RESUMO
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.
Assuntos
Betaína/metabolismo , Coenzima A Ligases/metabolismo , Coenzima A/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Microbiologia Industrial , Resíduos Industriais , Reatores Biológicos , Carnitina/biossíntese , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Coenzima A Ligases/análise , Coenzima A Ligases/genética , Escherichia coli/genética , Proteínas de Escherichia coli/análise , Proteínas de Escherichia coli/genética , Expressão Gênica , Genes Bacterianos , Vetores Genéticos , Reação em Cadeia da Polimerase/métodos , Espectrometria de Massas por Ionização por Electrospray , Especificidade por Substrato , Compostos de Trimetil Amônio/metabolismoRESUMO
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.
Assuntos
Carnitina/biossíntese , Glucose/metabolismo , Reatores Biológicos , Biotransformação , AMP Cíclico/farmacologia , Escherichia coli/metabolismo , Glicerol/metabolismo , Modelos Teóricos , Transdução de Sinais , Biologia de SistemasRESUMO
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.
Assuntos
Carbono/metabolismo , Carnitina/metabolismo , Escherichia coli/metabolismo , Redes e Vias Metabólicas , Cloreto de Sódio/farmacologia , Betaína/análogos & derivados , Betaína/metabolismo , Biotransformação/fisiologia , Escherichia coli/enzimologia , Pressão OsmóticaRESUMO
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).
Assuntos
Engenharia Biomédica/métodos , Reatores Biológicos , Vias Biossintéticas , Biotecnologia/métodos , Carnitina/biossíntese , Coenzimas/metabolismo , Escherichia coli/metabolismo , Acetatos/metabolismo , Trifosfato de Adenosina/metabolismo , Betaína/análogos & derivados , Betaína/metabolismo , Biotransformação/fisiologia , Escherichia coli/enzimologia , Fumaratos/metabolismo , Glicerol/metabolismo , NAD/metabolismo , Compostos de Trimetil Amônio/metabolismoRESUMO
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.
Assuntos
Carnitina/biossíntese , Escherichia coli/metabolismo , Biomassa , Reatores BiológicosRESUMO
The aim of this work was to understand the steps controlling the process of biotransformation of trimethylamonium compounds into L(-)-carnitine by Escherichia coli and the link between the central carbon or primary and the secondary metabolism expressed. Thus, the enzyme activities involved in the biotransformation process of crotonobetaine into L(-)-carnitine (crotonobetaine hydration reaction and crotonobetaine reduction reaction), in the synthesis of acetyl-CoA (pyruvate dehydrogenase, acetyl-CoA synthetase, and ATP:acetate phosphotransferase) and in the distribution of metabolites for the tricarboxylic acid (isocitrate dehydrogenase) and glyoxylate (isocitrate lyase) cycles, were followed in batch with both growing and resting cells and during continuous cell growth in stirred-tank and high-cell-density membrane reactors. In addition, the levels of carnitine, crotonobetaine, gamma-butyrobetaine, ATP, NADH/NAD(+), and acetyl-CoA/CoA ratios were measured to determine how metabolic fluxes were distributed in the catabolic system. The results provide the first experimental evidence demonstrating the important role of the glyoxylate shunt during biotransformation of resting cells and the need for high levels of ATP to maintain metabolite transport and biotransformation (2.1 to 16.0 mmol L cellular/mmol ATP L reactor h). Moreover, the results obtained for the pool of acetyl-CoA/CoA indicate that it also correlated with the biotransformation process. The main metabolic pathway operating during cell growth in the high cell-density membrane reactor was that related to isocitrate dehydrogenase (during start-up) and isocitrate lyase (during steady-state operation), together with phosphotransacetylase and acetyl-CoA synthetase. More importantly, the link between central carbon and L(-)-carnitine metabolism at the level of the ATP pool was also confirmed.
