Use of isolated cyclohexanone monooxygenase from recombinant Escherichia coli as a biocatalyst for Baeyer-Villiger and sulfide oxidations.

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.

Stereoselective synthesis of (S)-(+)-Naproxen catalyzed by carboxyl esterase in a multicompartment electrolyzer.

The stereoselective hydrolysis of racemic 2-substituted propionates, catalyzed by carboxyl esterase, provides a cost-competitive route to produce the optically pure, anti-inflammatory drug Naproxen. In the present work, we describe the application of the multicompartment electrolyzer reactor (ME) for the stereoselective hydrolysis of a racemic Naproxen ester, (R,S)-ethoxyethyl-[2-(6-methoxy-2-naphtyl)]propionate, catalyzed by a carboxyl esterase. The enzyme was trapped in a reactor chamber, delimited by two isoelectric membranes encompassing the pI value of the enzyme, together with the neutral substrate. After 90 min, a conversion of 45% was obtained with an enantiomeric excess of 84%. The reaction product, (S)-(+)-Naproxen, was electrophoretically removed in continuous from the reaction chamber and collected in a contiguous, more acidic chamber, separated from the enzyme and from the unreacted substrate. Moreover, at the end of the reaction, it was possible to recover the enzyme from the reactor and use it again.

Activity of different Candida antarctica lipase B formulations in organic solvents.

The activity of different formulations of Candida antarctica lipase B (CALB), such as crude CALB, purified CALB, purified CALB lyophilized with PEG (CALB + PEG) or oleic acid (CALB + OA), and the commercial formulation Novozym 435, was determined in toluene, carbon tetrachloride, and 1,4-dioxane at various water activities (a(w)). The reaction between vinylacetate and 1-octanol was used as the model reaction and both transesterification (formation of 1-octylacetate) and hydrolytic (formation of acetic acid from vinylacetate) activities were determined. For equal amounts of lipase protein, CALB + PEG (and to a lesser extent CALB + OA) displayed higher activity than that of the other formulations; for instance, in toluene (a(w) < 0.1), it was 260-, 13-, and 1.8-fold more active than crude CALB, purified CALB, and Novozym 435, respectively. Moreover, the transesterification activity of CALB + PEG was of the same order of magnitude (51%) of the activity shown by the enzyme in the hydrolysis of vinylacetate in aqueous buffer. These results suggest that PEG and oleic acid could act as lyoprotectants, preventing the formation of intermolecular interactions during the lyophilization process that might be responsible for protein denaturation. No diffusional limitation was observed for CALB + PEG-catalyzed reactions. Purified CALB, in contrast to the other formulations, showed a marked activity increase (2.1 to 7.8-fold) as a function of a(w) and, in 1,4-dioxane, it was 3.5-fold more active when it was added to the solvent after previous dissolution of the lyophilized powder in water.

Enantioselective synthesis of tert-butyl tert-butanethiosulfinate catalyzed by cyclohexanone monooxygenase.

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.

Ab initio study of oxygen atom transfer from hydrogen peroxide to trimethylamine.

Unlike what has been theoretically proposed for ammonia oxidation with hydrogen peroxide, trimethylamine oxidation occurs with a concerted mechanism, which is favored even when an explicit water molecule is added or continuum solvent (water) is simulated.

Characterization of an industrial biocatalyst: immobilized glutaryl-7-ACA acylase.

