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J M Cabral

Publications and source records attributed to J M Cabral.

At least 55 records · Page 3Linked to original sources
PubMed1996

Evaluation of affinity and pseudo-affinity adsorption processes for penicillin acylase purification.

Affinity ligand (6-Aminopenicillanic acid, Amoxycillin, Ampicillin, Benzylpenicillin and 4-Phenylbutylanzine) of penicillin acylase (EC 3.5.1.11) were attached to hydrophilic gels like Sepharose 4B-CNBr and Minileak 'medium'. Ampicillin and 4-Phenylbutylamine were the affinity ligands that presented the higher concentrations attached to both gels. Penicillin acylase adsorption on these affinity gels was mainly dependent on the activated group of the gel, the affinity ligand attached and the experimental conditions of enzyme adsorption. Under affinity conditions only the ligands Amoxycillin, Ampicillin and 4-Phenylbutylamine, immobilized on Minileak, adsorbed the enzyme from osmotic shock extracts at different pH values. These affinity ligand systems were characterized by low adsorption capacities of penicillin acylase activity (1.2-2.1 IU mL-1 gel) and specific activity (1.5-2.9 IU mg-1 prot). Under pseudo-affinity conditions all the ligands attached both activated to gels (Sepharose 4B-CNBr and Minileak) adsorbed the enzyme. The affinity gels were characterized by higher values of adsorption capacity (3.7 and 55.6 IU mL-1 gel) and adsorbed specific activity (2.0 and 6.1 IU mg-1 prot) than those observed under affinity conditions. The space arm of Minileak gel, shown to be fundamental to enzyme adsorption under affinity conditions, preferentially adsorbed proteins in relation to the enzyme under pseudo-affinity conditions. However, this effect was partially minimized when the gel was derivatized by the affinity ligands at concentrations higher than 6 mumol mL-1 gel. Ampicillin was the affinity ligand that presented the best results for specific adsorption of penicillin acylase under affinity and pseudo-affinity adsorption processes. The Sepharose 4B-CNBr derivatized gel also presented a good adsorption capacity of enzyme activity (26.8 IU mL-1 gel) under pseudo-affinity adsorption processes.

Adsorption↗
PubMed1995

Lipase from Chromobacterium viscosum: biochemical characterization indicating homology to the lipase from Pseudomonas glumae.

Previous purification of a commercial lipolytic preparation from Chromobacterium viscosum using gel filtration chromatography yielded two enzymatically active fractions, named lipases A and B. Characterization of these fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that lipase A consisted of a high molecular weight aggregate of lipase protein with lipopolysaccharides. This complex could be dissociated by treatment with EDTA-Tris buffer containing the non-ionic detergent n-octyl-beta-D-glucopyranoside and subsequent isoelectric focusing in an agarose gel containing the same detergent. Both lipases A and B revealed a major peak corresponding to an isoelectric point of 7.1. SDS-PAGE analysis of lipases A and B after purification by gel filtration or by IEF revealed one major protein band of M(r) of 33 K. Determination of N-terminal amino acid sequences confirmed that both fractions A and B contained the same lipase protein. Furthermore, the N-terminal amino acid sequence of the C. viscosum lipase was identical to the one of Pseudomonas glumae lipase.

Amino Acid Sequence↗
PubMed1995

Glyceride synthesis catalyzed by cutinase using the monomolecular film technique.

The monomolecular film technique previously used to study the kinetics of lipase hydrolysis was adapted to synthesizing oleoyl glycerides (monoolein, diolein, and triolein). The water subphase was replaced by glycerol, and a film of oleic acid was initially spread on the glycerol surface. In this system a recombinant cutinase from Fusarium solani was able to catalyze oleoyl glyceride synthesis. More than 50% of the oleic acid film was acylated after 7 min of reaction. The surface pressure applied to the monomolecular film acts as a physical selectivity factor since glyceride synthesis can be steered so as to produce either diolein or triolein.

Carboxylic Ester Hydrolases↗
PubMed1995

Superactivity of peroxidase solubilized in reversed micellar systems.