Assuntos
Trifosfato de Adenosina/metabolismo , Betaína/análogos & derivados , Betaína/metabolismo , Reatores Biológicos/microbiologia , Carnitina/biossíntese , Proteínas de Transporte/metabolismo , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Metilaminas/metabolismo , Biotransformação , Divisão Celular/fisiologia , Ciclo do Ácido Cítrico/fisiologia , Técnicas de Química Combinatória/métodos , Escherichia coli/citologia , Glioxilatos/metabolismo , Complexos Multienzimáticos/metabolismo , Transdução de Sinais/fisiologia , Estatística como AssuntoRESUMO
A simple unstructured model, which includes carbon source as the limiting and essential substrate and oxygen as an enhancing substrate for cell growth, has been implemented to depict cell population evolution of two Escherichia coli strains and the expression of their trimethylammonium metabolism in batch and continuous reactors. Although the model is applied to represent the trans-crotonobetaine to L-(-)-carnitine biotransformation, it is also useful for understanding the complete metabolic flow of trimethylammonium compounds in E. coli. Cell growth and biotransformation were studied in both anaerobic and aerobic conditions. For this reason we derived equations to modify the specific growth rate, mu, and the cell yield on the carbon source (glycerol), Y(xg), as oxygen increased the rate of growth. Inhibition functions representing an excess of the glycerol and oxygen were included to depict cell evolution during extreme conditions. As a result, the model fitted experimental data for various growth conditions, including different carbon source concentrations, initial oxygen levels, and the existence of a certain degree of cell death. Moreover, the production of enzymes involved within the E. coli trimethylammonium metabolism and related to trans-crotonobetaine biotransformation was also modeled as a function of both the cell and oxygen concentrations within the system. The model describes all the activities of the different enzymes within the transformed and wild strains, able to produce L-(-)-carnitine from trans-crotonobetaine under both anaerobic and aerobic conditions. Crotonobetaine reductase inhibition by either oxygen or the addition of fumarate as well as its non-reversible catalytic action was taken into consideration. The proposed model was useful for describing the whole set of variables under both growing and resting conditions. Both E. coli strains within membrane high-density reactors were well represented by the model as results matched the experimental data.
Assuntos
Betaína/análogos & derivados , Betaína/metabolismo , Reatores Biológicos , Carnitina/análise , Carnitina/biossíntese , Escherichia coli/crescimento & desenvolvimento , Modelos Biológicos , Algoritmos , Simulação por Computador , Escherichia coli/metabolismo , Fumaratos/metabolismo , Membranas/fisiologia , Complexos Multienzimáticos/antagonistas & inibidores , Oxirredutases/antagonistas & inibidores , Fatores de TempoRESUMO
Five different ionic liquids, based on dialkylimidazolium and quaternary ammonium cations associated with perfluorinated and bis (trifluoromethyl) sulfonyl amide anions, were used as reaction media to synthesize N-acetyl-L-tyrosine propyl ester by transesterification with alpha-chymotrypsin at 2% (v/v) water content at 50 degrees C. The synthetic activity was reduced by the increase in alkyl chains length of cations and by increases in anion size, which was related to the decrease in polarity. Incubation of the enzyme (with and without substrate) in ionic liquids exhibited first-order deactivation kinetics at 50 degrees C, allowing determination of deactivation rate constants and half-life times (1-3 h). Ionic liquids showed a clear relative stabilization effect on the enzyme, which was improved by increased chain length of the alkyl substituents on the imidazolium ring cations and the anion size. This effect was 10-times enhanced by the presence of substrate. For example, 1-butyl-3-methylimidazolium hexafluorophosphate increased the alpha-chymotrypsin half-life by 200 times in the presence of substrate with respect to the 1-propanol medium. These results show that ionic liquids are excellent enzyme-stabilizing agents and reaction media for clean biocatalysis in non-conventional conditions.
Assuntos
Quimotripsina/metabolismo , Íons/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismo , 1-Propanol/metabolismo , Animais , Catálise , Bovinos , Estabilidade Enzimática , Esterificação , Íons/síntese química , Íons/química , Soluções/metabolismoRESUMO
The L(-)-carnitine production by biotransformation using the recombinant strain Escherichia coli pT7-5KE32 has been studied and optimized with crotonobetaine and D(+)-carnitine as substrates. A resting rather than a growing cells system for L(-)-carnitine production was chosen, crotonobetaine being the best substrate. High biocatalytic activity was obtained after growing the cells under anaerobic conditions at 37 degrees C and with crotonobetaine or L(-)-carnitine as inducer. The growth incubation temperature (37 degrees C) was high enough as to activate the heat-inducible lambdap(L) promoter inserted in the plasmid pGP1-2. The best biotransformation conditions were with resting cells, under aerobiosis, with 4 g l(-1) and 100 mM biomass and substrate concentrations respectively. Under these conditions the biotransformation time (1 h) was shorter and the L(-)-carnitine yield (70%) higher than previously reported. Consequently productivity value (11.3 g l(-1)h(-1)) was highly improved when comparing with other published works. The resting cells could be reused until eight times maintaining product yield levels well over 50% that meant to increase ten times the L(-)-carnitine obtained per gram of biomass.
RESUMO
A flow injection analysis method for determining L-carnitine is reported. The system uses the enzyme L-carnitine dehydrogenase covalently immobilized to Eupergit C. The NADH produced by the action of the enzyme, which is proportional to the L-carnitine concentration, is quantified using fluorescence detection. The system response was rapid and had a wide range of linearity. At a flow rate of 0.2 ml/min, a detection limit of 1 microM (20 pmol) was obtained for L-carnitine, peak areas were linear up to 100 microM, and samples could be injected every 4 min. The method performed well as a routine assay, showing high sensitivity (54,000 AU/microM), a precision of 0.96%, and the ability to carry out 144 consecutive assays with an RSD of 1.47% (good stability). Comparisons were made with other known methods for L-carnitine determination. Presence of D-carnitine had no effect on L-carnitine assay. The analysis was valid for determining L-carnitine concentrations in commercial pharmaceutical preparations.
