Role of betaine:CoA ligase (CaiC) in the activation of betaines and the transfer of coenzyme A in Escherichia coli.

AIMS: Characterization of the role of CaiC in the biotransformation of trimethylammonium compounds into l(-)-carnitine in Escherichia coli. METHODS AND RESULTS: The caiC gene was cloned and overexpressed in E. coli and its effect on the production of l(-)-carnitine was analysed. Betaine:CoA ligase and CoA transferase activities were analysed in cell free extracts and products were studied by electrospray mass spectrometry (ESI-MS). Substrate specificity of the caiC gene product was high, reflecting the high specialization of the carnitine pathway. Although CoA-transferase activity was also detected in vitro, the main in vivo role of CaiC was found to be the synthesis of betainyl-CoAs. Overexpression of CaiC allowed the biotransformation of crotonobetaine to l(-)-carnitine to be enhanced nearly 20-fold, the yield reaching up to 30% (with growing cells). Higher yields were obtained using resting cells (up to 60%), even when d(+)-carnitine was used as substrate. CONCLUSIONS: The expression of CaiC is a control step in the biotransformation of trimethylammonium compounds in E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: A bacterial betaine:CoA ligase has been characterized for the first time, underlining its important role for the production of l-carnitine with Escherichia coli.

Impairing and monitoring glucose catabolite repression in L-carnitine biosynthesis.

Signal transduction pathways are usually avoided when optimizing a biotransformation process because they require complex mathematical formulations. The aim of this work was to use a Systems Biology approach to optimize and monitor the biotransformation of L-carnitine using signal transduction pathways. To this end, a dynamic model was constructed, integrating the metabolic pathways of L-carnitine biosynthesis as well as the expression of this metabolism by means of its regulation by transcription factors such as cAMP-CRP and CaiF. The model was validated using different C-sources as well as different reactor feeding approaches. A linear relationship between the external cellular cAMP and the L-carnitine production levels was predicted before being experimentally confirmed in several scenarios. Moreover, results of the model simulations and subsequent experimental findings demonstrated that the addition of exogenous cAMP was able to restore the L-carnitine production when glucose was used as C-source. Additionally, a way to monitor the L-carnitine biosynthesis by using the level of cAMP as a marker of the biotransformation state was in silico and experimentally demonstrated.

The existence of apotyrosinase in the cytosol of Harding-Passey mouse melanoma melanocytes and characteristics of enzyme reconstitution by Cu(II).

This paper reports the effect of Cu(II) supplementation on the tyrosinase isozymes from Harding-Passey mouse melanoma. The dopa-oxidase activity of the microsomal and soluble isozymes is increased by incubation with Cu(II), whereas the activity of the unique 'in vivo' melanin-forming isozyme, bound to melanosomes, is not. Other divalent cations are ineffective in increasing the dopa-oxidase activity of tyrosinases. These results indicate the existence of a mixture of tyrosinase and apotyrosinase in the cytosol of melanocytes before reaching the melanosome. The paucity of Cu(II) in the cytosol could be one of the mechanisms of regulation contributing to avoid the formation of melanin outside the melanosome. Some kinetic characteristics of the enzymatic reconstitution of soluble and microsomal isozymes by Cu(II) are also studied, and the results suggest that the glycosylation of apotyrosinase during its maturation yields a conformational change favouring the binding of Cu(II) at the enzyme active site, by lowering the activation energy of the reconstitution reaction.

Kinetic study of the interaction between frog epidermis tyrosinase and chloride.

The effect of halide ions on frog epidermis tyrosinase has been characterized with the trypsin-activated enzyme. At pH 7, the order of inhibition is I- greater than Br- greater than Cl- greater than F-. Chloride, the most extensively studied halide, shows a competitive pattern with respect to the substrate, L-DOPA. Inhibition is strongly pH-dependent, with a pKa of 6.12 for the responsible protonatable group. Other kinetic constants are also calculated using a novel approach. The mechanism of interaction between chloride and the enzyme is discussed, and a model is proposed in which chloride interferes the tyrosinase activity by displacing a catalytically important ligand, probably a histidine residue of the side-chain, from the copper at the enzyme-active site.

