Estrella Ferrer

Catabolism of 3- and 4-hydroxyphenylacetic acid by Klebsiella pneumoniae

Klebsiella pneumoniae catabolizes both 4-hydroxyphenylacetic acid and 3-hydroxyphenylacetic acid via meta-cleavage of 3,4-dihydroxyphenylacetic acid, ultimately yielding pyruvate and succinate. The organism can synthesize two hydroxylases catalysing 3,4-dihydroxyphenylacetic acid formation, which differ in substrate specificity, cofactor requirement, kinetics and regulation. Five enzymes sequentially involved in the catabolism of 3,4-dihydroxyphenylacetic acid are encoded on a 7 kbp fragment of the K. pneumoniae chromosome that has been isolated in a recombinant plasmid.

SIMAZINE DEGRADATION BY IMMOBILIZED AND SUSPENDED SOIL BACTERIUM

Abstract Simazine is one of the most heavily used herbicides for weed control in the production of a variety of agricultural crops. Few microorganisms have been isolated that metabolize S-triazines such as simazine at rates that are suitable for environmental remediation. DSZ1 strain cells were immobilized by adsorption onto ceramic supports. Kinetic parameters were estimated using nonlinear parameter estimation methods and compared between immobilized and suspended cells. The effect of substrate concentration and inoculum size/support ratio on kinetic parameters was investigated. Physiological status of immobilized cells was assessed by measuring their capacity for degrading the herbicide and this capacity was compared to that of free-living cells under different experimental conditions.

4-Hydroxybenzoate uptake in an isolated soil Acinetobacter sp.

Abstract. The isolated soil bacteria Acinetobacter strain BEM2 is able to utilize some xenobiotic aromatic compounds as a carbon source. In this study the metabolism of 4-hydroxybenzoate (4-HBA) by strain BEM2 was characterized. Degradation involved a meta-cleavage pathway yielding 3,4-dihydroxybenzoate (3,4-DHBA) as an intermediate and CO2 as the principal product from the C atoms in the aromatic ring. 4-HBA uptake was studied, and the kinetic parameters were determined. The uptake was shown to be directly coupled to ATP hydrolysis and its synthesis, according to the Mitchell chemiosmotic hypothesis.

Bioremediation of soil contaminated by propachlor using native bacteria

Abstract Soil from a herbicide disposal site was used to enrich for microorganisms that degraded 2-Chloro-N-isopropylacetanilide (propachlor). A bacterium, PEM1, able to mineralize the herbicide was isolated. A product of the microbial metabolism of propachlor was identified as N-isopropylacetanilide, suggesting that the first step in the mineralization process is dehalogenation. Strain PEM1 could grow on N-substituted acylanilides, but was incapable of growth on aniline and phenol. Rates of degradation of propachlor were monitored in low-organic-matter soil at field capacity, amended with 0·1–100 μg of herbicide per g of soil and inoculated with two different amounts of strain PEM1. Rates of degradation were comparable, and removal of propachlor close to 90% by 8 days.

Degradation of 3- and 4-hydroxybenzoate byKlebsiella pneumoniae

SummaryTwo different hydroxylases involved in benzenoid degradation have been isolated from Klebsiella pneumoniae and they can be differentially induced in the same strain. 3-Hydroxybenzoate-6-hydroxylase has an interesting biochemical mechanism and kinetic properties. 4-Hydroxybenzoate-1-hydroxylase activity is reported for the first time. The kinetic parameters of both enzymes have been studied.

Propachlor and alachlor degradation by immobilized and suspended Pseudomonas cells

Abstract The soil isolated Pseudomonas strain PEM1 has the ability to degrade propachlor (2-Cl-N-isopropylacetanilide) and alachlor (2-Cl-N-(2,6-diethylphenyl)N-methoxymethyl-acetamida). These cells were immobilized by adsorption onto ceramic support. Kinetic parameters were estimated using nonlinear parameter estimation methods and compared between immobilized and suspended Pseudomonas PEM1. The effect of inoculum/support ratio on kinetic parameters was investigated. Implications of these results for developing an industrial process with immobilized cells are discussed.

Purification and characterization of 4-hydroxybenzoate 3-hydroxylase from a Klebsiella pneumoniae mutant strain.

Unlike the parent wild-type strain, theKlebsiella pneumoniae mutant strain MAO4 has a 4-HBA+ phenotype. The capacity of this mutant to take up and metabolize 4-hydroxybenzoate (4-HBA) relies on the expression of a permease and an NADPH-linked monooxygenase (4-HBA-3-hydroxylase). Both enzymes are normally expressed at basal levels, and only the presence of 4-HBA in the media enhances their activities. Strikingly, when theAcinetobacter calcoaceticus pobA gene encoding 4-hydroxybenzoate-3-hydroxylase was expressed in hydroxybenzoateK. pneumoniae wild-type, the bacteria were unable to grow on 4-HBA, suggesting that the main difference between the wild-type and the mutant strain is the capability of the latter to take up 4-HBA. 4-HBA-3-hydroxylase was purified to homogeneity by affinity, gel-filtration, and anion-exchange chromatography. The native enzyme, which appeared to be a dimer of identical subunits, had an apparent molecular mass of 80 kDa and a pI of 4.6. Steady-state kinetics were analyzed; the initial velocity patterns were consistent with a concerted substitution mechanism. The purified enzyme had 362 amino acid residues, and a tyrosine seemed to be involved in substrate activation.

Comparison of Phenolic Substrate Utilization and Growth Kinetics Between Immobilized and Suspended Degradative-Bacteria

Establishing if immobilized cells have greater tolerance to aromatic pollutant chemicals than cells in suspension, is an important target in modelling and designing cell bioreactors. In this paper 4-hydroxyphenylacetic acid mineralization; the specific 4-hydroxyphenylacetic acid hydroxylase activity; and the viability in freely suspended and immobilized monocultures of two different strains, Escherichia coli W21 (pGA260) and Klebsiella pneumoniae are compared. The immobilized cells, growing on a ceramic support, exhibit viability and catalytic activity higher than suspended cells. By using a kinetic model for describing CO2 formation, it is demonstrated that immobilized K. pneumoniae cells are biocatalytically more active than the other systems studied.

Separation by counter-current distribution of young-chick erythrocyte populations and numerical resolution of distribution curves

The presence of two red cell populations in young chicks has been demonstrated after increasing the settling time used during counter-current distribution with charged 5% Dextran--4% poly(ethylene glycol) two-phase systems. A Fortran program using statistical methods was applied to show the resolution in two peaks (with two or three subpopulations assumed) of the counter-current distribution curves.

Partitioning behaviour in aqueous two-phase systems and fractionation by counter-current distribution of chick-embryo erythrocytes with numerical resolution of distributiion curves

The partition of chick-embryo and young-chick erythrocytes in dextran-poly-(ethylene glycol) two-phase systems depends on the interfacial tension and electrical potential differences between the phases. Counter-current distribution with charged 5% dextran-poly(ethylene glycol) systems has proved to be an adequate method for the separation of primitive and definitive erythrocytes present in chick embryos when a phase settling time of 20 min is used. The computer-aided numerical resolution of experimental curves has shown the existence of subpopulations which could not have been detected by using conventional methods.