Vaccinium mirtyllus peroxidase solubilized in reversed micelles was used for the oxidation of guaiacol. Some relevant parameters for the enzymatic activity, such as pH, w(o) (molar ratio water/surfactant), surfactant type and concentration, and cosurfactant concentration, were investigated. The peroxidase showed higher activities in reversed micelles than in aqueous solution. The stability of the peroxidase in reversed micelles was also studied, namely, the effect of w(o) and temperature on enzyme deactivation. The peroxidase displayed higher stabilities in CTAB/hexanol in isooctane reversed micelles, with half-life times higher than 500 h.

Cetrimonium↗
PubMed1995

Triglyceride hydrolysis and stability of a recombinant cutinase from Fusarium solani in AOT-iso-octane reversed micelles.

A recombinant cutinase from Fusarium solani was encapsulated in AOT reversed micelles. Physicochemical parameters of the system were optimized relative to triolein hydrolysis. Kinetic studies of triglyceride hydrolysis showed a decrease in specificity with increase of the acyl chain length. Stability of cutinase in the system under study is lower than in aqueous solution and decreases with increase in the water content in the system (W0 = [H2O]/[AOT]). The products of triolein hydrolysis had little effect on the cutinase stability. Although glycerol did not alter the stability, oleic acid decreases the enzyme stability. The increase in log P of solvent (from iso-octane to n-dodecane) decreased the stability. Deactivation profiles were fitted with the Henley and Sadana model (1).

Carboxylic Ester Hydrolases↗
PubMed1994

Liquid-liquid extraction of a recombinant protein, cytochrome b5, with aqueous two-phase systems of polyethylene glycol and potassium phosphate salts.

The partitioning of cytochrome b5 in aqueous two-phase systems of polyethylene glycol (PEG) and potassium phosphate salts was investigated. Cytochrome b5 partitioning is enhanced with decreasing polymer molecular mass and with increasing tieline length and pH. The effect of cytochrome b5 mutation, by substitution of the glutamic acid at positions 56 and 92 of the polypeptide chain by a lysine, on protein partitioning was also studied. Partitioning of cytochrome b5 mutants shows the same dependence on tieline length and pH, following the order cytochrome b5 > mutant 56 > mutant 92.

Animals↗
PubMed1994

Liquid-liquid extraction of a recombinant protein with reverse micelles.

Recombinant cytochrome b5 was extracted into the reversed micelle phase of an anionic surfactant (AOT) in octane and back-extracted to a final aqueous phase. The extraction of the protein was controlled by an electrostatic mechanism, since it was dependent on the global charge of the protein. This was directly demonstrated by experiments with native and mutant cytochromes obtained by site directed mutagenesis. The back-extraction of cytochrome b5 to a fresh aqueous phase was decreased by factors that reduced the size of the water pool of the organic phase, such as high salt concentrations (1-2 mol dm-3 NaCl) and low temperatures (4 degrees C), probably because of an increase in a favourable interaction of this protein with the surfactant at closer distances.

Animals↗
PubMed1994

Kinetic studies of the urease-catalyzed hydrolysis of urea in a buffer-free system.

The kinetics of urea hydrolysis catalyzed by urease, mainly in the absence of buffers by use of the self-buffer effect of the products, was investigated. The effect of pH, temperature, and concentration of enzyme, substrate, product, salt ions, and buffers on the kinetic behavior of urease was examined. A kinetic model of a modified Michaelis-Menten form, incorporating substrate and product inhibition, pH dependence, and temperature effect, was developed to describe the reaction rate. Experimental data indicated that urease in a buffer-free solution was less susceptible to the inhibition of substrate product. The Michaelis constant keeps almost constant with the variation of pH and temperature, and increases with the addition of buffers and salts. The data also suggested that the noncompetitive pattern of the product inhibition, which is not significantly affected by temperature, increases gently with increasing pH. A Monod form rate expression was proposed to analyze the pH effect on the maximum rate. The proposed kinetic model was also examined by the long-time experiments in which pH, substrate, and product concentration varied obviously during the reaction course.

Ammonia↗
PubMed1994

Sterol side-chain cleavage with immobilized Mycobacterium cells in water-immiscible organic solvents.