Kinetic study on the slow inhibition of epidermis tyrosinase by m-coumaric acid.

The inhibition by m-coumaric acid of oxidation of L-dopa by epidermis tyrosinase (monophenol,dihydroxy-L-phenylalanine:oxygen oxidoreductase, EC 1.14.18.1) is characterized by a prolonged transient phase. Kinetic data correspond to that for a postulated mechanism that involves rapid formation of a reduced enzyme-m-coumaric acid complex that subsequently undergoes a relatively slow reversible reaction. An overall inhibition constant for m-coumaric acid of 0.05 mM was calculated. The value of the Ki for the dissociation of m-coumaric acid from the rapidly formed complex was calculated as 0.53 mM. The first-order rate constants for the slow isomerization of the enzyme-inhibitor complex were calculated as 3.0 +/- 0.1 min-1 for the forward step and 0.31 +/- 0.06 min-1 for the reverse step.

Design of metabolic engineering strategies for maximizing L-(-)-carnitine production by Escherichia coli. Integration of the metabolic and bioreactor levels.

In this work metabolic engineering strategies for maximizing L-(-)-carnitine production by Escherichia coli based on the Biochemical System Theory and the Indirect Optimization Method are presented. The model integrates the metabolic and the bioreactor levels using power-law formalism. Based on the S-system model, the Indirect Optimization Method was applied, leading to profiles of parameter values that are compatible with both the physiology of the cells and the bioreactor system operating conditions. This guarantees their viability and fitness and yields higher rates of L-(-)-carnitine production. Experimental results using a high cell density reactor were compared with optimized predictions from the Indirect Optimization Method. When two parameters (the dilution rate and the initial crotonobetaine concentration) were directly changed in the real experimental system to the prescribed optimum values, the system showed better performance in L-(-)-carnitine production (74% increase in production rate), in close agreement with the model's predictions. The model shows control points at macroscopic (reactor operation) and microscopic (molecular) levels where conversion and productivity can be increased. In accordance with the optimized solution, the next logical step to improve the L-(-)-carnitine production rate will involve metabolic engineering of the E. coli strain by overexpressing the carnitine transferase, CaiB, activity and the protein carrier, CaiT, responsible for substrate and product transport in and out of the cell. By this means it is predicted production may be enhanced by up to three times the original value.

Kinetic study and intermediates identification of noradrenaline oxidation by tyrosinase.

Characterization of intermediates formed in the noradrenaline oxidation by mushroom tyrosinase and sodium periodate has been performed by rapid scanning spectrophotometry and graphical analysis of obtained spectra. In a pH range from 5.0 to 6.0, it has been possible to detect o-noradrenalinequinone-H+ as the first intermediate in these oxidations. The following steps for noradrenaline transformation into noradrenochrome would be: noradrenaline----o-noradrenalinequinone-H+----o- noradrenalinequinone----leukonoradrenochrome----noradreno chrome. It has been also verified that o-noradrenalinequinone-H+ is transformed into noradrenochrome at a constant ratio. The stoichiometry for this converstion followed the equation: 2-noradrenalinequinone-H+----noradrenaline + noradrenochrome. The pathway between noradrenaline and noradrenochrome has been studied as a system of various chemical reactions coupled to an enzymatic reaction. We have denominated this type of mechanism as an enzymatic-chemical-chemical mechanism, (E2CC). Whole rate constants for the implicated chemical steps at different pH and temperature values have been evaluated from measurement of the lag period arising from the accumulation of noradrenochrome that takes place when noradrenaline was oxidized at pH 5-6. The lag period was independent on enzyme concentration, but was increased when pH and/or temperature were increased. Rate constants pH independent for the deprotonation of noradrenalinequinone-H+ into noradrenalinequinone and for the internal cyclization of noradrenalinequinone into leukonoradrenochrome have been obtained. We conclude that this minor pathway of noradrenaline oxidation by tyrosinase follows a scheme similar to that established for L-dopa.

Effect of polyols on alpha-chymotrypsin thermostability: a mechanistic analysis of the enzyme stabilization.

