Summary Effect of initial sucrose concentration on the production of -fructofuranosidase from Aspergillus japonicus TIT-90076 were investigated in shaking batch cultures. The maximal enzyme production occurred at 25 % (w/v) sucrose and an inhibitory effect on cell growth was exhibited when the initial sucrose concentration was above 10 % (w/v). For evaluation of the process economy, a relationship between the process output (FFase production at 96-h cultivation, in units ml^–1) and the process input (initial sucrose concentration, S₀, in % (w/v)) can be simply expressed as follows: FFase production = (142.9S₀/(29.3 + S₀))(16.7 – 0.22S₀).     

Simultaneous production of citric acid and invertase by Yarrowia lipolytica SUC+ transformants

Simultaneous production of citric acid (CA) and invertase by Yarrowia lipolytica A-101-B56-5 (SUC⁺ clone) growing from sucrose, mixture of glucose and fructose, glucose or glycerol was investigated. Among the tested substrates the highest concentration of CA was reached from glycerol (57.15 g/L) with high yield (Y_CA/S = 0.6 g/g). When sucrose was used, comparable amount of CA was secreted (45 g/L) with slightly higher yield (Y_CA/S = 0.643 g/g). In all cultures amount of isocitrate (ICA) was below 2% of total citrates. Considering invertase production, the best carbon source appeared to be sucrose (72 380 U/L). The highest yield of CA and invertase biosynthesis calculated for 1 g of biomass was obtained for cells growing from glycerol (9.9 g/g and 4325 U/g, respectively). Concentrates of extra- and intracellular invertase of the highest activity were obtained from sucrose as substrate (0.5 and 1.8 × 10⁶ U/L, respectively).     

Separation of fructooligosaccharides using zeolite fixed bed columns

Recent studies have shown that the chromatographic separation of mixtures of monosaccharides and disaccharides may be improved by employing Y zeolites, a procedure which holds promise in the separation of oligosaccharides. In the present study, a column packed with zeolite was employed to study the separation of fructooligosaccharides (FOS). FOS were produced by an enzyme isolated from Rhodotorula sp., which produces GF₂ (kestose), GF₃ (nystose) and GF₄ (frutofuranosyl nystose). The identification and quantification of the sugars were carried out by ion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The separation of fructooligosaccharides was carried out using a fixed bed column packed with Ba²⁺-exchange Y zeolites. The effects of temperature (40–50 °C), injected volume per bed volume (2.55–7.64%), superficial velocity (0.1–0.15 cm min⁻¹) and eluent composition (40–60% ethanol) were investigated using a fractionary factorial design with separation efficiency as the response. The results showed that the most favorable conditions for the separation of the oligosaccharide–glucose mixture were 60% ethanol as eluent, temperature of 50 °C, superficial velocity of 0.1 cm min⁻¹ and 2.55% injection volume per bed volume of injection mixture, using two columns in series. The values for separation efficiency were 0.60 for oligosaccharide–glucose, 1.00 for oligosaccharide–fructose, 0.22 for oligosaccharide–sucrose, 0.43 for glucose–fructose, 0.82 for glucose–sucrose and 1.23 for fructose–sucrose.     

Production of fructooligosaccharides in high yield using a mixed enzyme system of β-fructofuranosidase and glucose oxidase

A mixed enzyme system, with -fructofuranosidase (obtained from Aspergillus japonicus) and commercial glucose oxidase (Gluzyme, Novo Nordisk), produced fructooligosaccharides (FOS) in high yield from sucrose. The reaction was performed in an aerated stirred tank reactor controlled at pH 5.5 by a slurry of CaCO₃. Glucose, an inhibitor of -fructofuranosidase, produced in the reaction was converted by glucose oxidase to gluconic acid, which was then precipitated to calcium gluconate in solution. The system produced more than 90% (w/w) FOS on a dry weight basis, the remainder was glucose, sucrose and a small amount of calcium gluconate. Most of the FOS and sucrose was hydrolyzed to fructose in the mixed enzyme system with glucose oxidase and -fructofuranosidase from Asp. niger.     