Assuntos
Carnitina/análise , Análise de Injeção de Fluxo/métodos , NAD/análise , Oxirredutases do Álcool/química , Calibragem , Enzimas Imobilizadas/química , Preparações Farmacêuticas/química , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrometria de FluorescênciaRESUMO
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.
Assuntos
Cromatografia Gasosa/métodos , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia/métodos , Cicloexanos , Liliaceae/química , Cicloexenos , Glicosídeos/análise , Glicosídeos/isolamento & purificação , Cinética , Espectrometria de Massas/métodos , Metanol/química , Modelos Químicos , Pressão , Temperatura , Terpenos/análise , Terpenos/isolamento & purificação , Fatores de Tempo , Água/químicaRESUMO
An enzymatic method for d-carnitine determination using the enzyme d-carnitine dehydrogenase is described. The assay is based on the amplified signal produced during NAD(+) cycling in the presence of a tetrazolium salt and using phenazine methosulfate as electron carrier. Optimum assay conditions were studied with two tetrazolium salt pairs: 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT)/MTT-formazan and 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride (INT)/INT-formazan. The first pair (MTT) showed higher sensitivity. The calibration curve was linear from 0.1 to 5 mM d-carnitine, with a quantification limit of 0.1 mM and a relative standard deviation of 1.51%. The procedure is simple, rapid, accurate, and easily automated. It was satisfactorily applied to following d-carnitine levels during the microbial transformation of d-carnitine into l-carnitine and to determining the d-carnitine content of pharmaceutical preparations.
Assuntos
Oxirredutases do Álcool , Carnitina/análise , Oxirredutases do Álcool/isolamento & purificação , Oxirredutases do Álcool/metabolismo , Automação , Biotransformação , Carnitina O-Acetiltransferase/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Formazans , Indicadores e Reagentes , Cinética , NAD/metabolismo , Reprodutibilidade dos Testes , Rhizobium/enzimologia , Sensibilidade e Especificidade , Espectrofotometria/métodos , EstereoisomerismoRESUMO
Two different immobilized chymotrypsin derivatives were used to synthesize kyotorphin, using N-benzoyl-L-tyrosine ethyl ester and L-arginine ethyl ester as substrates, in water-DMF media. The first was adsorbed onto Celite particles and the second was multipoint covalently attached into polyacrylamide gel. In all cases, the conversion of the carboxyl substrate was carried out in first-order reaction conditions. For the adsorbed enzyme, the reaction kinetics deviated from first-order likely due to a fast irreversible inactivation of enzyme during the reaction time even at low DMF concentration (15-20% v/v). The covalent attachment of enzyme resulted in elimination of irreversible activity loss by organic solvent up to 60% (v/v) of DMF. The catalytic activity of the covalent derivative was conserved as appropriate for performing a synthetic reaction up to 60% v/v of DMF (in comparison to 30% v/v for the adsorbed derivative), showing a clear improvement in its stability against reversible denaturation by this solvent. The selectivity of the synthetic reaction was slightly enhanced (from 40-50%) with the increase in DMF concentration to 80% v/v, but it was significantly improved (to 80%) when L-argininamide was used as nucleophile.
Assuntos
Quimotripsina/química , Endorfinas/síntese química , Enzimas Imobilizadas/química , Resinas Acrílicas/química , Animais , Bovinos , Terra de Diatomáceas/química , Dimetilformamida , Dipeptídeos/síntese química , Estabilidade Enzimática , Cinética , Solventes , Tirosina/análogos & derivados , Tirosina/químicaRESUMO
The use of a biological procedure for L-carnitine production as an alternative to chemical methods must be accompanied by an efficient and highly productive reaction system. Continuous L-carnitine production from crotonobetaine was studied in a cell-recycle reactor with Escherichia coli O44 K74 as biocatalyst. This bioreactor, running under the optimum medium composition (25 mM fumarate, 5 g/l peptone), was able to reach a high cell density (26 g dry weight/l) and therefore to obtain high productivity values (6.2 g L-carnitine l-1 h-1). This process showed its feasibility for industrial L-carnitine production. In addition, resting cells maintained in continuous operation, with crotonobetaine as the only medium component, kept their biocatalytic capacity for 4 days, but the biotransformation capacity decreased progressively when this particular method of cultivation was used.
