RESUMEN
The performance, in Baeyer-Villiger and heteroatom oxidations, of a partially purified preparation of cyclohexanone monooxygenase obtained from an Escherichia coli strain in which the gene of the enzyme was cloned and overexpressed was investigated. As model reactions, the oxidations of racemic bicyclo[3.2.0]hept-2-en-6-one into two regioisomeric lactones and of methyl phenyl sulphide into the corresponding (R)-sulphoxide were used. Enzyme stability and reuse, substrate and product inhibition, product removal, and cofactor recycling were evaluated. Of the various NADPH regeneration systems tested, 2-propanol/alcohol dehydrogenase from Thermoanerobium brockii appeared the most suitable because of the low cost of the second substrate and the high regeneration rate. Concerning enzyme stability, kosmotropic salts were the only additives able to improve it (e.g., half-life from 1 day in diluted buffer to 1 week in 1 M sodium sulphate) but only under storage conditions. Instead, significant stabilization under working conditions was obtained by immobilization on Eupergit C (half-life approximately 2.5 days), a procedure that made it possible to reuse the catalyst up to 16 times with complete substrate (5 g x L(-1)) conversion at each cycle. Reuse of free enzyme was also achieved in a membrane reactor but with lower efficiency. Water-organic solvent biphasic systems, which would overcome substrate inhibition and remove from the aqueous phase, where reaction takes place, the formed product, were unsuccessful because of their destabilizing effect on cyclohexanone monooxygenase. More satisfactory was continuous substrate feeding, which shortened reaction times and, very importantly, yielded in the case of bicyclo[3.2.0]hept-2-en-6-one (10 g x L(-1)) both lactone products with high optical purity (enantiomeric excess > or = 96%), which was not the case when all of the substrate was added in a single batch.
Asunto(s)
Alcohol Deshidrogenasa/química , Compuestos Bicíclicos Heterocíclicos con Puentes/química , Escherichia coli/metabolismo , Oxigenasas/química , Oxigenasas/aislamiento & purificación , Sulfuros/química , Catálisis , Estabilidad de Enzimas , Enzimas Inmovilizadas , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Oxidación-Reducción , Oxigenasas/biosíntesis , Oxigenasas/genética , Polímeros , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Sensibilidad y EspecificidadRESUMEN
Cyclohexanone monooxygenase from Acinetobacter calcoaceticus catalyzes the asymmetric oxidation of tert-butyl disulfide to enantiomerically pure (R)-tert-butyl tert-butanethiosulfinate. Lower enantioselectivities and conversions were observed in the oxidation of i-propyl, n-butyl, p-tolyl tert-butyl disulfides and alkylthiophosphonates.
Asunto(s)
Oxigenasas/química , Ácidos Sulfónicos/síntesis química , Acinetobacter calcoaceticus/enzimología , Catálisis , EstereoisomerismoRESUMEN
The chloroperoxidase catalyzed oxidation of methyl phenyl sulfide to (R)-methyl phenyl sulfoxide was investigated, both in batch and membrane reactors, using as oxidant H2O2, or O2 in the presence of either dihydroxyfumaric acid or ascorbic acid. The effects of pH and nature and concentration of the oxidants on the selectivity, stability, and productivity of the enzyme were evaluated. The highest selectivity was displayed by ascorbic acid/O2, even though the activity of chloroperoxidase with this system was lower than that obtained with the others. When the reaction was carried out in a membrane reactor, it was possible to reuse the enzyme for several conversion cycles. The results obtained with ascorbic acid/O2 and dihydroxyfumaric acid/O2 as oxidants do not seem to be compatible with either a mechanism involving hydroxyl radicals as the active species or with the hypothesis that oxidation occurs through the initial formation of H2O2. Copyright 1999 John Wiley & Sons, Inc.
RESUMEN
Peroxidases are ubiquitous oxidoreductases that use hydrogen peroxide or alkyl peroxides as oxidants. Advances have recently been made in using them to prepare, under mild and controlled conditions, chiral organic molecules that are valuable for the chemoenzymatic synthesis of a wide range of useful compounds. Horseradish peroxidase can be converted into a peroxygenative enzyme by molecular engineering. Chloroperoxidase, the most versatile peroxidase, behaves like a 'true' monooxygenase in sulfoxidations with molecular oxygen and an external reductant, with substantial increases in enantioselectivity and enzyme stability.