Sugar transport in isolated corn root protoplasts

Isolated corn (Zea mays L.) root protoplasts were used to study sucrose and hexose uptake. It is found that glucose was preferentially taken up by the protoplasts over sucrose and other hexoses. Glucose uptake showed a biphasic dependence on external glucose concentration with saturable (K_m of 7 millimolar) and linear components. In contrast, sucrose uptake only showed a linear kinetic curve. Sucrose and glucose uptake were linear over a minimum of 1 hour at pH 6.0 and 1 millimolar exogenous sugar concentration. Glucose uptake showed a sharp 42°C temperature optimum, while sucrose uptake showed a lower temperature sensitivity which did not reach a maximum below 50°C. Uptake of both sugars was sensitive to several metabolic inhibitors and external pH. Differences between sucrose and glucose uptake in two different sink tissue (i.e. protoplasts from corn roots and soybean cotyledons) are discussed.     

Thermodynamics of the Binding of Chymotrypsin with the Black-eyed Pea Trypsin and Chymotrypsin Inhibitor (BTCI)

The binding of -chymotrypsin to black-eyed pea trypsin/chymotrypsin inhibitor (BTCI) has been studied using the inhibitory activity against the enzyme and the formation of the complex enzyme/inhibitor followed by measurements of fluorescence polarization. Apparent equilibrium constants were estimated for several temperatures and the values obtained range from 0.32 × 10⁷ to 1.36 × 10⁷ M^–1. The following values were found from van't Hoff plots: H _vh ^° = 10.8 kcal mol^-1 (from inhibitory assays) and 11.1 kcal mol^–1 (from fluorescence polarization); S° = 67.9 and = 67.8 kcal K^–1 mol^–1, respectively. Calorimetric binding enthalpy was determined (corrected for the ionization heat of the buffer) and the resulting value was H _cal ^° = 4.9 kcal mol^-1. These results indicate that the binding of chymotrypsin to BTCI is an entropically driven process.     

Simultaneous estimation ofV max,Km, and the rate of endogenous substrate production (R) from substrate depletion data

The nonlinear and 3 linearized forms of the integrated Michaelis-Menten equation were evaluated for their ability to provide reliable estimates of uptake kinetic parameters, when the initial substrate concentration (S₀) is not error-free. Of the 3 linearized forms, the one where t/(S₀–S) is regressed against ln(S₀/S)/(S₀–S) gave estimates ofV _max and K_m closest to the true population means of these parameters. Further, this linearization was the least sensitive of the 3 to errors (±1%) in S₀. Our results illustrate the danger of relying on r² values for choosing among the 3 linearized forms of the integrated Michaelis-Menten equation. Nonlinear regression analysis of progress curve data, when S₀ is not free of error, was superior to even the best of the 3 linearized forms. The integrated Michaelis-Menten equation should not be used to estimateV _max and K_m when substrate production occurs concomitant with consumption of added substrate. We propose the use of a new equation for estimation of these parameters along with a parameter describing endogenous substrate production (R) for kinetic studies done with samples from natural habitats, in which the substrate of interest is an intermediate. The application of this new equation was illustrated for both simulated data and previously obtained H₂ depletion data. The only means by whichV _max, K_m, and R may be evaluated from progress curve data using this new equation is via nonlinear regression, since a linearized form of this equation could not be derived. Mathematical components of computer programs written for fitting data to either of the above nonlinear models using nonlinear least squares analysis are presented.     

Purification and characterization ofGardnerella vaginalis hemolysin

The extracellular hemolysin fromGardnerella vaginalis (GVH) was purified 88-fold and partially characterized. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a molecular weight of 59 kD. The hemolysin is most active at pH 6. The mechanism of hemolysis involves one temperature-dependent binding step followed by cell lysis. GVH does not act like an enzyme but is firmly bound to the erythrocyte membrane and used up during the reaction. Osmotic protectants like sucrose and dextran 4 (4000–6000 D) do not protect the toxin-treated erythrocytes from lysis, but dextran 8 (8000–12,000 D) provides full protection. GVH is inactivated by cholesterol,- and-glucosidase. A partial inactivation was found with-mannosidase and N-acetylglucosaminidase. Human lymphocytes are about 100 times less susceptible than human erythrocytes. The specificity of GVH to human erythrocytes is very high. Red cells from monkey, horse, cow, sheep, and rabbit show less than 1% of human erythrocyte susceptibility.     

