Modification of galactitol dehydrogenase from Rhodobacter sphaeroides D for immobilization on polycrystalline gold surfaces.

Galactitol dehydrogenase (GatDH) from Rhodobacter sphaeroides is a multifunctional enzyme that catalyzes in the presence of oxidized beta-nicotinamide adenine dinucleotide (NAD(+)) the interconversion of various multivalent aliphatic alcohols to the corresponding ketones. The recombinant GatDH was provided with an N-terminal His(6)-tag to which distally up to three cysteine residues were attached. This protein construct maintained nearly full enzymatic activity, and it could be covalently immobilized via thiol bonds onto the surface of a gold electrode. Binding of GatDH onto the gold electrode was verified by SPR measurements, and residual enzyme activity was measured by cyclic voltammetry using 1,2-hexanediol as substrate, the cofactor NAD(+) and the redox mediator CTFM (4-carboxy-2,5,7-trinitrofluorenyliden-malonnitrile) in solute form. The results demonstrate the possibility of a directed functional immobilization of proteins on gold surfaces, which represents a proof-of-concept for the development of reactors for electrochemical synthon preparation using dehydrogenases.

[Bacillus cereus is a microbial decomposer of 2,4-dichlorophenol].

A microorganism degrading 2,4-dichlorophenol isolated from an aeration pond of the Baikal Pulp and Paper Mill was identified as Bacillus cereus BIP507 based on morphological and physiological characters as well as 16s rDNA sequencing. This microorganism proved able to degrade high 2,4-dichlorophenol concentrations (up to 560 microM).

GEMSS: grid-infrastructure for medical service provision.

OBJECTIVES: The European GEMSS Project is concerned with the creation of medical Grid service prototypes and their evaluation in a secure service-oriented infrastructure for distributed on demand/supercomputing. Key aspects of the GEMSS Grid middleware include negotiable QoS support for time-critical service provision, flexible support for business models, and security at all levels in order to ensure privacy of patient data as well as compliance to EU law. METHODS: The GEMSS Grid infrastructure is based on a service-oriented architecture and is being built on top of existing standard Grid and Web technologies. The GEMSS infrastructure offers a generic Grid service provision framework that hides the complexity of transforming existing applications into Grid services. For the development of client-side applications or portals, a pluggable component framework has been developed, providing developers with full control over business processes, service discovery, QoS negotiation, and workflow, while keeping their underlying implementation hidden from view. RESULTS: A first version of the GEMSS Grid infrastructure is operational and has been used for the set-up of a Grid test-bed deploying six medical Grid service prototypes including maxillo-facial surgery simulation, neuro-surgery support, radio-surgery planning, inhaled drug-delivery simulation, cardiovascular simulation and advanced image reconstruction. CONCLUSIONS: The GEMSS Grid infrastructure is based on standard Web Services technology with an anticipated future transition path towards the OGSA standard proposed by the Global Grid Forum. GEMSS demonstrates that the Grid can be used to provide medical practitioners and researchers with access to advanced simulation and image processing services for improved preoperative planning and near real-time surgical support.

Mycobacterium fluoranthenivorans sp. nov., a fluoranthene and aflatoxin B1 degrading bacterium from contaminated soil of a former coal gas plant.

Mycobacterium strain FA4T was isolated with fluoranthene as the single carbon source from soil of a former coal gas plant, polluted with polycyclic aromatic hydrocarbons. The physiological properties, fatty acid pattern, and the 16S ribosomal RNA gene sequence indicated membership to the genus Mycobacterium, but were different from all type strains of Mycobacterium species. Based on comparative 16S rRNA gene sequence analyses strain FA4T could be assigned to the Mycobacterium neoaurum taxon showing 98% sequence similarity to M. diernhoferi as its closest neighbour. The occurrence of epoxymycolate in the cell wall differentiates FA4 from all members of this taxon which synthesize wax-ester mycolates in addition to alpha-mycolates. Strain FA4T is able to degrade aflatoxin B1. This biological attribute might be useful in biological detoxification processes of foods and feeds. From the investigated characteristics it is concluded that strain FA4T represents a new species, for which we propose the name Mycobacterium fluoranthenivorans sp. nov. The type strain of Mycobacterium fluoranthenivorans is FA4T (DSM 44556T = CIP 108203T).

Stereoselective oxidation of aliphatic diols and reduction of hydroxy-ketones with galactitol dehydrogenase from Rhodobacter sphaeroides D.