Asunto(s)
Biotecnología/métodos , Peroxidasas/metabolismo , Alcoholes/química , Alcoholes/metabolismo , Catálisis , Estabilidad de Enzimas , Hidroxilación , Indoles/química , Indoles/metabolismo , Nitrógeno/química , Nitrógeno/metabolismo , Oxidación-Reducción , Peroxidasas/química , Azufre/química , Azufre/metabolismoRESUMEN
The oxidation of alkyl aryl sulphides by myeloperoxidase (MPO) at the expense of hydrogen peroxide was investigated under steady-state conditions. The sulphide concentration effect was studied under saturating H2O2 concentrations at pH 5.0 and 20 degreesC. The kinetic constants, kcat and Km, of the different substrates were determined and the values were in the 1-10 s-1 range and around 43+/-26 microM respectively, whatever the sulphide considered. In the case of p-substituted thioanisoles, the oxidation rate was dependent upon the substituent effect. The correlation of log(kcat) with the substituent constants (sigma+ values) (Hammett equation) could be explained by a reaction mechanism involving the enzyme compound II and a sulphenium radical cation. This conclusion was also supported by spectrophotometric analysis of catalytic intermediates of the enzyme, showing the accumulation of compound II. Moreover, chiral HPLC analyses showed that MPO oxidation of alkyl aryl sulphides produced the corresponding (R)-sulphoxides with a low enantioselectivity (4-8%). Chloride ion effects on the MPO-catalysed oxygenation of sulphides were also studied. Chloride acted as a substrate for MPO and as an activator in MPO-catalysed sulphoxidation. Inhibition occurred at chloride concentrations above 120 mM, whereas below 120 mM, chloride increased the reaction rate when using p-tolyl methyl sulphide as the substrate. In the presence of 100 mM chloride the catalytic efficiency (kcat/Km) of MPO increased 3-4-fold, whatever the sulphide considered, but racemic products were obtained. These data have been interpreted in the light of known structural information on the accessibility of the distal haem cavity.
Asunto(s)
Neutrófilos/enzimología , Peroxidasa/metabolismo , Ésteres del Ácido Sulfúrico/metabolismo , Catálisis , Cloruros/metabolismo , Humanos , Peróxido de Hidrógeno/metabolismo , Cinética , Oxidación-Reducción , Estereoisomerismo , Sulfuros/metabolismoRESUMEN
To obtain conformational data on the monomeric form of insulin, which is believed to be the physiologically active form of the hormone, insulin in sodium dodecyl sulfate solution was studied by Fourier-transform infrared (FTIR) spectroscopy and circular dichroism, and results were compared with those obtained with des(B23-30) octapeptide insulin (DOI) and dimeric insulin in buffer. The FTIR amide I band (1600-1700 cm-1) was examined, and a quantitative evaluation of the secondary structure fractions of the various conformations showed less of a beta-sheet component for both insulin in SDS and DOI in buffer than for insulin in buffer, corresponding to a lack of monomers binding to form dimers. At the concentrations used for FTIR (> or = 2 mg/mL), the CD spectra of insulin in SDS and DOI in buffer were qualitatively identical but different from that of insulin in buffer, which is associated at these concentrations. The CD spectrum pattern of insulin in very dilute solution (80 nM), where it is prevalently monomeric, is very similar to that of monomeric insulin in SDS, which suggests that the conformation of the hormone in the two cases is very similar.
Asunto(s)
Insulina/química , Animales , Bovinos , Dicroismo Circular , Conformación Proteica , Dodecil Sulfato de Sodio , Soluciones , Espectrofotometría Ultravioleta , Espectroscopía Infrarroja por Transformada de Fourier , TensoactivosRESUMEN
At pH 3, chloride dramatically influenced both the Km of chloroperoxidase (CPO) for methyl p-tolyl sulfide, which decreased, and its activity, which increased. The Km value changed from 75 microM in the absence of chloride to < or = 1.2 microM in > or = 0.9 mM chloride, and the kcat from 53 s-1 in 0 to 1750 s-1 in 50 mM halide. The kcat/Km value at 0.9 mM chloride was 414 microM -1s-1 compared to 0.7 microM-1s-1 in the absence of the halide. At pH 5, the activating effect was less pronounced. Chloride also acted as inhibitor versus hydrogen peroxide. The data are consistent with a reaction mechanism in which, on hand, chloride competes with hydrogen peroxide for the native enzyme and, on the other hand, activates sulfide oxidation by binding to CPO Compound I to give a CPO-chlorinating intermediate (EOCl-). However, contrary to what happened in the absence of chloride, where the oxidation was enantioselective and an oxygen atom of H2O2 was incorporated in the sulfoxide (from experiments with 18O-labeled H2O2), in the presence of the halide the oxidation was not enantioselective and there was no incorporation of oxygen from H2O2. The data suggest that sulfide oxidation takes place through an enzyme-generated freely dissociable oxidized halogen intermediate formed by the interaction of EOCl- with Cl-.
