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M Busquets

Publications and source records attributed to M Busquets.

At least 19 recordsLinked to original sources
PubMed2004

Isolation and characterization of a lipoxygenase from Pseudomonas 42A2 responsible for the biotransformation of oleic acid into ( S )-( E )-10-hydroxy-8-octadecenoic acid.

The isolation of a new lipoxygenase-like (LOX-like) enzyme from Pseudomonas 42A2 and its characterization is described. The enzyme, located in the periplasm of the cell, which contained 0.55 mol of Fe2+ per mol of protein, is monomeric and has a molecular mass of 45 kDa. In the presence of oxygen, the enzyme converts oleic acid into (E)-10-hydroperoxy-8-octadecenoic acid (HPOD), which decomposes to the corresponding (E)-10-hydroxy-8-octadecenoic acid (HOD). The absolute configuration of this acid was determined as S on the basis of exciton-coupled CD data, and specific rotation and NMR analysis of the corresponding p -bromobenzoate derivative. The reaction in vivo leads to the dihydroxy derivative (E)-7,10-dihydroxy-8-octadecenoic acid (DHOD), so that the three hydroxy-fatty acids can be isolated from the culture medium. The activity of the enzyme was optimal between 25 and 30 degrees C and 44% of its activity still remained at 55 degrees C. Its optimal pH is 8.5-9; and the presence of magnesium ions increased LOX activity by 1.5. The activity of the LOX is highest in unsaturated fatty acids containing double bonds in position 9 (oleic, linoleic and linolenic acids), linoleic acid being preferred (100% activity) over linolenic (60.4%) and oleic acids (46%). However, kinetic studies showed that the affinity of the enzyme is similar for the three substrates.

Biotransformation↗
PubMed2001

Contribution of engineered electrostatic interactions to the stability of cytosolic malate dehydrogenase.

Protein engineering is a promising tool to obtain stable proteins. Comparison between homologous thermophilic and mesophilic enzymes from a given structural family can reveal structural features responsible for the enhanced stability of thermophilic proteins. Structures from pig heart cytosolic and Thermus flavus malate dehydrogenases (cMDH, Tf MDH), two proteins showing a 55% sequence homology, were compared with the aim of increasing cMDH stability using features from the Thermus flavus enzyme. Three potential salt bridges from Tf MDH were selected on the basis of their location in the protein (surface R176-D200, inter-subunit E57-K168 and intrasubunit R149-E275) and implemented on cMDH using site-directed mutagenesis. Mutants containing E275 were not produced in any detectable amount, which shows that the energy penalty of introducing a charge imbalance in a region that was not exposed to solvent was too unfavourable to allow proper folding of the protein. The salt bridge R149-E275, if formed, would not enhance stability enough to overcome this effect. The remaining mutants were expressed and active and no differences from wild-type other than stability were found. Of the mutants assayed, Q57E/L168K led to a stability increase of 0.4 kcal/mol, as determined by either guanidinium chloride denaturalization or thermal inactivation experiments. This results in a 15 degrees C shift in the optimal temperature, thus confirming that the inter-subunit salt bridge initially present in the T.flavus enzyme was formed in the cMDH structure and that the extra energy obtained is transformed into an increase in protein stability. These results indicate that the use of structural features of thermophilic enzymes, revealed by a detailed comparison of three-dimensional structures, is a valid strategy to improve the stability of mesophilic malate dehydrogenases.

Catalysis↗
PubMed2000

Screening and production of rhamnolipids by Pseudomonas aeruginosa 47T2 NCIB 40044 from waste frying oils.

World production of oils and fats is about 2.5 million tonnes, 75% of which are derived from plants. Most of them are used in the food industry for the manufacture of different products, or directly as salad oil. Great quantities of waste are generated by the oil and fat industries: residual oils, tallow, marine oils, soap stock, frying oils. It is well known that the disposal of wastes is a growing problem and new alternatives for the use of fatty wastes should be studied. Used frying oils, due to their composition, have great potential for microbial growth and transformation. The use of economic substrates such as hydrophobic wastes meets one of the requirements for a competitive process for biosurfactant production. In the Mediterranean countries, the most used vegetable oils are sunflower and olive oil. Here we present a screening process is described for the selection of micro-organism strains with the capacity to grow on these frying oils and accumulate surface-active compounds in the culture media. From the 36 strains screened, nine Pseudomonas strains decreased the surface tension of the medium to 34-36 mN/M; the emulsions with kerosene remained stable for three months. Two Bacillus strains accumulated lipopeptide and decreased the surface tension to 32-34 mN/m. Strain Ps. aeruginosa 47T2 was selected for further studies. The effect of nitrogen and a C/N of 8. 0 gave a final production of rhamnolipid of 2.7 g l-1 as rhamnose, and a production yield of 0.34 g g-1.

Bacteria↗
PubMed1997

Oxydation of oleic acid to (E)-10-hydroperoxy-8-octadecenoic and (E)-10-hydroxy-8-octadecenoic acids by Pseudomonas sp. 42A2.

