Kinetics of Pyruvate Decarboxylase Deactivation by Benzaldehyde

Based on experimental data, a kinetic model for the deactivation of partially purified pyruvate decarboxylase (PDC) by benzaldehyde (0–200 mM) in MOPS buffer (2.5 M) has been developed. An initial lag period prior to deactivation was found to occur. With first order dependencies of PDC deactivation on exposure time and on benzaldehyde concentration, a reaction time deactivation constant of 2.64×10^?3 h^?1 and a benzaldehyde deactivation coefficient of 1.98×10^?4 mM^?1 h^?1 were determined for benzaldehyde concentrations up to 200 mM. The PDC deactivation kinetic equations established in this study are an essential component in an overall model being developed to describe the enzymatic biotransformation of benzaldehyde and pyruvate to produce the pharmaceutical intermediate (R)-phenylacetylcarbinol (R-PAC).     

Deactivation studies of immobilized glucose oxidase

Studies have been performed in a tubular flow reactor to characterize the deactivation of immobilized glucose oxidase. The effects of oxygen concentration in the range of 0.09 to 0.467mM and hydrogen peroxide concentrations in the range of 0.1 to 10mM were studied. A simple mathematical model assuming first-order reaction and deactivation was found to describe the deactivation behavior adequately. The deactivation rate constant was found to increase with increasing levels of feed oxygen. Hydrogen peroxide was found to deactivate the enzyme severely and the deactivation rate constants were higher than those for oxygen deactivation. The influence of external and internal diffusion effects on the deactivation rate constant were examined. Although diffusional restrictions were negligible for oxygen transfer to the pellet, they were significant for transfer of hydrogen peroxide to the bulk stream. Increasing deactivation rates. Severe internal diffusion limitations were observed for the glucose oxidase system. However, for particle sizes in the range of 500 to 2000 μm, no effect on the rate of deactivation of the enzyme was observed.     

Length-dependent deactivation of ventricular trabeculae in the bivalve, Spisula solidissima

Shortening-deactivation has been identified and characterized in ventricular trabeculae of the bivalve, Spisula solidissima (Heterodonta, Mactridae). This muscle had ultrastructural similarities to vertebrate smooth muscle. Deactivation was defined as the fraction of maximal force lost during a contraction when a muscle is shortened rapidly (by a quick-release, QR) to a known length, relative to a control isometric contraction at that same length. The magnitude of deactivation was dependent on the size of the release and the point at which the release was applied during the cycle of contraction. QR/quick-stretch (QS) perturbations at the same point during the contraction resulted in negligible deactivation. The magnitude of deactivation was independent of shortening rate. Deactivation was attenuated by applying caffeine (100 μM) and blocked with high extracellular Ca²⁺ (56 mM). The Ca²⁺ ionophore, A23187 (10 μM), augmented deactivation as did the positive inotrope serotonin (100 nM). Treatment with ryanodine (5 μM) had no significant effect on deactivation. These results suggest that a reduction in Ca²⁺ at the contractile element and/or sequestration of Ca²⁺ may occur during shortening. Deactivation may minimize the magnitude of work done during active shortening of bivalve cardiac muscle, particularly against the low afterload exhibited in the bivalve peripheral circulatory system. Intracellular Ca²⁺ fluxes during sudden length perturbations may explain the effect of stretch on action potential duration in the bivalve heart, as shown previously.     

Polarized Photoacoustic Spectra of the Cyanobacterium Synechococcus Oriented in Polymer Film

Photoacoustic spectra (PAS) were obtained for the cyanobacterium Synechococcus (Anacystis nidulans) cells embedded in isotropic and stretched polyvinyl alcohol films. The polarized radiation with the electric vector changing in 30° intervals with respect to given direction in a sample plane was used. Two cyanobacterium strains, one with very low biliprotein content, second with normal amount of biliproteins were investigated. The polarized absorption and fluorescence spectra were also measured. Conclusions were drawn about the thermal deactivation occurring in differently oriented pools of chromophores and about mutual orientation of their transition moments. Thermal deactivation in carotenoids (Cars) of both strains was different. The ratio of Car thermal deactivation to the thermal deactivation of chlorophyll (Chl) was higher in cyanobacteria with lower content of biliproteins than in the strain with normal amount of these complexes. Hence biliproteins can play the role in excitation energy transfer from Cars to Chls. For complex biological samples, polarized PAS can be a more sensitive method to investigate the directions of the absorption transition moments than the widely used polarized absorption spectra.     