Asunto(s)
Cloruro Peroxidasa/química , Cloruros/farmacología , Sulfuros/química , Cinética , Oxidación-Reducción , EstereoisomerismoRESUMEN
The chloroperoxidase-catalyzed and horseradish peroxidase catalyzed oxidations of sulfides by tert-butyl and other peroxides have been investigated. The former metal enzyme afforded the corresponding sulfoxides having R absolute configuration in up to 92% enantiomeric excess (ee), whereas the latter gave racemic products. The various factors that control the enantioselectivity of the oxygenation have been examined.
Asunto(s)
Cloruro Peroxidasa/metabolismo , Peroxidasa de Rábano Silvestre/metabolismo , Sulfuros/metabolismo , Cinética , Oxidación-Reducción , Estereoisomerismo , Especificidad por Sustrato , Sulfóxidos/aislamiento & purificaciónRESUMEN
The influence of micelles of sodium dodecyl sulfate, cetyltrimethylammonium bromide, lysophosphatidylcholine and dodecylphosphorylcholine on the content and stability of the ordered structure of human beta-endorphin and its 12-26 fragment has been investigated. The structure was determined by far-ultraviolet circular dichroism and the stability by the resistance of the polypeptide to proteolysis with trypsin and chymotrypsin, monitored by HPLC. The alpha-helix inducing effects of the amphipathic compounds were in the order anionic greater than zwitterionic greater than cationic. The protection against proteolysis was very marked, especially for trypsin, and it was proportional to the alpha-helix inducing potential of amphipathic compounds. However, the lower resistance to proteolysis of the highly structured 12-26 fragment suggests that factors other than secondary structure may be responsible for the resistance to proteolysis.
Asunto(s)
Dicroismo Circular , Lípidos/farmacología , Péptido Hidrolasas/metabolismo , Análisis Espectral , Tensoactivos/farmacología , betaendorfina/metabolismo , Cetrimonio , Compuestos de Cetrimonio , Cromatografía Líquida de Alta Presión , Quimotripsina/metabolismo , Humanos , Lisofosfatidilcolinas , Micelas , Fosforilcolina/análogos & derivados , Conformación Proteica , Dodecil Sulfato de Sodio , Soluciones , Tripsina/metabolismoRESUMEN
The denaturation by urea and renaturation of 20 beta-hydroxysteroid dehydrogenase, a tetrameric enzyme consisting of four identical subunits, were followed by high-performance size exclusion chromatography to detect intermediates in the processes. During the denaturation process no intermediate form (structured monomers or dimers) between the tetramer and the denatured monomer was observed. During the renaturation process, carried out either with or without NADH, high molecular weight aggregates, native tetramers, and low molecular weight intermediates were evidenced and quantified. The contemporaneous measurement of recovery of activity unambiguously demonstrated that the tetrameric structure is essential for enzymatic activity.
Asunto(s)
20-Hidroxiesteroide Deshidrogenasas/análisis , Cortisona Reductasa/análisis , Urea/farmacología , Cromatografía en Gel/métodos , Cromatografía Líquida de Alta Presión/métodos , Desnaturalización Proteica/efectos de los fármacos , Especificidad por Sustrato/efectos de los fármacosRESUMEN
Subtilisin Carlsberg has been made soluble in organic solvents such as dioxane and acetonitrile by covalent linking to polyethylene glycol. Far-ultraviolet circular dichroism and intrinsic protein fluorescence have shown that subtilisin dissolved in dioxane, in which the enzyme is active and highly stable, maintains the native secondary structure as well as the native microenvironment for tyrosyl residues. In acetonitrile subtilisin undergoes conformational changes that cause enzyme inactivation and precipitation.