Erythrocyte morphological states, phases, transitions and trajectories

Morphological response (MR) of red blood cells represents a triphasic sequence of spontaneously occurring shape transformation between different shape states upon transfer the cells into isotonic sucrose solution in the order: S₀ (initial discoid shape in physiological saline) → S₁ (echinocytic shape at the beginning of MR, phase 1) → S₂ (intermediate discoid shape, phase 2) → S₃ (final stomatocytic shape, phase 3). In this paper, the dynamics of cell shape changes was investigated by non-invasive light fluctuation method and optical microscopy. Among 12 possible transitions between four main shape states, we experimentally demonstrate here an existence of nine transitions between neighbour or remote states in this sequence. Based on these findings and data from the literature, we may conclude that red blood cells are able to change their shape through direct transitions between four main states except transition S₁ → S₀, which has not been identified yet. Some shape transitions and their temporal sequence are in accord with predictions of bilayer couple concept, whereas others for example transitions between remote states S₃ → S₁, S₁ → S₃ and S₃ → S₀ are difficult to explain based solely on the difference in relative surface areas of both leaflets of membrane suggesting more complex mechanisms involved. Our data show that MR could represents a phenomenon in which the major role can play pH and chloride-sensitive sensor and switching mechanisms coupled with transmembrane signaling thus involving both cytoskeleton and membrane in coordinated shape response on changes in cell ionic environment.     

Heat resistance ofCitrobacter freundii in media with various water activities

Summary The heat resistance ofCitrobacter freundii NCTC 9750 between 45–65°C in media with various water activities has been determined.At a water activity of nearly 1.00, the Arrhenius plot of the death rate shows a sharp breakpoint at 56.5°C, suggesting the existence of at least two different thermal inactivation processes causing lethality of the bacterial cell. The activation energy below 56.5°C is 0.4186 MJ/mol (100 000 cal/mol), above 56.5°C it is 0.1863 MJ/mol (44 500 cal/mol). After addition of sucrose (1.8 mol/l) or NaCl (0.77 mol/l) to the heating medium, such a breakpoint is not observed. The activation energy for these processes are, for sucrose; 0.2097 MJ/mol, for NaCl; 0.3641 MJ/mol. However, at an NaCl concentration of 1.54 mol/l there is a breakpoint at 53.3°C.The influence of the sucrose concentration on the heat resistance can be described by the formula: ln k_S=ln k_O–a [sucrose]. Such a simple correlation does not exist for the influence of NaCl or glycerol.The heat inactivation of whole cells ofC. freundii was also measured with a differential scanning calorimeter. The first irreversible conformation change took place at 323 K, the main conformation change at 343 K.     

Heat resistance of Salmonella senftenberg 775W at various sucrose concentrations in distilled water

Summary The heat resistance of Salmonella senftenberg 775 W, NCTC 9959, has been determined in distilled water pH 6.5 at sucrose concentrations up to 2.20 mol l^–1 at temperatures between 63 and 70°C. Surviving cells were counted on minimal and enriched agar media to investigate the influence of the various nutrients on the recovery of heat injured cells. At various sucrose concentrations and temperatures multiphasic exponential parts of inactivation curves were found. Systematic differences between the recovery media depended on sucrose concentration, temperature and phase of exponential inactivation. At 60°C and sucrose concentrations between 0.52 and 1.82 mol l^–1 the relationship between inactivation rate and sucrose concentration could be described by the equation ln k₅=ln k₀-_T [sucrose]. The activation energy of thermal inactivation reactions, substantially decreased when sucrose (1.82 mol l^–1) was added to the heating menstruum. The activation energies in different recovery agars were of the same order, which suggests that the critical sites in heat inactivation are not key enzymes of the synthetic pathways of amino-acids and nucleotides. The differences between activation energies, calculated for cells of the various exponential phases of inactivation in both non-sucrose and 1.82 mol sucrose per 1 heating media, were also small, further suggesting that these critical sites are the same in cells from the various phases. Compared to published data on the heat resistance of S. senftenberg 775 W, we found a decreased resistance in a non-sucrose medium but an equal or increased resistance, depending on the phase of exponential inactivation, at a sucrose concentration of 1.82 mol l^–1.     