The influence of the synthetic substrate (N-acetyl-L-tyrosine ethyl ester) and the different polyols (ethylene glycol, glycerol, erythritol, xylitol and sorbitol) on the thermostability of alpha-chymotrypsin at 60 degrees C have been studied. The results obtained showed an important stabilizing effect in the presence of both additives. In order to describe the kinetics of enzyme stabilization, the experimental results were analyzed by a four-parameters deactivation model with excellent agreement. In all cases, alpha-chymotrypsin exhibited non-first-order deactivation kinetics, corresponding to a two-step unimolecular mechanism, where the main protective effect of polyols was observed in the first-step of the deactivation profile. Thus, the presence of polyols increased the level of activity stabilization (alpha 1), and decreased the first-order deactivation rate constant (k1). Additionally, the experimental results were analyzed as a function of both, the change in the standard free energy of denaturation (delta(delta Gzero)), and a protective effect, defined as the ratio of alpha-chymotrypsin half-lives (with and without polyols), showing in both cases a clear stabilizing effect of these polyhydroxylic cosolvents for the enzyme. The overall protective effect of polyols was also simultaneously related to their concentration and their water-activity depressing power.

Incorporation of bovine thyroid peroxidase in liposomes.

Bovine thyroid peroxidase (TPO), an enzyme requiring lipids for demonstrating catalytic activity, was incorporated in liposomes made of pure phospholipids. The enzyme did not show high differences in activity when bilayer thickness was changed, but dipalmitoyl phosphatidyl choline (DPPC) seemed to be more appropriate for activity. The perturbation caused on lipid fluidity by enzyme incorporation was studied by differential scanning calorimetry (DSC) and fluorescence polarization of the apolar probe 1,6-diphenyl-1,3,5-hexatriene (DPH). The complexes of TPO with dimyristoyl phosphatidyl choline (DMPC), DPPC, and distearoyl phosphatidyl choline (DSPC) bilayers showed transition temperatures (Tc) which were lower than the characteristic ones shown by liposomes with the respective phospholipids alone. The microsomal fraction from which TPO was extracted was in the fluid state at 37 degrees C, the temperature at which thyroid peroxidase works 'in vivo'. Since the effect of the protein in lowering the transition temperature of the phospholipids was so low, the contribution of phospholipids containing unsaturated fatty acids has to be essential for obtaining a fluid bilayer at body temperature.

A non-destructive method to determine the safranal content of saffron (Crocus sativus L.) by supercritical carbon dioxide extraction combined with high-performance liquid chromatography and gas chromatography.

A supercritical carbon dioxide extraction method to obtain selectively volatile compounds of saffron without sample destruction has been developed. The influence of both pressure and temperature was studied, 20 MPa and 100 degrees C being the best conditions to extract the total safranal content. A decrease in supercritical fluid density was shown to be a critical parameter for enhancing the extraction power of carbon dioxide. For all the assay conditions, the extracts mainly contained safranal and HTCC, as demonstrated by gas chromatography and high-performance liquid chromatography analyses. Both chromatographic methods were suitable for safranal quantification and showed excellent agreement. Supercritical extracts from five different saffron types were studied by high-performance liquid chromatography and their safranal contents were determined.

Activity of soluble and immobilized hesperidinase on insoluble hesperidin.

A michaelian kinetic behaviour was found when the α-ramnosidase activity, both of native and immobilized hesperidinase, was determined on hesperidin suspensions. In spite of the low hesperidin solubility in the reaction medium, the maximum rates overtook the expected values, thus pointing to the enzyme ability to degrade insoluble hesperidin.

A cross-flow reactor with immobilized pectolytic enzymes for juice clarification.

A new system for continuous juices clarification is presented. The bioreactor combines microporous plates commercially available and industrial pectinases immobilized on nylon membranes in a cross-flow configuration. The kinetic behaviour of the reactor for different recirculation flow rates has been determined. Fresh apricot juice has been continuously clarified in the bioreactor with excellent results.

Influence of polyhydroxylic cosolvents on papain thermostability.