A batch of the immobilized industrial biocatalyst glutaryl-7-ACA acylase (GA), one of the two enzymes involved in the biotransformation of cephalosporin C (CefC) into 7-aminocephalosporanic acid (7-ACA), was characterized. K(m) value for glutaryl-7-ACA was 5 mM. Enzyme activity was found to be optimal at pH between 7 and 9.5 and to increase with temperature and in buffered solutions. To avoid product degradation, optimal reaction conditions were obtained working at 25 degrees C using a 50-mM phosphate buffer, pH 8.0. Immobilized GA showed good stability at pH value below 9 and at temperature up to 30 degrees C. The inactivation of immobilized GA in the presence of different amounts of H(2)O(2), a side product that might be present in the plant-scale industrial solutions of glutaryl-7-ACA, was also investigated, but the deactivation rates were negligible at H(2)O(2) concentration that might be reached under operative conditions. Finally, biocatalyst performance in the complete two-step enzymatic conversion process from CefC to 7-ACA was determined on a laboratory scale. Following the complete conversion of a 75 mM solution of CefC into glutaryl-7-ACA catalyzed by an immobilized D-amino acid oxidase (DAAO), immobilized GA was used for the transformation of this intermediate into the final product 7-ACA. This reaction was repeated for 42 cycles. An estimation of the residual activity of the biocatalyst showed that 50% inactivation of immobilized GA was reached after approximately 300 cycles, corresponding to an enzyme consumption of 0.4 kU per kg of isolated 7-ACA.

Properties and Synthetic Applications of Enzymes in Organic Solvents.

Biotransformations already represent an effective and sometimes preferable alternative to chemical synthesis for the production of fine chemicals and optically active compounds. To further widen the versatility of the biological approach, the so-called "nonaqueous enzymology", which now represents an important area of research and biotechnological development, has emerged in the last ten years or so. This new methodology is especially suitable for the modification of precursors of pharmaceutical compounds and fine chemicals, which, in most cases, are insoluble or poorly soluble in water. Even though the idea of carrying out an enzymatic process in organic solvent was initially considered with scepticism, biocatalysis in such media is now investigated and exploited in numerous academic and industrial laboratories. One of the reasons that makes enzymatic catalysis in nonaqueous media so appealing, is the important new properties that enzymes exhibit in organic solvents. For example, they are often more stable and can catalyze reactions that are impossible or difficult in water. Furthermore, enzyme selectivity can also differ from that in water and can change, or even reverse, from one solvent to another. This phenomenon, which can be called "medium engineering", can be exploited as a valid alternative to protein engineering. The first part of this review examines the thermodynamic, kinetic, spectroscopic, and physical approaches that have been adopted to investigate the factors that affect activity, stability, structure, and selectivity of enzymes in organic solvents. These combined studies have brought the understanding of enzyme catalysis in organic solvents to a level almost comparable to that reached for biocatalysis in aqueous media. The second part surveys a number of the synthetic applications of enzymes in organic media, which span from the preparation of milligrams of specifically labeled compounds to the modification of fats on multiton scale and from the preparation of complex key intermediates for the pharmaceutical industry to the synthesis of polymers.

Crystallization and preliminary X-ray diffraction studies of phospholipase D from Streptomyces sp.

Crystals of purified phospholipase D (E.C. 3.1.4.4) from Streptomyces sp. strain PMF have been grown under two different crystallization conditions using vapour diffusion. Both conditions gave monoclinic crystals in space group P2(1). The unit-cell parameters were a = 57.28, b = 57.42, c = 68.70 A, beta = 93.17 degrees. The crystals diffract at 110 K to a resolution beyond 1.4 A using synchrotron radiation.

Cloning, overexpression, and properties of a new thermophilic and thermostable esterase with sequence similarity to hormone-sensitive lipase subfamily from the archaeon Archaeoglobus fulgidus.