Asunto(s)
Polietilenglicoles , Subtilisinas , Acetonitrilos , Dicroismo Circular , Dioxanos , Conformación Proteica , Solventes , Espectrometría de Fluorescencia , TirosinaRESUMEN
The effects of micelles of nonionic, zwitterionic, anionic and cationic surfactants and lipids on the conformation of glucagon and insulin have been investigated by circular dichroism and intrinsic protein fluorescence. The influence of these amphipathic compounds on the hydrolysis, monitored by HPLC, of glucagon and insulin by trypsin and chymotrypsin has also been studied. The alpha-helix content of glucagon was increased to a similar extent by all the micelles, irrespective of their charge and of whether they were synthetic surfactants or phospholipids. The amphipathic compounds always induced a blue-shift in the wavelength of maximum emission of fluorescence of glucagon of about 9 nm, whereas the fluorescence intensity was increased in some cases and decreased in others. The circular dichroism of insulin was also modified in some cases. Some amphipathic compounds protected glucagon against proteolysis by trypsin and chymotrypsin very markedly, whereas others did not protect at all or only slightly protected the hormone. Two hypotheses have been formulated to explain the different results. Hydrolysis of insulin was generally not influenced by surfactants and lipids.
Asunto(s)
Glucagón , Insulina , Lípidos/farmacología , Tensoactivos/farmacología , Aniones , Cationes , Cetrimonio , Compuestos de Cetrimonio/farmacología , Cromatografía Líquida de Alta Presión , Quimotripsina/metabolismo , Dicroismo Circular , Glucagón/metabolismo , Hidrólisis , Insulina/metabolismo , Lisofosfatidilcolinas/farmacología , Conformación Proteica/efectos de los fármacos , Dodecil Sulfato de Sodio/farmacología , Soluciones , Tripsina/metabolismoRESUMEN
We investigated the denaturation of tetrameric 20 beta-hydroxysteroid dehydrogenase (20R)-17 beta,20 beta,21-trihydroxysteroid:NAD+ oxidoreductase, EC 1.1.1.53) to find out whether intermediate states are formed during the process. The denaturation process was studied in the presence and absence of stabilizers, both specific, such as NADH, and non-specific, such as the salting-out anion phosphate. Changes in enzymatic activity, intrinsic protein fluorescence and far-ultraviolet circular dichroism were monitored. When NADH was present, denaturation of the enzyme by urea was a one-step transition between the native and the completely denatured state. In dilute phosphate, and even more so in concentrated phosphate, the existence of intermediate states with different stability is evidenced by the noncoincidence of the transition curves that probe for different functional and conformational aspects of the enzyme. Therefore, for 20 beta-hydroxysteroid dehydrogenase the formation of intermediates can be prevented by adding NADH, or enhanced by adding concentrated phosphate.
Asunto(s)
20-Hidroxiesteroide Deshidrogenasas/antagonistas & inhibidores , Cortisona Reductasa/antagonistas & inhibidores , Urea/farmacología , Dicroismo Circular , Estabilidad de Enzimas , Cinética , Desnaturalización Proteica , Espectrometría de FluorescenciaRESUMEN
Diethyl pyrocarbonate inactivated the tetrameric 3 alpha,20 beta-hydroxysteroid dehydrogenase with second-order rate constants of 1.63 M-1 s-1 at pH 6 and 25 degrees C or 190 M-1 s-1 at pH 9.4 and 25 degrees C. The activity was slowly and partially restored by incubation with hydroxylamine (81% reactivation after 28 h with 0.1 M hydroxylamine, pH 9, 25 degrees C). NADH protected the enzyme against inactivation with a Kd (10 microM) very close to the Km (7 microM) for the coenzyme. The ultraviolet difference spectrum of inactivated vs. native enzyme indicated that a single histidyl residue per enzyme subunit was modified by diethyl pyrocarbonate, with a second-order rate constant of 1.8 M-1 s-1 at pH 6 and 25 degrees C. The histidyl residue, however, was not essential for activity because in the presence of NADH it was modified without enzyme inactivation and modification of inactivated enzyme was rapidly reversed by hydroxylamine without concomitant reactivation. Progesterone, in the presence of NAD+, protected the histidyl residue against modification, and this suggests that the residue is located in or near the steroid binding site of the enzyme. Diethyl pyrocarbonate also modified, with unusually high reaction rate, one lysyl residue per enzyme subunit, as demonstrated by dinitrophenylation experiments carried out on the treated enzyme. The correlation between inactivation and modification of lysyl residues at different pHs and the protection by NADH against both inactivation and modification of lysyl residues indicate that this residue is essential for activity and is located in or near the NADH binding site of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Dietil Pirocarbonato , Formiatos , Hidroxiesteroide Deshidrogenasas , Sitios de Unión , Unión Competitiva , Fenómenos Químicos , Química , Dietil Pirocarbonato/farmacología , Formiatos/farmacología , Histidina , Concentración de Iones de Hidrógeno , Hidroxilaminas , Hidroxiesteroide Deshidrogenasas/antagonistas & inhibidores , Lisina , NAD/metabolismo , Progesterona/metabolismo , Conformación Proteica/efectos de los fármacos , Streptomyces/enzimologíaRESUMEN