Prediction of continuous reactors performance based on batch reactor deactivation kinetics data of immobilized lipase

Experiments on deactivation kinetics of immobilized lipase enzyme fromCandida cylindracea were performed in stirred batch reactor using rice bran oil as the substrate and temperature as the deactivation parameter. The data were fitted in first order deactivation model. The effect of temperature on deactivation rate was represented by Arrhenius equation. Theoretical equations were developed based on pseudo-steady state approximation and Michaelis-Menten rate expression to predict the time course of conversion due to enzyme deactivation and apparent half-life of the immobilized enzyme activity in PFR and CSTR under constant feed rate policy for no diffusion limitation and diffusion limitation of first order. Stability of enzyme in these continuous reactors was predicted and factors affecting the stability were analyzed.     

Optimising diquat use for submerged aquatic weed management

An experimentally derived prediction tool is under development which aims to assess potential deactivation of diquat caused by water and deposits on plant leaf surfaces in New Zealand water bodies, where aquatic weeds are targeted for diquat treatment. Optimising the use and success of diquat is important not only in managing public confidence in the use of aquatic herbicides, but also in minimising financial risk from failed treatments. Our approach focuses on characterising lake water quality and plant condition factors in these lakes to identify parameters that might be useful indicators of diquat deactivation potential. Water samples have been collected at 3-month intervals from lakes receiving large scale treatment for weed control. Samples have been analysed for turbidity, suspended solids, chlorophyll a, conductivity and dissolved anions. Samples have also been spiked with 1 mg l⁻¹ diquat to measure loss from adsorption and/or absorption. Shoot samples were also collected from targeted weed species at each sampling site and the amount of organic and inorganic deposits on plants has been measured and then added to a second diquat spiked sample to assess potential additional diquat loss from these deposits. Our results have shown deactivation from deposits on plant surfaces which is highly correlated with turbidity, including inorganic suspended solids and total suspended solids. A plant “dirtiness” scale has been devised to help predict the likely success or risk of diquat failure prior to any decision to proceed with treatment. Deactivation in water was only weakly linked to total suspended solids. Our failure to find significant correlation with the water quality factors measured may reflect the need for more detailed analysis of the particle size and composition of suspended solids and future research will address these issues.     

Rate of activation and deactivation of K∶Cl cotransport by changes in cell volume in hemoglobin SS,CC and AA red cells

Red blood cells (RBC) of subjects homozygous for hemoglobin A (AA), C (CC) and S (SS) exhibit different cell volumes which might be related to differences in cell volume regulation. We have investigated how rapidly K:Cl cotransport is activated and deactivated to regulate the cell volume in these cells. We measured the time course of net K⁺ efflux after step changes in cell volume and determined two delay times: one for activation by cell swelling and a second for deactivation by cell shrinkage. Cell swelling induced by 220 mOsm media activated K⁺ efflux to high values (10–20 mmol/ liter cell x hr) in CC and SS; normal AA had a threefold lower activity. The delay time for activation was very short in blood with a high percentage of reticulocytes (retics): (SS, 10% retics, 1.7±0.3 min delay, n=8; AA, 10% retics, 4±1.5 min, n=3; CC, 11.6% retics, 4±0.3, n=3) and long in cells with a smaller percentage of reticulocytes: (AA, 1.5% retics, 10±1.4 min, n=8; CC whole blood 6% retics, 10±2.0 min, n=10, P<0.02 vs. SS). The delay times for deactivation by cell shrinking were very short in SS (3.6±0.4 min, n=8, P<0.02) and AA cells with high retics (2.7±1 min, n=3) and normal retics (2.8±1 min, n=3), but 8–15-fold longer in CC cells (29±2.8 min, n=9).Density fractionation of CC cells (n=3) resulted in coenrichment of the top fraction in reticulocytes and in swelling-activated cotransport (fourfold) with short delay time for activation (4±0.3 min) and long delay for deactivation (14±4 min). The delay time for activation, but not for deactivation, increased markedly with increasing cell density. These findings indicate that all CC cells do not promptly shut off cotransport with cell shrinkage and high rates of cellular K⁺ loss persist after return to isotonic conditions.In summary, (i) K:Cl cotransport is not only very active in young cells but it is also very rapidly activated and deactivated in young AA and SS cells by changes in cell volume. (ii) Delay times for cotransport activation markedly increased with RBC age and in mature cells with low cotransport rates, long delay times for activation were observed. (iii) The long delay time for deactivation exhibited even by young CC cells induces a persistent loss of K⁺ after cell shrinkage which may contribute in vivo to the uniformly low cell volume, low K⁺ and water content of CC cells.This research was supported by National Institutes of Health grants Shannon Award HL-35664, HL-42120, Sickle Cell Center grant HL-38655, and a Grant-in-Aid of the New York Branch of the American Heart Association. The technical help of Sandra M. Suzuka, M.S. is gratefully acknowledged.     