Heat resistance of Klebsiella pneumoniae in media with various sucrose concentrations

Summary The heat resistance of Klebsiella pneumoniae, an organism of widespread occurrence in nature has been determined in media containing various amounts of sucrose at temperatures between 47° and 59°C.In the presence of sucrose and at all temperatures the inactivation curves show a fast initial drop (logarithmic phase) in the number of survivors followed by a less rapid one (tail phase). The influence of the sucrose concentration can be described withln k _s = ln k _O – _T [sucrose] for media with more than 0.52 mol/l sucrose for the logarithmic as well as for the tail phase of inactivation.The heat-injured cells were recovered on various media to investigate the influence of the presence of small metabolites and nutrients on the shape of the inactivation curves and on the death rate. For cells heated in media without sucrose, the recovery on a rich medium was much better than on a poor one; for cells heated in media with more than 0.26 mol/l sucrose, no difference was observed between the various recovery media.The activation energies as determined on the various media are always nearly the same, which strongly suggests that the critical sites in the heat inactivation were not enzymes playing a key role in the synthesis of small molecules such as amino acids or nucleotides.     

Kinetic resolution of ketoprofen ester catalyzed by lipase from a mutant of CBS 5791

A biotransformation process was developed for the production of (S)-ketoprofen by enantioseletive hydrolysis of racemic ketoprofen ester using the mutant Trichosporon laibacchii strain CBS 5791. A satisfactory result was obtained, in which the E was 82.5, with an ee of 0.94 and a conversion of 0.47 under the optimum hydrolysis conditions [E is enantiomeric ratio, E=ln[1–X(1+ee)]/ln[1–X(1–ee)]; ee is enantiomeric excess, ee=(C_S–C_R)/(C_S+C_R): temperature of hydrolysis was 23°C]. The medium used in biotransformation was a mixture of growth broth and biotransformation broth at a ratio of 1:9, the concentration of Tween 80 was 15 g/l, the time of hydrolysis, 72 h. These results are promising for further scale-up. Tween 80 significantly improved lipase enantioselectivity and activity at the optimum concentration.     

Biospeciation, by potentiometry and computer simulation, of Sm-EDTMP, a bone tumor palliative agent

¹⁵³Sm-EDTMP (ethylenediaminetetra(methylenephosphonic) acid) is of considerable interest as a bone therapeutic radiopharmaceutical but its properties in solution are not yet well characterized. The protonation constants of EDTMP and the formation constants of the complexes of Sm-EDTMP have accordingly been measured potentiometrically by glass electrode titrations at 25°C in 0.15 M NaCl. Six protonation constants (log ₀₁₁ = 9.638, log ₀₁₂ = 17.330, log ₀₁₃ = 23.597, log ₀₁₄ = 28.636, log ₀₁₅ = 31.501, log ₀₁₆ = 32.624) and the formation constants of the [Sm(EDTMP)H_-1]^6- (log _11-1 = 4.865), [SmEDTMP]^5- (log ₁₁₀ = 12.018), [Sm(EDTMP)H]^4- (log ₁₁₁ = 17.892) and [Sm(EDTMP)H₂]^3- (log ₁₁₂ = 23.437) complexes were determined. Computer simulations indicate that the [SmEDTMP]^5- and the hydroxy [Sm(EDTMP)H_-1]^6- species are the major Sm(III) complexes formed in blood plasma, which explains the high degree of localization in the kidney and urine observed in biodistribution studies. Calcium ions are probably the maior competitor for EDTMP in blood plasma. As the presence of secondary skeletal metastases results in a high rate of bone turnover, it is possible that the high concentration of calcium at these sites encourages localization of ¹⁵³Sm-EDTMP.     