Biotransformation of oleic acid with Pseudomonas sp. 42A2 has been found to produce(E)-10-hydroxy-8-octadecenoic acid (2a), (E)-10-hydroperoxy-8-octadecenoic acid (3a), and (E)-7,10-dihydroxy-8-octadecenoic acid (4a). Structures of the metabolites were fully characterized by infrared and 1H and 13C NMR spectra of the acids, by fast atom bombardment (FAB) and electron impact (EI) and chemical ionization (CI) mass spectrometry of the corresponding methyl esters. This is the first time that the two former compounds of trans stereochemistry have been described to have originated from a Pseudomonas sp. cell culture. Time course of products accumulation showed that biotransformation started with bacterial growth, the amount of products 2a (5.58 g/l) and 4a (2.63 g/l) being optimum after 24 h of incubation while hydroperoxide 3a (1.15 g/l) reached its maximum after 16 h of the biotransformation process. Experiments conducted to ascertain whether the conversion enzyme(s) was cell-bound or extracellular, showed that the enzyme(s) is cell bound, located in the periplasmic space and has lipoxygenase activity.

Biotransformation↗
PubMed1996

Cloning, sequencing and functional expression of a DNA encoding pig cytosolic malate dehydrogenase: purification and characterization of the recombinant enzyme.

Using the polymerase chain reaction, DNA encoding cytosolic malate dehydrogenase (cMDH) has been cloned from a pig heart cDNA library. Large amounts of the enzyme (30 mg per litre of original culture) have been produced in Escherichia coli using an inducible expression vector (pKK223-3) in which the 5'-non-coding region of the gene was replaced with the tac promoter. The complete nucleotide sequence of the DNA is reported for the first time. The recombinant cMDH purified was shown to be identical to the native enzyme according to: chromatographic behaviour, isoelectric point, N-terminal amino acid sequence, and physiochemical and catalytic properties.

Amino Acid Sequence↗
PubMed1993

[Practicability of panoramic hysteroscopy with CO2. Clinical experience: 923 cases].

Between 1987 and 1990, Diagnostic Panoramic Hysteroscopies with CO2 as distending medium was proposed in 890 cases. Age groups, complications and failures are discussed. The notion of "insufficient" hysteroscopy is defined as well as the attitude toward this situation. A technique, vaginoscopy is described, making possible to perform the examination in patients that never had sexual intercourse or senile patients. Vaginoscopy and repetitive hysteroscopy after the first hysteroscopy found insufficient are important tools to achieve good diagnosis and fewer than 1% failure of the procedure among patients with narrow introitus.

Adult↗
PubMed1991

Heterogeneity of chicken liver cytosolic aspartate aminotransferase.

The isolated native molecular forms of chicken liver cytosolic aspartate aminotransferase give rise to two kinds of generation processes: (a) on storage, molecular forms are transformed into a series of variants with increasing anodic mobilities; and (b) addition of thiol reagents not only avoids the process, but causes the partial transformation of minor subforms into variants with higher isoelectric point values. In both cases the mobilities of each generated variant coincide with that of the corresponding native molecular form. The variants generated either by storage or in the presence of thiol reagents were separated by chromatofocusing. Several comparative studies have demonstrated the structural and functional identity between native molecular forms and 'in vitro' active generated variants of the enzyme. The results obtained suggest that native minor subforms arise from the major alpha form due to oxidation process and might represent intermediate species in the intracellular cytosolic aspartate aminotransferase turnover.

Animals↗
PubMed1991

Heterogeneity of human liver alanine aminotransferase due to sulfhydryl groups oxidation.

The cytosolic isoenzyme of human liver alanine aminotransferase exhibited a progressive change in its chromatographic behavior on DEAE-Sepharose when partially purified preparations were stored for up to 8 days at 4 degrees C. This change was characterized by the appearance of an additional chromatographic variant and was avoided by addition of 2-mercaptoethanol. The experimental evidence presented indicates that the progressive oxidation of free sulfhydryl groups of the enzyme is responsible for the charge modifications and heterogeneity observed.

Alanine Transaminase↗
PubMed1990

Comparative analysis of the reduction of oxaloacetate by human hepatoma and normal liver extracts.

An apparent activation of the malate dehydrogenase activity is observed in the double-reciprocal plot at high oxaloacetate concentrations when human hepatoma extracts are analyzed. This phenomenon does not occur in healthy liver samples. In hepatoma extracts, the ratio of lactate dehydrogenase to malate dehydrogenase activities becomes five-fold higher than that of normal liver. Experiments performed with mixtures of both purified enzymes and, conversely, by using oxamate, a specific inhibitor of lactate dehydrogenase, reveal that the deviation in Michaelis-Menten behavior observed is due to the oxaloacetate reductase activity of lactate dehydrogenase instead of the presence of a novel malate dehydrogenase isoenzyme.

Carcinoma, Hepatocellular↗
PubMed1989

Purification and characterization of chicken brain cytosolic aspartate aminotransferase.