Naringin in Ganshuang Granule suppresses activation of hepatic stellate cells for anti‐fibrosis effect by inhibition of mammalian target of rapamycin

A previous study has demonstrated that Ganshuang granule (GSG) plays an anti‐fibrotic role partially by deactivation of hepatic stellate cells (HSCs). In HSCs activation, mammalian target of rapamycin (mTOR)‐autophagy plays an important role. We attempted to investigate the role of mTOR‐autophagy in anti‐fibrotic effect of GSG. The cirrhotic mouse model was prepared to demonstrate the anti‐fibrosis effect of GSG. High performance liquid chromatography (HPLC) analyses were used to identify the active component of GSG. The primary mouse HSCs were isolated and naringin was added into activated HSCs to observe its anti‐fibrotic effect. 3‐methyladenine (3‐MA) and Insulin‐like growth factor‐1 (IGF‐1) was added, respectively, into fully activated HSCs to explore the role of autophagy and mTOR. GSG played an anti‐fibrotic role through deactivation of HSCs in cirrhotic mouse model. The concentration of naringin was highest in GSG by HPLC analyses and naringin markedly suppressed HSCs activation in vitro, which suggested that naringin was the main active component of GSG. The deactivation of HSCs caused by naringin was not because of the autophagic activation but mTOR inhibition, which was supported by the following evidence: first, naringin induced autophagic activation, but when autophagy was blocked by 3‐MA, deactivation of HSCs was not attenuated or reversed. Second, naringin inhibited mTOR pathway, meanwhile when mTOR was activated by IGF‐1, deactivation of HSCs was reversed. In conclusion, we have demonstrated naringin in GSG suppressed activation of HSCs for anti‐fibrosis effect by inhibition of mTOR, indicating a potential therapeutic application for liver cirrhosis.     

Deactivation of macrophage oxidative burstin vitro by different strains ofHistoplasma capsulatum

It was previously reported thatHistoplasma capsulatum (Hc) yeast not only failed to stimulate a murine macrophage oxidative burst (OB), but they also blunted or abolished OB stimulation by a subsequent encounter with potent stimuli such as zymosan or phorbol 12-myristate 13-acetate (PMA). The present studies show that macrophage deactivation is proportional to the time of incubation and the dose of Hc yeast that induce the deactivated state. Hc yeast derived from a virulent strain (G217B) are more efficient inducers of macrophage deactivation than similar preparations derived from the avirulent Downs Hc strain. Yeast cells of two other pathogenic fungi,Candida albicans andCryptococcus neoformans are shown to stimulate rather than deactivate a macrophage OB.     

A kinetic model for the deactivation of pyruvate decarboxylase (PDC) by benzaldehyde

To provide further understanding of the biotransformation of benzaldehyde to L-phenylacetyl carbinol (L-PAC), an intermediate in L-ephedrine production, a kinetic model has been developed for the deactivation of pyruvate decarboxylase (PDC) by benzaldehyde. The model confirms that deactivation is first order with respect to benzaldehyde concentration and exhibits a square root dependency on time. The model covers the range of benzaldehyde concentrations 100–300 mM, as it has been shown previously that 200 mM benzaldehyde can produce L-PAC concentrations up to 190 mM (28.6 g/L) using partially purified PDC from Candida utilis.     