A new esterase gene from the hyperthermophilic archaeon Archaeoglobus fulgidus, reported to show homology with the mammalian hormone-sensitive lipase (HSL)-like group of the esterase/lipase family, was cloned by means of the polymerase chain reaction from the A. fulgidus genome. In order to compare the biochemical properties of this putative hyperthermophilic enzyme with those of the homologous, thermophilic member of HSL group, namely Alicyclobacillus (formerly Bacillus) acidocaldarius esterase 2 (EST2), an overexpression system in Escherichia coli was established. The recombinant protein, expressed in soluble and active form at 20 mg/liter of E. coli culture, was purified to homogeneity and characterized. The enzyme, a 35.5-kDa monomeric protein, was demonstrated to be a B"-type carboxylesterase (EC 3.1.1.1) on the basis of substrate specificity and the action of inhibitors. Among the p-nitrophenyl (PNP) esters tested the best substrate was PNP-hexanoate with K(m) and k(cat) values of 11 +/- 3 microM (mean +/- SD, n = 3) and 1014 +/- 38 s(-1) (mean +/- SD, n = 3), respectively, at 70 degrees C and pH 7.1. Inactivation by diethylpyrocarbonate, phenylmethylsulfonylfluoride, diisopropylfosfofluoridate (DFP), and physostigmine, as well as labeling with [(3)H]DFP, supported our previous suggestion of a catalytic triad made up of Ser(160)-His(285)-Asp(255). The sequence identity with the thermostable A. acidocaldarius EST2 was 42.5%. The enzyme proved to be much more stable than its Alicyclobacillus counterpart. The conformational dynamics of the two proteins were investigated by frequency-domain fluorometry and anisotropy decay and the activity/stability/temperature relationship was discussed.

Fourier-transform infrared spectroscopy study of dehydrated lipases from candida antarctica B and pseudomonas cepacia

Fourier-transform infrared (FT-IR) spectroscopy was employed to investigate potential lyophilization-induced changes in the secondary structure of lipases from Candida antarctica B and Pseudomonas cepacia. The secondary structure elements were determined by curve fitting of the amide III bands of the two lipases in the lyophilized state in KBr pellets and in solution. It was found that lyophilization decreased the alpha-helix and increased the beta-sheet content. However, FT-IR analysis of crosslinked enzyme crystals of Pseudomonas cepacia lipase also indicated an increase in the beta-sheet content, which appears despite the fact that the enzyme, being in the crystallized state, should possess native conformation. This result partially questions the suitability of FT-IR for analysis of the structure of solid proteins, at least as far as the beta-sheet content is concerned, because it is possible that the method overestimates the beta-sheets by measuring other hydrogen-bonded nonperiodic intermolecular structures. No significant modification was observed when lipase from Pseudomonas cepacia was lyophilized in the presence of methoxypoly(ethylene glycol). Copyright 1999 John Wiley & Sons, Inc.

Spectroscopic investigation of lipase from pseudomonas cepacia solubilized in 1,4-dioxane by non-covalent complexation with methoxypoly(ethylene glycol)

Lipase from Pseudomonas cepacia was made soluble in 1,4-dioxane by lyophilization of the enzyme from aqueous solutions containing methoxypoly(ethylene glycol) (PEG). The solubility of the enzyme-PEG complex depended both on protein concentration and PEG protein ratio. Intrinsic protein fluorescence and far- and near-UV circular dichroism revealed that not only did the enzyme not unfold in the organic solvent, but rather became more compact. This was seen by the slight quenching of fluorescence intensity and by the enhancement of the near-UV circular dichroism negative signals, which are indicative of stronger interactions of tryptophanyl and/or tyrosyl residues among themselves or with other parts of the enzyme molecule. The specific activity of the lipase-PEG complex in the organic solvent was at least 2 orders of magnitude higher than that of the enzyme powder. This can be attributed both to the maintenance of native conformation and to enzyme dissolution in the reaction medium which should minimize possible limitations to enzyme-substrate interactions. © 1999 John Wiley & Sons, Inc.,

Chloroperoxidase-catalyzed enantioselective oxidation of methyl phenyl sulfide with dihydroxyfumaric acid/oxygen or ascorbic acid/oxygen as oxidants.

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.

Optimization of Pseudomonas cepacia lipase preparations for catalysis in organic solvents.