Aspartate aminotransferase from the cytosolic fraction of chicken brain was isolated with acceptable yield and high degree of purity. The enzyme appeared in multiple molecular forms: alpha, beta, gamma, and delta (alpha predominates), as detected by polyacrylamide gel electrophoresis with specific staining. These different forms of the enzyme were separated by DEAE-Sephacel chromatography, and showed different isoelectric points and maximal velocities values, whereas their molecular weight, optimum pH and Michaelis constants were very similar. Generation process studies suggest that minors subforms of the enzyme could be raised from alpha form by a mechanism in which the oxidation of particular amino acid groups are involved.

Animals↗
PubMed1989

Cytosolic aspartate aminotransferases from different chicken tissues: purification and characterization of their multiple forms.

Cytosolic aspartate aminotransferases from chicken heart, liver, spleen, skeletal muscle and breast muscle differed in number of their molecular forms, detected by polyacrylamide gel electrophoresis and specific staining. The number of molecular forms varied from tissue to tissue but the electrophoretic mobilities of a given form in all tissues were analogous. Within a single tissue most of the enzyme activity was present as the lowest-running band (alpha form) and the rest was distributed in minor bands termed (B,tau, alpha and epsilon forms). We report a method for the purification of cytosolic aspartate aminotransferases from various chicken tissues. The procedure can be carried out in one week and allows the obtention of isolated molecular forms of the enzyme, independently of the tissue under study. Separation of multiple forms was also achieved by chromatofocusing. The isoelectric points determined by this method for a given form in all five tissues were analogous and differed from those of the molecular forms of the enzyme from other origins. An Mr of 100,000 was obtained for all molecular forms of the five chicken tissues studied.

Animals↗
PubMed1989

[Selective and ambulatory salpingography].

A new technique is here proposed to enhance, in a selective or direct way, the fallopian tubes. This new approach is suggested because hysterosalpingography (with the filling of the uterine cavity) is an indirect procedure, and therefore not completely reliable. Since intrauterine pressure cannot exceed 200 mmHg, and more pressure is needed to reach the tube ostia, false negatives are produced; up to 50% of the fallopian tubes not enhanced during hysterosalpingography are normal. Thurmond et al. were the first to introduce selective salpingography. They used a blind procedure with fluoroscopy passing through the uterine cavity, catheterized, with probes, and performed repeated fluoroscopic examinations of the uterine cavity. We suggest a different technique consisting in the use of hysteroscopy to visualize the ostium, and of a direct image to place the probe into the fallopian tube. The intubation is then repeated, if needed, in the other tube, which is then enhanced with the conventional means. X-ray pictures of both tubes will then be made. The technique is thus a simple and quick one, which can be carried out on an out-patient basis at the Radiology Department, without premedication, and with no need for previous preparation. Neither is it necessary to dilate the cervix nor to anesthesize the patient if the Karl-Storz chorionscope is used, which has proved the most useful instrument for this purpose. No complications were observed and the contraindications were similar to those of hysterosalpingography and hysteroscopy. To optimize the method, 16 direct fallopian tube intubations were carried out on a selected group of patients suffering from different kinds of infertility. The procedure lasted no longer than 20 minutes, was well tolerated, and allowed the patients to immediately resume their usual activities. We believe this practical technique to be the procedure of choice when one/both fallopian tubes are not enhanced during conventional hysterosalpingography.

Female↗
PubMed1988

Generation process of cytosolic aspartate aminotransferase molecular forms by several treatments.

Alpha-, beta-, and gamma-forms of chicken liver cytosolic aspartate aminotransferase generate variants on storage (4 degrees C, 25 days). The variants developed from each isolated form appeared as evenly spaced bands with increasing anodic mobilities after polyacrylamide gel electrophoresis (PAGE), pH 8.8, and specific staining. Their mobilities coincided with those of the more negatively charged forms present in fresh tissue. Development of faster-running variants on storage was avoided by addition of thiol reagents to the freshly isolated forms. In their presence, beta- and gamma-forms were partially transformed into one and two variants with lower anodic mobilities analogous to those of native alpha- and beta-forms. Short pH and heat treatments did not modify the electrophoretic patterns of the alpha-, beta-, and gamma-forms, but the incubation with 5 mM L-ascorbic acid (37 degrees C, 7 h) produced more anodic active bands. The formation of these variants was inhibited by the presence, in the incubation mixture, of superoxide dismutase and catalase. The kinetic parameters of the forms submitted to the different treatments were similar to those of the freshly isolated subforms. The results obtained suggest that minor subforms of the enzyme could be generated in vivo by a mechanism in which the oxidation of particular amino acid groups is involved.

Animals↗
PubMed1988

Simultaneous purification and characterization of aspartate aminotransferase isoenzymes from chicken liver.

Cytosolic and mitochondrial isoenzymes of aspartate aminotransferase (EC 2.6.1.1) were purified to homogeneity from chicken liver, without previous fractionation of the subcellular components. The procedure includes initial heat treatment and ammonium sulfate fractionation. The two isoenzymes can then be separated by a DEAE-Sepharose chromatography using a linear gradient of L-aspartate (reaction substrate). The separated fractions can be further purified by a parallel step with HA-Ultrogel prior to octyl-Sepharose (c-AAT) and CM-Sepharose (m-AAT) chromatographies. Michaelis constants, pI values, inhibition by adipate and subforms generation with time were studied for both isoenzymes.

Animals↗