The effect of phloretin on single potassium channels in myelinated nerve

The effect of phloretin, a dipolar organic compound, on single potassium channel currents of myelinateed nerve fibres of Xenopus laevis has been investigated, using inside-out patches prepared by the method of Jonas et al. (1989). The I channel, a potential dependent K channel with intermediate deactivation kinetics, was reversibly blocked by 20 µM phloretin applied on the inside; the block was strongest at negative membrane potentials and less pronounced at positive potentials. Phloretin shifted the curve relating open probability to membrane potential towards more positive potentials and reduced its slope and maximum. This confirms previous findings on the effect of phloretin on the voltage dependence of the fast macroscopic K conductance. Single channel conductance and deactivation kinetics were not altered by phloretin. Offprint requests to: Correspondence to: H. Meves     

Kinetics of deactivation for thermostable DNA polymerase enzymes

Summary The kinetics of thermal deactivation for thermostable DNA polymerase enzymes were investigated by using the experimental data published elsewhere (Nielson et al. 1996. Strategies. 9, 7–8). The order of deactivation (a) and the deactivation rate constants (k _d) were determined for different Taq DNA polymerase enzymes and were found to be of first order.     

Production of inulinase by solid-state fermentation: effect of process parameters on production and preliminary characterization of enzyme preparations

This work was aimed at producing inulinase by solid-state fermentation of sugarcane bagasse, using factorial design to identify the effect of corn steep liquor (CSL) and soybean bran concentration, particle size of bagasse and size of inoculum. Maximum inulinase activity achieved was 250 U per g of dry substrate (gds) at 20% (w/w) of CSL, 5% (w/w) of soybean bran, 1 × 10¹⁰ cells mL⁻¹ and particle size of bagasse in the range 9/32 mesh. The use of soybean bran decreased the time to reach maximum activity from 96 to 24 h and the maximum productivity achieved was 8.87 U gds⁻¹ h⁻¹. The maximum activity was obtained at pH 5.0 and 55.0°C. Within the investigated range, the enzyme extract was more thermostable at 50.0°C, showing a D-value of 123.1 h and deactivation energy of 343.9 kJ gmol⁻¹. The extract showed highest stability from pH 4.5 to 4.8. Apparent K _m and V _max are 7.1 mM and 17.79 M min⁻¹, respectively.     

A caveat for the use of log P values for the assessment of the biocompatibility of organic solvents

The degree of enzyme deactivation for lipases from Candida rugosa and Pseudomonas sp., hydroxynitrile lyase and mandelate racemase upon exposure to organic solvents can be correlated to their respective partition coefficients (log P values). However, three unexpected results were obtained: (1) the deactivation exerted by protic solvents, e.g., methanol, is severely underestimated; (2) little deactivation by an organic solvent cannot neccessarily be correlated to catalytic activity in this medium, and (3) in contrast to other enzymes, hydroxynitrile lyase is exceptionally stable towards deactivation by DMF.     

Studies on pH and thermal stability of novel purified L-asparaginase from <Emphasis Type="Italic">Pectobacterium carotovorum</Emphasis> MTCC 1428

Glutaminase free L-asparaginase is known to be an excellent anticancer agent. In the present study, the combined effect of pH and temperature on the performance of purified novel L-asparaginase from Pectobacterium carotovorum MTCC 1428 was studied under assay conditions using response surface methodology (RSM). Deactivation studies and thermodynamic parameters of this therapeutically important enzyme were also investigated. The optimum pH and temperature of the purified L-asparaginase were found to be 8.49 and 39.3°C, respectively. The minimum deactivation rate constant (k _d ) and maximum half life (t _1/2) were found to be 0.041 min⁻¹ and 16.9 h, respectively at pH of 8.6 and 40°C. Thermodynamic parameters (ΔG, ΔH, ΔS, and activation energies) were also evaluated for purified L-asparaginase. The probable mechanism of deactivation of purified L-asparaginase was explained to an extent on the basis of deactivation studies and thermodynamic parameters.     