The possibility of using whole Mycobacterium sp. cells for the selective degradation of the side-chain of sitosterol in an organic bioconversion medium was investigated. Sterol solubility limits were estimated and free-cell biocompatibility tests were carried out, using a range of water-immiscible solvents. Among these, phthalates exhibited good biocompatibility and sterol-solubilizing capacities. Several organic and inorganic matrices were tested for the immobilization of Mycobacterium sp. cells by surface adhesion. Celite led to the best results, being thus selected for beta-sitosterol side-chain degradation tests in phthalates and in aqueous medium. Cells entrapped in kappa-carrageenan and in polyurethane foams were also used in these tests. The highest degradation activities were obtained with cells immobilized in Celite, with bis(2-ethylhexyl)phthalate as the conversion medium, resulting in molar conversion yields up to 70%, with respect to beta-sitosterol (5 g l-1). Further activity and stability tests revealed that this bioconversion system is markedly temperature dependent.

Androstenedione↗
PubMed1994

Dipeptide synthesis and separation in a reversed micellar membrane reactor.

The synthesis of dipeptide AcPheLeuNH2 catalyzed by alpha-chymotrypsin encapsulated in TTAB/octanol/heptane reversed micelles was investigated in a tubular ceramic membrane reactor, operated in a batch mode. The reaction medium conditions (TTAB concentration, buffer molarity, and pH) were optimized using a factorial design in order to achieve maximum synthesis rates. Hydrated reversed micelles permeated through the membrane together with the substrate ester, dipeptide, and by-products. However, as a result of the low solubility of the peptide in the reaction medium, selective precipitation occurred, thus enabling the complete retention of the solid product by the ultrafiltration membrane and therefore an integration of a separation step in the biotransformation process. In spite of the continuous accumulation of solids inside the reactor, constant permeation flow rates could be maintained throughout the operation. The influence of alpha-chymotrypsin, TTAB, and water concentration on the kinetics and mass transfer of the system was also investigated. The behavior of the system during a continuous experiment was also evaluated.

Biotechnology↗
PubMed1994

Experimental modulation of the reactivity of pleural milky spots (Kampmeier's foci) by Freund's adjuvants, betamethasone and mycobacterial infection.

We studied the response of milky spots in the parietal pleura of the rat and mouse to intrapleural instillation of immunomodulatory agents such as complete or incomplete Freund's adjuvants and betamethasone, and also to infection by mycobacteria (M. avium). Both incomplete (mineral oil) and complete (mineral oil plus dead mycobacteria) adjuvants, as well as M. avium infection, induced a striking increase in the size and cellularity of the pleural milky spots whereas betamethasone caused a slight atrophy. The extensive inflammatory infiltrates observed after adjuvant injection differed between milky spots reactive to complete and incomplete Freund's adjuvants. Fifteen days after adjuvant administration, the pleural milky spots of rats were still enlarged and hypercellular but differences were noted in the size of milky spots of the pleura between the 2 adjuvant treatments: animals submitted to injection of complete Freund's adjuvant showed an increase in the size of milky spots from d 3 to d 15, while the size of milky spots of the incomplete Freund's adjuvant treated group showed a decrease in size from d 3 to d 15. The milky spots at d 15 were well organised: reticulin fibres permeated the whole area of the milky spot and the different cell types were evenly distributed. Histiocytes, which were previously confined to the inner layer, were now the main cell type in all areas of milky spots. A moderate number of mast cells and a few eosinophils were also seen. Complete Freund's adjuvant caused the formation of granulomas in the milky spots, a change that was not detected in animals treated with incomplete adjuvant.(ABSTRACT TRUNCATED AT 250 WORDS)

Adjuvants, Immunologic↗
PubMed1993

Kinetic studies of Mucor miehei lipase in phosphatidylcholine microemulsions.

Ethanol-oleic acid esterification by a free and microencapsulated lipase from Mucor miehei, using dodecane as solvent and phosphatidylcholine as surfactant, was studied. The initial esterification rate was influenced by the water content in the biphasic system. Kinetic studies with free and microencapsulated enzyme showed that the microencapsulation led to an increase of the kinetic parameters (Vmax and Km), probably due to an increase of the interfacial area. The reaction rate was also affected by the shaking rate, the temperature and the pH. The optimal temperature and pH achieved were, respectively, 40 degrees C and 4.5 using free enzyme, and 50 degrees C and 6 using microencapsulated enzyme.