The activity of different lipase (from Pseudomonas cepacia) forms, such as crude powder (crude PC), purified and lyophilized with PEG (PEG + PC), covalently linked to PEG (PEG-PC), cross-linked enzyme crystals (CLEC-PC), and immobilized in Sol-Gel-AK (Sol-Gel-AK-PC) was determined, at various water activities (aw), in carbon tetrachloride, benzene and 1,4-dioxane. The reaction of vinyl butyrate with 1-octanol was employed as a model and both transesterification (formation of 1-octyl butyrate) and hydrolysis (formation of butyric acid from vinyl butyrate) rates were determined. Both rates depended on the lipase form, solvent employed, and aw value. Hydrolysis rates always increased as a function of aw, while the optimum of aw for transesterification depended on the enzyme form and nature of the solvent. At proper aw, some lipase forms such as PEG + PC, PEG-PC, and Sol-Gel-AK-PC had a total activity in organic solvents (transesterification plus hydrolysis) which was close to (39 and 48%) or even higher than (130%) that displayed by the same amount of lipase protein in the hydrolysis of tributyrin-one of the substrates most commonly used as standard for the assay of lipase activity-in aqueous buffer. Instead, CLEC-PC and crude PC were much less active in organic solvents (2 and 12%) than in buffer. The results suggest that enzyme dispersion and/or proper enzyme conformation (favored by interaction with PEG or the hydrophobic Sol-Gel-AK matrix) are essential for the expression of high lipase activity in organic media.

Sulphoxidation reaction catalysed by myeloperoxidase from human leucocytes.

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.

Overexpression and properties of a new thermophilic and thermostable esterase from Bacillus acidocaldarius with sequence similarity to hormone-sensitive lipase subfamily.

We previously purified a new esterase from the thermoacidophilic eubacterium Bacillus acidocaldarius whose N-terminal sequence corresponds to an open reading frame (ORF3) reported to show homology with the mammalian hormone-sensitive lipase (HSL)-like group of the esterase/lipase family. To compare the biochemical properties of this thermophilic enzyme with those of the homologous mesophilic and psychrophilic members of the HSL group, an overexpression system in Escherichia coli was established. The protein, expressed in soluble and active form at 10 mg/l E. coli culture, was purified to homogeneity and characterized biochemically. The enzyme, a 34 kDa monomeric protein, was demonstrated to be a B'-type carboxylesterase (EC 3.1.1.1) on the basis of substrate specificity and the action of inhibitors. Among the p-nitrophenyl (PNP) esters tested the best substrate was PNP-exanoate with Km and kcat values of 11+/-2 microM (mean+/-S.D., n=3) and 6610+/-880 s-1 (mean+/-S.D., n=3) respectively at 70 degreesC and pH7.1. In spite of relatively high sequence identity with the mammalian HSLs, the psychrophilic Moraxella TA144 lipase 2 and the human liver arylacetamide deacetylase, no lipase or amidase activity was detected. A series of substrates were tested for enantioselectivity. Substantial enantioselectivity was observed only in the resolution of (+/-)-3-bromo-5-(hydroxymethyl)-Delta2-isoxazoline, where the (R)-product was obtained with an 84% enantiomeric excess at 36% conversion. The enzyme was also able to synthesize acetyl esters when tested in vinyl acetate and toluene. Inactivation by diethylpyrocarbonate, diethyl-p-nitrophenyl phosphate, di-isopropylphosphofluoridate (DFP) and physostigmine, as well as labelling with [3H]DFP, supported our previous suggestion of a catalytic triad made up of Ser-His-Asp. The activity-stability-temperature relationship is discussed in relation to those of the homologous members of the HSL group.

Comparison of antibody and albumin catalyzed hydrolysis of steroidal p-nitrophenylcarbonates.

A monoclonal antibody (MAb) was produced against the p-nitrophenylphosphate derivative of 3 alpha, 5 beta-lithocholic acid, a transition-state analog for hydrolysis of a steroidal p-nitrophenylcarbonate. The indicated reaction was catalyzed by this Ab with kinetic constants kcat = 4.0 x 10(-2)/min and K(m) = 3.3 microM at pH 9.0 and 35 degrees C. The Ab also hydrolyzed the isomeric p-nitrophenylcarbonate of 3 beta, 5 beta-lithocholic acid with kcat = 8.4 x 10(-2)/min and K(m) = 1.0 microM. Bovine serum albumin (BSA) was found to catalyze the same reactions with similar turnover rates and Michaelis constants of 15 and 14 microM, respectively. Although the BSA-catalyzed reaction was only weakly inhibited by the phosphate ester TSA (IC50 ca. 40 microM), the Ab-catalyzed reaction was completely inhibited at less than 1 microM of the TSA. The relative rates and efficiencies of the MAb-catalyzed and BSA-catalyzed reactions are discussed in the context of the hydrophobic sites and intrinsic reactivity of the protein surfaces, and the induction of groups on the Ab to enhance the enzymatic function.