Femtosecond pump-supercontinuum probe and transient lens spectroscopy of adonixanthin

The ultrafast internal conversion (IC) dynamics of adonixanthin in organic solvents were studied by pump-supercontinuum probe (PSCP) and transient lens (TL) spectroscopy after photoexcitation to the S₂ state. Transient PSCP spectra in the range 344-768 nm provided the spectral evolution of the S₀ → S₂ ground state bleach and S₁ → S_n excited state absorption. Time constants were τ₂ = 115 and 111 fs for the S₂ → S₁ IC and τ₁ = 6.4 and 5.8 ps for the S₁ → S₀ IC in acetone and methanol, respectively. There was only an insignificant polarity dependence of τ₁, underlining the negligible importance of intramolecular charge transfer (ICT) in the lowest-lying excited state of C₄₀ carotenoids with carbonyl substitution on the β-ionone ring. A blueshift and a spectral narrowing of the S₁ → S_n ESA band, likely due to solvation dynamics, and formation of the adonixanthin radial cation at high pump energies via resonant two-photon ionization were found.     

Photolysis of the ion pair [Pt(NH3)5Cl]S2O8: reduction of platinum(IV) induced by outer-sphere charge transfer excitation

The ion pair [Pt^IV(NH₃)₅Cl]³⁺S₂O₈²⁻ shows a S₂O₈²⁻ → [Pt(NH₃)₅Cl]³⁺ outer-sphere charge transfer (OSCT) absorption at λ_max=267 nm. OSCT excitation leads to the reduction of Pt(IV) by S₂O₈²⁻ to Pt(II) with φ=3×10⁻³ at λ_irr=280 nm.     

On indicator dilution and perfusion heterogeneity: A stochastic model

Unidirectional extraction of a substrate S in the capillaries following the arterial injection of a bolus containing S and a reference tracer R is assumed to follow first-order kinetics. If C_R and C_S denote normalized venous effluent concentrations of R and S, respectively, let L(t)=ln[C_R(t)⧸C_S(t)]. We derive a formula which expresses the experimental L(t) data in terms of the mean μ(t) and variance of the transit times of those capillaries which are contributing indicators at each sample time t. We examine the information thus contained in the L data about capillary and noncapillary transit times under several kinematic assumptions. We show that if the capillary and noncapillary transit times are stochastically independent with frequency functions h_c(t) and h_av(t), respectively, then the shapes of the graphs of L(t) and μ(t) depend on the variances and skewnesses of h_c(t) and h_av(t). Specifically, let r₂ be the ratio of the variance of h_c(t) to the variance of h_av(t), and let r₃ be the ratio of skewnesses in the same order. Then the graph of μ(t) is concave downward if r₂⧸r₃ > 1, concave upward if r₂⧸r₃< 1, and linear if r₂⧸r₃ = 1. If the fraction of S extracted is not too large, L(t) has nearly the same shape as μ(t), and therefore, L(t) contains information about h_c(t) and h_av(t).     

Immobilization of <Emphasis Type="Bold">β</Emphasis>-fructofuranosidase from <Emphasis Type="Italic">Aspergillus japonicus</Emphasis> on chitosan using tris(hydroxymethyl)phosphine or glutaraldehyde as a coupling agent

A partially purified -fructofuranosidase from Aspergillus japonicus was covalently immobilized on to chitosan beads using either glutaraldehyde or tris(hydroxymethyl)phosphine (THP) as a coupling agent. Compared with the glutaraldehyde-immobilized and the free enzyme, the THP-immobilized enzyme had the highest thermal stability with 78% activity retained after 12 days at 37 ° C. The THP-immobilized enzyme also had higher reusability than that immobilized by glutaraldehyde, 75% activity was retained after 11 batches (or 11 days) at 37° C for the THP immobilized enzyme system. Less yield (48%) of fructooligosaccharides (FOS) were produced by the THP-immobilized enzyme compared with the free enzyme system (58%) from 50 (w/v) sucrose at 50 ° C.     