Chromatography, Gas↗
PubMed1993

Immobilization studies of an industrial penicillin acylase preparation on a silica carrier.

Penicillin acylase (EC 3.5.1.11) was immobilized by covalent binding on a silica gel carrier activated by silanization. High immobilization yields, operational stability, enzyme loading and support reutilization capacity were obtained. The effects of several variables on the activation of the support and on the immobilization method were studied. Other supports and immobilization methods were assessed for the immobilization of penicillin acylase and compared with the basic process used. Activity versus temperature, pH, buffer molarity and penicillin concentration profiles were determined and compared for the free, crosslinked and covalently-bound silica carrier penicillin acylase preparations. The hydrolysis performance of the latter preparation was assessed in a batch basket reactor and the time course of the hydrolysis reaction modelled by a two parameter model equation. The operational stabilities of penicillin acylase coupled covalently to the silica gel support and immobilized by a crosslinking procedure using glutaraldehyde were also compared.

Biotechnology↗
PubMed1992

Kinetics and stability of a Chromobacterium viscosum lipase in reversed micellar and aqueous media.

The lipolytic activity of Chromobacterium viscosum lipase B (EC 3.1.1.3.; triacylglycerol hydrolase) solubilized both in water and AOT/isooctane reversed micelles has been investigated using triolein as a substrate. The influence of relevant parameters in the catalytic activity such as temperature, pH, surfactant and substrate concentrations, and water content was tested and compared in both media. A study of stability of the lipase was carried out, with particular reference to the influence of pH. Three major effects of the encapsulation of the lipase in the micelles were observed: increased activity (up to 7 times higher than in water), greater stability, specially at pH 7, and higher resistance to thermal deactivation.

Chromobacterium↗
PubMed1992

Screening of whole-cell immobilization procedures for the delta 1-dehydrogenation of steroids in organic medium.

Different whole-cell immobilization methods and conditions were tested for the delta 1-dehydrogenation of 6-alpha-methyl-hydrocortisone-21-acetate with Arthrobacter simplex cells, in n-decane-1-ol and chloroform. Among the entrapment methods, polyurethane foams gave the best productivity of n-decane-1-ol, but could not be used in chloroform. Partition and diffusional barriers appeared to be the factors limiting productivity in entrapped-cell systems. The coentrapment of enzyme-stabilizing (glycerol, sucrose, sodium sulfate, monosodium glutamate) or hydrophobic additives did not significantly improve dehydrogenation rates in chloroform, although in some cases higher activities resulted in n-decane-1-ol. Dehydration of kappa-carrageenan-immobilized cells lowered the productivity in both solvents. The use of cell adsorption methods on silica-based carriers produced biocatalysts that in some cases were more active in chloroform than the entrapped cells. However, the surface-attached cells appeared to be more sensitive to solvent toxicity, more hydrophilic supports generally giving higher dehydrogenation rates. The estimated partition coefficients for the substrate between several of the tested supports and the two solvents are also presented. High partition ratios followed high measured activities in entrapment matrices but not in Celite (cell adsorption matrix). Activities in chloroform were always very low (less than 1 mumol product per hour and gram of cell dry weight). Polyurethane entrapment with n-decane-1-ol as a reaction medium was the most promising system, with measured dehydrogenation rates up to 40 times higher.

Agar↗
PubMed1992

Purification of lipases.

Interest on lipases from different sources (microorganisms, animals and plants) has markedly increased in the last decade due to the potential applications of lipases in industry and in medicine. Microbial and mammalian lipases have been purified to homogeneity, allowing the successful determination of their primary aminoacid sequence and, more recently, of the three-dimensional structure. The X-ray studies of pure lipases will enable the establishment of the structure-function relationships and contribute for a better understanding of the kinetic mechanisms of lipase action on hydrolysis, synthesis and group exchange of esters. This article reviews the separation and purification techniques that were used in the recovery of microbial, mammalian and plant lipases. Several purification procedures are analysed taking into account the sequence of the methods and the number of times each method is used. Novel purification methods based on liquid-liquid extraction, membrane processes and immunopurification are also reviewed.

Animals↗