Papain thermostability was studied, and non-first-order deactivation kinetics were observed. The results obtained were analyzed by a two-step series-type deactivation model involving the native and active enzyme, an active intermediate enzyme state, and a final inactive state, with excellent agreement. The influence of different polyhydroxylic cosolvents (ethylene glycol, glycerol, erythritol, xylitol and sorbitol) on the thermostability of papain at 60 degrees C was also studied. Analysis of the results by the assayed model showed that the main protective effect of cosolvents was observed in the second step of the deactivation profile. The results obtained were analyzed as a function of both the thermodynamic parameters and a protective effect, defined as the ratio of papain half-lives (with and without cosolvents) for the second deactivation step, showing in both cases an important stabilizing effect of these cosolvents on the enzyme. The overall protective effect of cosolvents was also related simultaneously to their concentration and their water activity-depressing power.

Kinetic and operational study of a cross-flow reactor with immobilized pectolytic enzymes.

The kinetics and operational behavior of a nylon membrane derivative with immobilized pectolytic enzymes in a cross-flow reactor were analyzed. A high dependence on the recycling flow rate was observed. A design equation of the system has been proposed by taking into account both the shear stress deactivation and the control of the external diffusional limitations. Integration of the resulting design equation allowed us to study the effect of different operational parameters on substrate conversion. The catalytic efficiency of the immobilized derivative in a cross-flow reactor showed the highest pectin hydrolysis capability when it was compared with two different configurations of packed-bed reactors.

Conformational studies of soluble and immobilized frog epidermis tyrosinase by fluorescence.

Fluorescence spectra and soluble quenching of intrinsic protein fluorescence were used as indexes of conformational changes suffered by frog epidermis tyrosinase. The activation process and the immobilization of the enzyme involving either free amino groups or its carbohydrate moiety were studied. The conformational changes resulting from denaturation of each one of the protein derivatives, as well as the effect of active center copper extraction, were followed by fluorescence studies. The results showed that: both activation and immobilization were accompanied by conformational changes of the protein leading to more unfolded states; neither enzyme nor immobilized enzyme were fully unfolded upon denaturation although enzymic activity was lost; the enzyme immobilized through its carbohydrate moiety was more unfolded upon denaturation than the enzyme immobilized through amino groups, thus pointing to a higher conformational stabilization in the last situation; and that tryptophyl residues moved to a localization near the active site upon activation.

FT-IR spectroscopy of natural melanins isolated from Harding-Passey mouse melanoma.

The alterations caused on the structure of melanins by acid and alkaline extraction methods were studied by means of techniques such as Fourier Transform Infrared spectroscopy and thermogravimetry. Acid extraction results in the hydrolysis of aromatic monomers of the pigment, leading to uncyclicized residues and to a less conjugated polymer. Alkaline methods catalyse the covalent binding of proteins present in the melanin extract, thus leading to melanoprotein artifacts. A new method based on the delipidation with ether and deproteinization with SDS of previously isolated melanosomes is proposed. This method shows that melanins present in Harding-Passey mouse melanoma have a bound protein moiety, so that they should exist "in vivo" as melanoprotein complexes. Thermogravimetric data suggest that the chromogen moiety of the melanoproteins in Harding-Passey mouse melanoma is a mixture of eu- and pheomelanins.

Salt stress effects on the central and carnitine metabolisms of Escherichia coli.