From the Rhodobacter sphaeroides mutant D a galactitol dehydrogenase (GDH) was isolated and characterized in an earlier investigation (1). The enzyme expressed activity with a wide spread substrate spectrum, like sugars, sugar alcohols, secondary alcohols or the corresponding ketones and it can be used for the production of the rare sugar L-tagatose by regioselective oxidation of galactitol (2). This study focuses on the preparation of optically pure aliphatic diols by oxidation of one enantiomer or stereospecific reduction of keto-alcohols and diketones. The oxidation of 1,2-propanediol, 1,2-butanediol, 1,2-pentanediol and 1,2-hexanediol occurred highly specific with the S-enantiomer leaving the R-enantiomer of the diols in the reaction vessel. Also (S)-1,2,6-hexanetriol was oxidized by GDH to 1,6-dihydroxy-2-hexanone. The Km values of these reactions decreased with increasing length of the carbon chain. Reduction of hydroxyacetone or 1-hydroxy-2-butanone resulted in an excess of 93% (S)-1,2-propanediol and more than 98% of (S)-1,2-butanediol, respectively. The diketone 2,3-hexanedione was only reduced to (2R,3S)-2,3-hexanediol, one of the possible four configurations. The wide substrate spectrum on one hand and the selectivity in the reaction on the other hand make GDH a very interesting enzyme for the production of optically pure building blocks in the chemical synthesis of bioactive compounds.

Phototrophic transformation of phenol to 4-hydroxyphenylacetate by Rhodopseudomonas palustris.

Newly isolated and culture collection strains of Rhodopseudomonas palustris were able to transform phenol to 4-hydroxyphenylacetate under phototrophic conditions in the presence of acetate, malate, benzoate, or cinnamate as growth substrates. The reaction was examined with uniformly (14)C-labelled phenol and the product was identified by HPLC retention time, UV-scans, and (1)H- and (13)C-NMR analysis. The transformation reaction was detectable in cell-free extracts in the presence of NAD(+) and acetyl-CoA. For further degradation of 4-hydroxyphenylacetate by R. palustris, low partial pressures of oxygen were essential, presumably for aerobic aromatic ring fission reactions by mono- and di-oxygenases.

Purification by Immunoaffinity Chromatography, Characterization, and Structural Analysis of a Thermostable Pyranose Oxidase from the White Rot Fungus Phlebiopsis gigantea.

A moderately thermostable pyranose oxidase (PROD) was purified to apparent homogeneity with a yield of 71% from mycelium extracts of the white rot fungus Phlebiopsis gigantea by an efficient three-step procedure that included heat treatment, immunoaffinity chromatography, and gel filtration on Superdex 200. PROD of P. gigantea is a glycoprotein with a pI between pH 5.3 and 5.7. The relative molecular weight (M(infr)) of native PROD is 295,600 (plusmn) 5% as determined by four independent methods. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of PROD revealed two distinct but similar stained bands corresponding to polypeptides with M(infr)s of 77,000 and 70,000, suggesting a heterotetrameric enzyme structure. The tetrameric structure of PROD was confirmed by electron microscopic examinations, which additionally showed the ellipsoidal shape (4.6 by 10 nm) of each subunit. Spectral analyses and direct determinations showed the presence of covalently bound flavin adenine dinucleotide with a stoichiometry of 3.12 mol/mol of enzyme. A broad pH optimum was determined in the range pH 5.0 to 8.0 in 100 mM sodium phosphate, and the activation energy for d-glucose oxidation was 24.7 kJ/mol. The main substrates of PROD are d-glucose, l-sorbose, and d-xylose, for which K(infm) values 1.2, 16.5, and 22.2 mM were determined, respectively. PROD showed high stability during storage. In 100 mM sodium phosphate (pH 6.0 to 8.0), the half-life of PROD activity was >300 days at 40(deg)C, >110 days at 50(deg)C (pH 7.0), and 1 h at 65(deg)C.

Structure and dynamics of the complex of single stranded DNA binding protein of Escherichia coli with circular single stranded DNA of filamentous phages.

We have analyzed the equilibrium and nonequilibrium properties of the complex of the single stranded DNA binding protein of Escherichia coli (EcoSSB) and circular single stranded DNA of filamentous phages M13mp8 and F1 using static and dynamic light scattering, analytical ultracentrifugation and electron microscopy. Upon binding to the single stranded DNA the EcoSSB tetramer replaces an equivalent volume of water trapped within the coiled single stranded DNA and hinders the folding of the single stranded DNA into secondary structures at all salt concentrations. The salt dependent compaction of the stoichiometric complex can be described assuming a flexible polyelectrolyte chain. The solution structure of the macromolecular complex is a random coil and in the electron microscope a beaded flexible structure of the complex with a bead diameter of 6 nm appears at all salt concentrations used. The internal motions of the stoichiometric complex can be described by the Rouse-Zimm model of polymer dynamics. The segmental mobility of the complex can be correlated with changes in the binding site size of the EcoSSB tetramer; it indicates the presence of interactions between EcoSSB tetramers bound to single stranded DNA.