Intracellular activity of morphologically identified neurons of the grass frog's optic tectum in response to moving configurational visual stimuli

Summary In the grass frogRana temporaria, various classes of tectal neurons were identified by means of intracellular recording and iontophoretic staining using potassium-citrate/Co³⁺-lysine-filled micropipettes, which have been defined previously by extracellular recording methods. Class T5(1) neurons had receptive fields (RF) of 33°±5° diameter. In response to a moving 8°×8° square (S), a 2°×16° worm-like (W), or a 16°×2° antiworm-like (A) moving stripe, these cells showed excitatory postsynaptic potentials (EPSPs) and spikes which were interrupted occasionally by small inhibitory postsynaptic potentials (IPSPs). The excitatory responses (R) were strongest towards the square (R_S) and less to the worm (R_W). For the antiworm (R_A) the responses were smallest or equal to the worm stimulus yielding the relationship R_S>R_WR_A. Some of these cells were identified as pear-shaped or large ganglionic neurons, whose somata were located in the tectal cell layer 8. The somata of other large ganglionic neurons were found in layer 7 and the somata of other pear-shaped neurons at the top of layer 6, both displaying T5(1) properties. Class T5(2) neurons (RF=34°±3°) responded with large EPSPs and spikes, often interrupted by small IPSPs, when their RF was traversed by the square stimulus. The excitatory activity was somewhat less to the worm stimulus, whereas no activity at all, or only IPSPs, were recorded in response to the antiworm-stimulus; thus yielding the relationship for the excitatory activity R_S>R_W>R_A 0. Such a cell was identified as pyramidal neuron; the soma was located at the top of layer 6, with the long axon travelling into layer 7 to the medulla oblongata. Class T5(3) neurons (RF=29°±6°) showing EPSPs and spikes according to the relationship R_S>R_A>R_W have been identified as large ganglionic neurons. Their somata were located in layer 8. Class T5(4) neurons (RF=24±7°) responded only to the square stimulus with EPSPs and spikes, sometimes interrupted by IPSPs and yielding the relationship R_S>R_AR_W0. The somata of these large ganglionic or pear-shaped neurons were located in layer 8. Class T1(1) neurons (RF=30°–40°) were most responsive to stimuli moving at a relatively long distance in the binocular visual field, and have been identified as pear-shaped neurons. Their somata were located in layer 6.Further neurons are described and morphologically identified which have not yet been classified by extracellular recording methods. For example,IPSP neurons (RF=20°–30°) responded (R) with IPSPs only according to the relationship R_S>R_A R_W. The somata of these pear-shaped neurons were located in layer 6.The properties of tectal cells in response to electrical stimulation of the optic tract and to brisk changes of diffuse illumination suggest certain neuronal connectivity patterns. The results support the idea ofintegrative functional units (assemblies) of connected cells which are involved in various perceptual processes, such as configurational prey selection expressed by T5(2) prey-selective neurons.Abbreviations A antiworm-like 16°×2° stripe stimulus with long axis perpendicular to the direction of movement - W wormlike 2°×16° stripe stimulus with long axis oriented parallel to the direction of movement - S square 8°×8° moving stimulus - ERF excitatory receptive field - IRF inhibitory receptive field - RF receptive field - EPSP excitatory postsynaptic potential - IPSP inhibitory postsynaptic potential     

Electrochemically produced hydrogen peroxide affects Joliot-type oxygen-evolution measurements of photosystem II

The main technique employed to characterize the efficiency of water-splitting in photosynthetic preparations in terms of miss and double hit parameters and for the determination of S_i (i = 2,3,0) state lifetimes is the measurement of flash-induced oxygen oscillation pattern on bare platinum (Joliot-type) electrodes. We demonstrate here that this technique is not innocent. Polarization of the electrode against an Ag/AgCl electrode leads to a time-dependent formation of hydrogen peroxide by two-electron reduction of dissolved oxygen continuously supplied by the flow buffer. While the miss and double hit parameters are almost unaffected by H₂O₂, a time dependent reduction of S₁ to S_− 1 occurs over a time period of 20 min. The S₁ reduction can be largely prevented by adding catalase or by removing O₂ from the flow buffer with N₂. Importantly, we demonstrate that even at the shortest possible polarization times (40 s in our set up) the S₂ and S₀ decays are significantly accelerated by the side reaction with H₂O₂. The removal of hydrogen peroxide leads to unperturbed S₂ state data that reveal three instead of the traditionally reported two phases of decay. In addition, even under the best conditions (catalase + N₂; 40 s polarization) about 4% of S_− 1 state is observed in well dark-adapted samples, likely indicating limitations of the equal fit approach. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.