The aim was to understand how interaction of the central carbon and the secondary carnitine metabolisms is affected under salt stress and its effect on the production of L-carnitine by Escherichia coli. The biotransformation of crotonobetaine into L-carnitine by resting cells of E. coli O44 K74 was improved by salt stress, a yield of nearly twofold that for the control being obtained with 0.5 M NaCl. Crotonobetaine and the L-carnitine formed acted as an osmoprotectant during cell growth and biotransformation in the presence of NaCl. The enzyme activities involved in the biotransformation process (crotonobetaine hydration reaction and crotonobetaine reduction reaction), in the synthesis of acetyl-CoA/acetate (pyruvate dehydrogenase, acetyl-CoA synthetase [ACS] and ATP/acetate phosphotransferase) and in the distribution of metabolites for the tricarboxylic acid cycle (isocitrate dehydrogenase [ICDH]) and glyoxylate shunt (isocitrate lyase [ICL]) were followed in batch with resting cells both in the presence and absence of NaCl and in perturbation experiments performed on growing cells in a high density cell recycle membrane reactor. Further, the levels of carnitine, crotonobetaine, gamma-butyrobetaine and ATP and the NADH/NAD(+) ratio were measured in order to know how the metabolic state was modified and coenzyme pools redistributed as a result of NaCl's effect on the energy content of the cell. The results provided the first experimental evidence of the important role played by salt stress during resting and growing cell biotransformation (0.5 M NaCl increased the L-carnitine production in nearly 85%), and the need for high levels of ATP to maintain metabolite transport and biotransformation. Moreover, the main metabolic pathways and carbon flow operating during cell biotransformation was that controlled by the ICDH/ICL ratio, which decreased from 8.0 to 2.5, and the phosphotransferase/ACS ratio, which increased from 2.1 to 5.2, after a NaCl pulse fivefold the steady-state level. Resting E. coli cells were seen to be made up of heterogeneous populations consisting of several types of subpopulation (intact, depolarized, and permeabilized cells) differing in viability and metabolic activity as biotransformation run-time and the NaCl concentration increased. The results are discussed in relation with the general stress response of E. coli, which alters the NADH/NAD(+) ratio, ATP content, and central carbon enzyme activities.

Role of energetic coenzyme pools in the production of L-carnitine by Escherichia coli.

The aim of this work was to understand the steps controlling the biotransformation of trimethylammonium compounds into L(-)-carnitine by Escherichia coli. The high-cell density reactor steady-state levels of carbon source (glycerol), biotransformation substrate (crotonobetaine), acetate (anaerobiosis product) and fumarate (as an electron acceptor) were pulsed by increasing them fivefold. Following the pulse, the evolution of the enzyme activities involved in the biotransformation process of crotonobetaine into L(-)-carnitine (crotonobetaine hydration), in the synthesis of acetyl-CoA (ACS: acetyl-CoA synthetase and PTA: ATP: acetate phosphotransferase) and in the distribution of metabolites for the tricarboxylic acid (ICDH: isocitrate dehydrogenase) and glyoxylate (ICL: isocitrate lyase) cycles was monitored. In addition, the levels of carnitine, the cell ATP content and the NADH/NAD(+) ratio were measured in order to assess the importance and participation of these energetic coenzymes in the catabolic system. The results provided an experimental demonstration of the important role of the glyoxylate shunt during biotransformation and the need for high levels of ATP to maintain metabolite transport and biotransformation. Moreover, the results obtained for the NADH/NAD(+) pool indicated that it is correlated with the biotransformation process at the NAD(+) regeneration and ATP production level in anaerobiosis. More importantly, a linear correlation between the NADH/NAD(+) ratio and the levels of the ICDH and ICL (carbon and electron flows) and the PTA and ACS (acetate and ATP production and acetyl-CoA synthesis) activity levels was assessed. The main metabolic pathway operating during cell metabolic perturbation with a pulse of glycerol and acetate in the high-cell density membrane reactor was that related to ICDH and ICL, both regulating the carbon metabolism, together with PTA and ACS enzymes (regulating ATP production).

A model that links growth and secondary metabolite production in plant cell suspension cultures.

Plant cell suspensions of grape cells (Vitis vinifera L. cv. Gamay Fréaux) were grown in shake flasks operated both in the batch and semicontinuous mode. A mathematical model was developed to describe grape cell growth, sucrose uptake, and secondary metabolite (anthocyanin) production. Parameters were estimated from batch studies data. The model was able to predict results for semicontinuous experiments by only modifying the value of four of these parameters. The modified parameters (maximum specific rate of biomass production, maximum specific rate of substrate consumption for maintenance, maximum specific rate of anthocyanin production, and degradation constant of anthocyanins) were related to the kinetics rather than to the yield of the process. The model introduces the concept of primary and secondary metabolism substrate concentration-dependent competition for precursors. Further, the model was able to predict the evolution of the cell system when substrate is scarce, as the value of the different kinetic constants determines the portion of substrate that is used for biomass production, secondary metabolite production, and cell maintenance. (c) 1995 John Wiley & Sons, Inc.