Anaerobic dechlorination of 2,4-dichlorophenol in freshwater sediments in the presence of sulfate.

In the presence of added sulfate, 2,4-dichlorophenol and 4-chlorophenol were transformed stoichiometrically to 4-chlorophenol and phenol, respectively, in anaerobic freshwater lake sediments between 18 and 40 degrees C. The concomitantly occurring sulfate reduction reduced the initial sulfate concentration from 25 mM to about 6 to 8 mM and depressed methane formation.

Anaerobic biodegradation of 2,4-dichlorophenol in freshwater lake sediments at different temperatures.

Anaerobic degradation of 2,4-dichlorophenol (2,4-DCP) between 5 and 72 degrees C was investigated. Anaerobic sediment slurries prepared from local freshwater pond sediments were partitioned into anaerobic tubes or serum vials, which then were incubated separately at the various temperatures. Reductive 2,4-DCP dechlorination occurred only in the temperature range between 5 and 50 degrees C, although methane was formed up to 60 degrees C. In sediment samples from two sites and at all tested temperatures from 5 to 50 degrees C, 2,4-DCP was transformed to 4-chlorophenol (4-CP). The 4-CP intermediate was subsequently degraded after an extended lag period in the temperature range from 15 to 40 degrees C. Adaptation periods for 2,4-DCP transformation decreased between 5 and 25 degrees C, were essentially constant between 25 and 35 degrees C, and increased in the tubes incubated at temperatures between 35 and 40 degrees C. The degradation rates increased exponentially between 15 and 30 degrees C, had a second peak at 35 degrees C, and decreased to about 5% of the peak activity by 40 degrees C. In tubes from one sediment sample, incubated at temperatures above 40 degrees C, an increase in the degradation rate was observed following the minimum at 40 degrees C. This suggests that at least two different organisms were involved in the transformation of 2,4-DCP to 4-CP. Storage of the original sediment slurries for 2 months at 12 degrees C resulted in increased adaptation times, but did not affect the degradation rates.

In situ distribution of EcoRI methylase and restriction endonuclease in cells of Escherichia coli Bs 5.

Specific IgG antibodies were raised in rabbits against purified EcoRI methylase and restriction endonuclease. Post embedding labeling experiments, using the protein A-gold technique, were made with paraformaldehyde-glutaraldehyde fixed cells, embedded in Lowicryl K4M resin at low temperatures. Labeling with methylase-specific antibodies showed 60-70% of gold particles in the cytoplasm and 30-40% at the cell envelope, whereas the use of restriction enzyme-specific antibodies led to a distribution of 10-30% in the cytoplasm and 70-90% in the cell envelope. The results coincide with the proposed function of the enzymes: in the cytoplasm methylase protects the cells' own DNA from self-destruction, and the restriction endonuclease cuts foreign DNA when entering the cell.

Immunocytochemical localization of component C of the methylreductase system in Methanococcus voltae and Methanobacterium thermoautotrophicum.

Antibodies were raised against homogeneous preparations of component C of the methylreductase system from Methanococcus voltae and Methanobacterium thermoautotrophicum. Cells of these organisms were fixed with paraformaldehyde and/or glutaraldehyde, sectioned, and labeled with antibodies and colloidal gold-labeled protein A. In M. voltae the gold particles were predominantly located in the vicinity of the cytoplasmic membrane. In rare cases a similar result was obtained also with M. thermoautotrophicum. However, in all but a few of the ultrathin sections of this bacterium, the label was randomly distributed in the cell interior. If one assumes a reliable fixation of all cell components, these results would suggest that the two distantly related methanogens studied have distinctive patterns for the localization of component C. The results with M. voltae are in agreement with recent findings that the methylreductase system is involved in the generation of a proton-motive force at the membrane.

Immunoelectron microscopic localization of the restriction endonuclease EcoRI in Escherichia coli BS 5.

Purified restriction endonuclease EcoRI isolated from Escherichia coli BS 5 was used for the production of enzyme-specific IgG antibodies in rabbits. For enzyme localization experiments, paraformaldehyde-glutaraldehyde-fixed cells were embedded and polymerized by a low-temperature procedure using Lowicryl K4M. The immuno electron microscopic protein A-gold technique and an immuno-gold method revealed that 70% of the enzyme-specific labeling were located in the cell envelope whereas 30% were found in the cytoplasm. In metal-shadowed preparations, no indications for the presence of the enzyme could be found on the cell surface; however, on the surface of cell protoplasts enzyme specific labeling could be detected. The results indicate the presence of the major amount of EcoRI in the periplasmic space of the cell where it might be loosely bound or even freely diffusible.