Activity, stability, and conformation of methoxypoly(ethylene glycol)-subtilisin at different concentrations of water in dioxane.

The transesterification activity, autolysis, thermal stability and conformation of subtilisin Carlsberg, made soluble in dioxane by covalent linking to methoxypoly(ethylene glycol) (PEG), were investigated as a function of the concentration of water in the medium. Electrospray mass spectrometry showed that the modified enzyme preparation was a mixture of proteins containing from 2 to 5 covalently linked PEG chains per subtilisin molecule. PEG-subtilisin catalyzed transesterification between vinyl butyrate and 1-hexanol was optimum at 0.55 MH(2)O, while hydrolysis prevailed above 2 MH(2)O. There was a decrease in the overall enzyme activity with increasing water concentration because of autolysis and denaturation of the enzyme. Subtilisin powder and celite-immobilized subtilisin were more stable and less susceptible to autolysis than the PEG-modified enzyme. Circular dichroism and intrinsic protein-fluorescence studies showed that the conformation of PEG-subtilisin did not change as a function of water concentrations between 0 and 9 M. The K(m,app) value of PEG-subtilisin for 1-hexanol was highly influenced by water, which behaved as a competitive inhibitor in the transesterification reaction with an affinity for the enzyme similar to that of the alcohol. The K(m,app) for the acylating agent was not significantly modified by water. Lyoprotectants such as sorbitol and free PEG did not influence the activity of PEG-subtilisin but notably increased the activity of subtilisin powder and celite-immobilized subtilisin. The addition of 1.7-5.5 M water, however, rendered enzyme preparations containing no additives as active as those containing the lyoprotectants.

A chemiluminescent flow sensing device for determination of choline and phospholipase D activity in biological samples.

A chemiluminescent flow-sensing device for the determination of phospholipase D (PLD) activity and/or choline (Ch) in biological samples using choline oxidase (ChO) and horseradish peroxidase (HRP) immobilized on Eupergit C (polymer beads of methacrylamide, N-methylene-bis-methacrylamide, and allyl-glycidyl-ether) was developed. The best results were obtained with immobilized ChO and HRP at a polymer beads wet weight ratio of 16:1. The optimized parameters of the developed sensing device were 56 microM luminol in working solution; sample volume, 60 microliters; flow rate, 0.3 ml/min; and sample throughput, 15/h. The detection limit (3 SD) using a luminescent enhancer was 1.2 microM for Ch, corresponding to 0.167 mIU of PLD activity per milliliter. Without enhancer the values were 3.0 microM and 0.417 mIU, respectively. The Ch recovery varied between 80.4 and 109%. The biological samples quenched the luminescent light to different extents, and this matrix effect was readily overcome by measuring the luminescent signal of added Ch standard. The flow biosensor was used for the determination of PLD in samples of different origin, including rape seeds during maturation.

Preparation of 3-deacetyl cephalosporins by Aspergillus niger lipase.

Lipase from Aspergillus niger was used for the selective hydrolysis of the 3-O-acetate of cephalosporin C to give an intermediate useful for further chemical elaborations. This lipase was purified to homogeneity and its properties compared with previously published data that present some discrepancies. The lipase proved to be very effective in catalyzing 3-O-acetate hydrolysis and versatile toward substitution on the beta-lactamic ring. In fact, as an example, two other cephalosporinic derivatives, cephalotin and cefotaxime, were efficiently deacetylated. The lipase was immobilized on Eupergit C and employed continuously in either a column or a batch reactor for 2 months without appreciable loss of activity. (c) 1996 John Wiley & Sons, Inc.