Assuntos
Aciltransferases , Reatores Biológicos , Carnitina/biossíntese , Escherichia coli/enzimologia , Escherichia coli/crescimento & desenvolvimento , Microbiologia Industrial , Anaerobiose , Betaína/análogos & derivados , Betaína/metabolismo , Meios de Cultura , Indução Enzimática , Hidroliases/metabolismoRESUMO
L(-)-carnitine was produced from D(+)-carnitine by resting cells of Escherichia coli O44 K74. Oxygen did not inhibit either the carnitine transport system or the enzymes involved in the biotransformation process. Aerobic conditions led to higher product yield than anaerobic conditions. The biotransformation yield depended both on biomass and initial substrate concentrations used; the selected values for these variables were 4.30 g l-1 cells and 100 mmol l-1 D(+)-carnitine. Under these conditions the L(-)-carnitine production rate was 0.55 g l-1 h-1, the process yield was 44%, and the productivity was 0.22 g l-1 h-1 after a 30 h incubation period. Crotonobetaine production, besides L(-)-carnitine, showed that the action of more than one enzyme occurred during the biotransformation process. On the other hand, the addition of fumarate at high substrate concentrations (250 and 500 mmol l-1) led to a higher metabolic activity, which meant an increment of L(-)-carnitine production.
Assuntos
Carnitina/farmacocinética , Escherichia coli/enzimologia , Aerobiose , Anaerobiose , Betaína/análogos & derivados , Betaína/análise , Biomassa , Biotransformação , Carnitina/química , Cromatografia Líquida de Alta Pressão , Fumaratos/metabolismo , Concentração de Íons de Hidrogênio , Temperatura , Fatores de TempoRESUMO
NAD(H) was retained in a noncharged ultrafiltration membrane reactor for the simultaneous and continuous production of L-lactate and gluconate with coenzyme regeneration. Polyethyleneimine (PEI), a 50-kDa cationic polymer, achieved coenzyme retentions above 0.8 for PEI/NAD(H) molar ratios higher than 5. The ionic strength of the inlet medium caused a decrease of NAD(H) retention that can be counterbalanced by an initial addition of 1% bovine serum albumin (BSA). Continuous reactor performance in the presence of PEI and BSA showed that NAD(H), glucose dehydrogenase, and lactate dehydrogenase were retained by 10-kDa ultrafiltration membranes; L-lactate and gluconate were produced at conversions higher than 95%. PEI enhanced the thermal stability of the enzymes used and increased the catalytic efficiency of glucose dehydrogenase, while no effect was found on the kinetic parameters of lactate dehydrogenase. A model that implements the kinetic equations of the two enzymes describes the reactor behavior satisfactorily. In brief, the use of PEI to retain NAD(H) is a new interesting approach to be widely applied in continuous synthesis with the large number of known dehydrogenases.
Assuntos
Reatores Biológicos , Gluconatos/metabolismo , Ácido Láctico/biossíntese , NAD/metabolismo , Ultrafiltração/métodos , Biotecnologia/métodos , Estabilidade Enzimática/efeitos dos fármacos , Glucose 1-Desidrogenase , Glucose Desidrogenase/química , Glucose Desidrogenase/metabolismo , Cinética , L-Lactato Desidrogenase/química , L-Lactato Desidrogenase/metabolismo , Membranas , Modelos Biológicos , NAD/química , Polietilenoimina/química , Polietilenoimina/metabolismo , Polietilenoimina/farmacologiaRESUMO
The activity decay of alpha-chymotrypsin due to temperature and pH has been related to the associated structural changes of the protein that could be studied with the help of melting temperature which was determined by using ultraviolet absorption spectroscopy, and fluorescence spectra measurements. The kinetic behaviour in activity loss of alpha-chymotrypsin followed a two-step deactivation model, involving an intermediate state. The increase in temperature and pH showed a clear deactivation effect, reducing exponentially the half-life of the enzyme. At pH 7.0, this two-step deactivation process was also observed with both the maximum fluorescence intensity (I(max)) and emission wavelength (lambda(max)). The series-type kinetic model allowed to establish a clear correlation between the activity and the fluorescence spectral normalized data: the intermediate state of the enzyme occurred at an identical deactivation denaturation level (alpha1), and a proportionality between the decay rate constants was observed. As a function of the incubation temperature, another correlation was observed between the alpha1 profile, initial lambda(max) and thermal unfolding transition, allowing to identify the intermediate state of the kinetic model as that obtained at the melting temperature (43.9 degrees C).
Assuntos
Quimotripsina/antagonistas & inibidores , Quimotripsina/química , Animais , Bovinos , Quimotripsina/metabolismo , Estabilidade Enzimática , Temperatura Alta , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Cinética , Modelos Químicos , Desnaturação Proteica , Espectrometria de Fluorescência , Relação Estrutura-AtividadeRESUMO
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.
RESUMO
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.