Modulation of monocyte–tumour cell interactions by <Emphasis Type="Italic">Mycobacterium vaccae</Emphasis>

Immunotherapy with Mycobacterium vaccae as an adjuvant to chemotherapy has recently been applied to treatment of patients with cancer. One of the mechanisms of antitumour activity of Mycobacterium bovis bacillus Calmette-Guérin (BCG), the prototype immunomodulator, is associated with activation of monocytes/macrophages. These studies were undertaken to determine how M. vaccae affects monocyte–tumour cell interactions and, in particular, whether it can prevent or reverse deactivation of monocytes that occurrs following their contact with tumour cells during coculture in vitro. Deactivation is characterised by the impaired ability of monocytes to produce tumour necrosis factor (TNF-), interleukin 12 (IL-12), and enhanced IL-10 secretion following their restimulation with tumour cells. To see whether deactivation of monocytes can be either prevented or reversed, three different strains of M. vaccae—B 3805, MB 3683, and SN 920—and BCG were used to stimulate monocytes before or after exposure to tumour cells. Pretreatment of monocytes with M. vaccae MB 3683, SN 920 and BCG before coculture resulted in increased TNF- and decreased IL-10 production. All strains of M. vaccae and BCG used for treatment of deactivated monocytes enhanced depressed TNF- secretion. Strain SN 920 and BCG increased IL-12 release but only BCG treatment inhibited an enhanced IL-10 production by deactivated monocytes. Thus, although some strains of M. vaccae may either prevent or reverse tumour-induced monocyte deactivation, none of them appears to be more effective than BCG.     

Effect of carbon starvation on toluene degradation activity by toluene monooxygenase-expressing bacteria

Subsurface bacteria commonly exist in a starvation state with only periodic exposure to utilizable sources of carbon and energy. In this study, the effect of carbon starvation on aerobic toluene degradation was quantitatively evaluated with a selection of bacteria representing all the known toluene oxygenase enzyme pathways. For all the investigated strains, the rate of toluene biodegradation decreased exponentially with starvation time. First-order deactivation rate constants for TMO-expressing bacteria were approximately an order of magnitude greater than those for other oxygenase-expressing bacteria. When growth conditions (the type of growth substrate and the type and concentration of toluene oxygenase inducer) were varied in the cultures prior to the deactivation experiments, the rate of deactivation was not significantly affected, suggesting that the rate of deactivation is independent of previous substrate/inducer conditions. Because TMO-expressing bacteria are known to efficiently detoxify TCE in subsurface environments, these findings have significant implications for in situ TCE bioremediation, specifically for environments experiencing variable growth-substrate exposure conditions.     

Mechanistic model of the growth of Streptococcus cremoris HP at super optimal temperatures

The kinetic response of Streptococcus cremoris HP to growth at super optimal temperatures is reported. The response to a step increase in temperature was shown to be transient and to result from an increased metabolic rate caused by the raised temperature combined with thermal deactivation of the cell mass present. The catabolic and anabolic activities of the cell were shown to decay at different rates resulting in an accumulation of cells capable of catabolism (energy production) but unable to reproduce. The proposed mechanism was confirmed by independent estimates of the catabolic and anabolic activities of the culture. A mathematical model based on the proposed mechanism and incorporating simultaneous exponential growth, thermal death, and catabolic uncoupling of anabolically inactive cells was developed. Experimental evaluation of the model indicated the presence of a delay in deactivation of metabolic activity in response to a temperature transient. After the inclusion of this delay in death, it was confirmed that the model was capable of prediction of the balanced growth and transient response of this organism to changes in growth temperature. The delay in death was shown to be of major significance to the control of a simulated cheddar cheese fermentation.     

Immobilization ofArthrobacter simplex cells in thermally reversible hydrogels: Comparative effects of organic solvent and polymeric surfactant on steroid conversion

Summary Organic solvents have sometimes been used to increase the solubility of water insoluble substrates for steroid transformation using immobilized whole cells, even though the cell viability is often damaged. Polymeric surfactants which form micelles in aqueous solutions could be used instead of organic solvents to solubilize the steroid. We have successfully utilized this approach by employing a poly(dimethyl siloxane)-poly(ethyleneoxide) (PDMS-PEO) block copolymer surfactant to enhance conversion of hydrocortisone to prednisolone by immobilizedArthrobacter simplex cells, without deactivation of the immobilized cells.