Jose Luis Allende

Molecular cloning and analysis of the genes encoding the 4-hydroxyphenylacetate hydroxylase from Klebsiella pneumoniae

Abstract The Klebsiella pneumoniae genes encoding the hydroxylase involved in the meta-cleavage pathway of 4-hydroxyphenylacetic acid (4-HPA) were cloned, and the DNA fragment from the region essential for hydroxylase activity was sequenced. K. pneumoniae 4-HPA hydroxylase was composed of two proteins (HpaA and HpaH) with different molecular masses. HpaA seems to be a flavin-containing hydroxylase with a molecular mass of 58,781 Da. HpaH, with a molecular mass of 18,680 Da, seems to be a “helper” protein required for productive hydroxylation of the substrate. The hpa genes were expressed and the hydroxylase was active in Escherichia coli. Comparison of the enzyme with other monooxygenases indicates that K. pneumoniae 4-HPA hydroxylase is a member of a new family of hydroxylases.

Transport of 4-hydroxyphenylacetic acid in Klebsiella pneumoniae

Klebsiella pneumoniae M5a1 has been shown to possess an inducible transport system for 4-hydroxyphenylacetate (4-HPA). This transport system has a Kt of 16.3 microM and a maximal velocity of 31.2 nmol/min (milligrams dry weight). The transport system has been inhibited by inhibitors of energy metabolism with a concomitant decrease in cellular ATP concentrations, and the 4-HPA binding activity has been detected in the crude shock extracts. All these observations indicate that 4-HPA uptake is an active transport which involves a periplasmic binding protein and it seems to be energized by phosphate bond energy.

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.

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.

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.

Thermodynamic equilibrium and excess properties of alkylsulphates in water by vapour pressure osmometry

Abstract In this paper we have studied the thermodynamic behaviour of sodium dodecylsulphate (SDS) and sodium tetradecylsulphate (STS) in aqueous micellar solutions over a wide range of temperatures (298–363 K). From vapour pressure osmometry measurements we have determined the osmotic and activity coefficients, the excess Gibbs free energy, excess enthalpy and excess entropy functions. Using several association models we have determined the type of association of the two detergents studied. Finally, self-association constants have been calculated in the temperature range 298–363 K, for the different association models.

Improving dioxygenase stability by gene chromosome insertion: implementation in immobilized-cell systems.

The immobilization of recombinant cells by using the unstable 3,4-dihydroxyphenylacetate 2,3-dioxygenase was studied as a model. Dioxygenase activity and cell viability were compared in immobilized-cell systems and cells in suspension. Immobilization increased enzyme stability and the efficient degradation of 3,4-dihydroxyphenylacetate. The stability of the cloned enzyme and the viability of the immobilized recombinant cells were well maintained for at least 15 days. We used the strain Escherichia coli CC118-D in which the hpaB gene from Klebsiella pneumoniae, coding for the subunit of 3,4-dihydroxyphenylacetate 2,3-dioxygenase, was inserted into the chromosome. This study has demonstrated that the implementation of E. coli CC118-D in a pilot-scale bioreactor resulted in a 100% stabilization of dioxygenase activity, and could be a useful tool for bioremediation processes.

4-Hydroxybenzoate Uptake in Klebsiella planticola Strain DSZ1 Is Driven by ΔpH

Klebsiella planticola strain DSZ1 has the ability to degrade different aromatic compounds such as benzoate and organochlorinated as propachlor and alachlor. DSZ1 strain cells mineralised 4-hydroxybenzoate (4HBA) through a meta-cleavage pathway, yielding protocatechuate as dihydroxylated intermediate, with a specific rate of CO2 formation 0.12 × 10−6 (cpm/OD) h−1, and a rate of 4-HBA utilisation of 0.75 mmol h−1. Aerobically the 4HBA transport system is driven by gradient of protons (ΔpH), but is not ATP-driven. Under anaerobic conditions, the system can use the nitrate reduction as a final electron acceptor in respiration. A kinetic analysis of the 4HBA transport system revealed a Kt value of 16 μM with a Vmax value of 25 nmol/min.mg at pH 7.

Three Different Topics for Bioremediation Application on Soils and Waters Contaminated with Aromatic Compounds, by Using Immobilized Bacteria

Molecular techniques and modelling are presented as powerful tools required in the performance of efficient soil and water bioremediation systems. An Escherichia coli CC118-D strain was constructed by inserting the Klebsiella pneumoniae hpa B gene, coding for the unstable 3,4-dihydroxyphenylacetate 2,3-dioxygenase, into its chromosome. When the constructed strain was immobilized, both enzyme stability and viability increased along the studied period, in absence of antibiotic. We proposed this strategy as an approach to overcoming plasmid instability and to enhance enzyme activity and stability, avoiding antibiotic utilization. A model was developed to understand and predict the behaviour of bacteria and pollutants in a bioreactor system, considering: fluid dynamics, molecular/cellular scale processes and biofilm formation.

Thermodynamic Behaviour of Sodium Deoxycholate, Sodium Dodecylsulphate and Sodium Tetradecylsulphate Micellar Solutions

In this paper we have studied the thermodynamic behavior in aqueous solution of three anionic surfactants: Sodium dodecylsulphate (SDS), sodium tetra- decylsulfate (STS and sodium deoxycholate (SDC). From osmotic pressure data and theoretical association models WE have calculated several excess functions (SE , GE and HE ) in the temperature range 298–363 K.