Glucagon was immobilized onto Sepharose matrices activated with CNBr or tresyl chloride, as a function of several parameters including pH of coupling, concentration of added polypeptide, and presence or absence of urea. The hormone was linked to the matrix through a single point per molecule, namely, the epsilon -amino group of Lys(12) when the coupling was carried out at alkaline pH, or the imidazole group of His(1) when the coupling was carried out at acidic pH. Glucagon immobilized at alkaline pH interacted specifically with soluble glucogon. The extent of self-association was similar to that of free glucagon, which exists in solution in a monomer-trimer equilibrium whose association constant is highly dependent on the characteristics of the buffer (pH, ionic strength, and nature of anions). The immobilized hormone proved to be suitable for the purification of the free one from a pancreatic extract. After a preliminary treatment with charcoal-dextran, the extract was percolated on a glucagon-Sepharose column under associating conditions (high concentrations of salting out anions and alkaline pH) and then, following a washing to remove extraneous compounds, the specifically bound hormone was eluted under dissociating conditions (low ionic strength). The subunit exchange chromatography of the extract gave a ca. 90% pure product. The overall recovery of the process was ca. 66%. The leakage of immobilized hormone was 40% in the case of CNBr activation of Sepharose and 15% in the case of tresyl chloride activation, after an eight-day treatment under working conditions.
RESUMEN
The effect of the lyotropic series of anions on the stability and renaturation of tetrameric 20 beta-hydroxysteroid dehydrogenase (17,20 beta,21-trihydroxysteroid:NAD+ oxidoreductase, EC 1.1.1.53) was investigated. The variations in enzymatic activity were correlated with the changes in protein fluorescence, circular dichroism, reactivity of histidine residues and molecular weight. High concentrations of salting-out anions (phosphate, citrate, sulphate) were found to stabilize the enzyme markedly and increase the renaturation yield of the urea-denatured enzyme. Phosphate, for instance, induced the highest stabilization at about 1.2 M and the maximum reactivation (66%) at 0.5 M. At low anion concentration (0.01 M), the reactivation was only 7%. The renaturation property of salting-out anions seems to be due to their stabilizing effect on the end-product, i.e., the assembled tetramer. Salting-in anions (perchlorate, thiocyanate, iodide) inactivated the enzyme. At moderate anion concentrations (no greater than 0.25 M) the activation, which occurred slowly, without tetramer dissociation and with minor modifications of enzyme conformation, was fully reversed by concentrated phosphate or by saturating concentrations of NADH. In contrast, the inactivation induced by high anion concentrations (1-2 M) was rapid, irreversible and linked to considerable modifications of enzyme conformation.
Asunto(s)
20-Hidroxiesteroide Deshidrogenasas/metabolismo , Aniones/farmacología , Cortisona Reductasa/metabolismo , Dicroismo Circular , Cinética , Fosfatos/farmacología , Desnaturalización Proteica , Espectrometría de Fluorescencia , Urea/farmacologíaRESUMEN
The renaturation of free and Sepharose-immobilized D-amino-acid oxidase (D-amino-acid:oxygen oxidoreductase (deaminating), EC 1.4.3.3), after its denaturation with 6 M guanidine hydrochloride, was investigated. No reactivation, or extremely limited reactivation (less than or equal to 4+), was obtained with the free enzyme, is spite of various attempts including the use of dialysis or buffers containing cofactors, different types of anions, surfactants and low concentrations of denaturing agents. The main obstacle to renaturation appeared to be the interaction among denatured or partially renatured monomers giving rise to inactive aggregates. In contrast, using the immobilized enzyme approach, substantial renaturation (up to 50%) of D-amino-acid oxidase was achieved. The denaturation-renaturation process was followed by monitoring the catalytic activity as well as the intrinsic protein fluorescence. An inverse correlation was found to exist between the degree of matrix activation by CNBr and the yield of enzyme reactivation. The anions of the lyotropic series markedly influenced the reactivation, showing an effectiveness opposite to their salting-out potential (thiocyanate congruent to iodide greater than chloride greater than phosphate congruent to sulphate congruent to citrate). Instead, the anions considerably increased the activity and stability of free and immobilized enzyme, according to their salting-out potential. Immobilized monomers of D-amino-acid oxidase, which in solution undergoes self-association, showed poor capacity to interact with the free enzyme: thus they appear unsuitable for analytical and preparative purposes.