Gert-Wieland Kohring

Studies on dissimilatory sulfate-reducing bacteria that decompose fatty acids

Three strains (2ac9, 3ac10 and 4ac11) of oval to rodshaped, Gram negative, nonsporing sulfate-reducing bacteria were isolated from brackish water and marine mud samples with acetate as sole electron donor. All three strains grew in simple defined media supplemented with biotin and 4-aminobenzoic acid as growth factors. Acetate was the only electron donor utilized by strain 2ac9, while the other two strains used in addition ethanol and/or lactate. Sulfate served as electron acceptor and was reduced to H2S. Complete oxidation of acetate to CO2 was shown by stoichiometric measurements with strain 2ac9 in batch cultures using sulfate, sulfite or thiosulfate as electron acceptors. With sulfate an average growth yield of 4.8 g cell dry weight was obtained per mol of acetate oxidized; with sulfite or thiosulfate the growth yield on acetate was about twice as high. None of the strains contained desulfoviridin. In strain 2ac9 cytochromes of the b- and c-type were detected. Strain 2ac9 is described as type strain of the new species and genus, Desulfobacter postgatei.

Production, purification and characterization of glucose oxidase from a newly isolated strain of Penicillium pinophilum

Abstract A number of nutritional factors influencing growth and glucose oxidase (EC 1.1.3.4) production by a newly isolated strain of Penicillium pinophilum were investigated. The most important factors for glucose oxidase production were the use of sucrose as the carbon source, and growth of the fungus at non-optimal pH 6.5. The enzyme was purified to apparent homogeneity with a yield of 74%, including an efficient extraction step of the mycelium mass at pH 3.0, cation-exchange chromatography and gel filtration. The relative molecular mass (Mr) of native glucose oxidase was determined to be 154 700 ± 4970, and 77 700 for the denatured subunit. Electron-microscopic examinations revealed a sandwich-shaped dimeric molecule with subunit dimensions of 5.0 × 8.0 nm. Glucose oxidase is a glycoprotein that contains tightly bound FAD with an estimated stoichiometry of 1.76 mol/mol enzyme. The enzyme is specific for d-glucose, for which a Km value of 6.2 mM was determined. The pH optimum was determined in the range pH 4.0–6.0. Glucose oxidase showed high stability on storage in sodium citrate (pH 5.0) and in potassium phosphate (pH 6.0), each 100 mM. The half-life of the activity was considerably more than 305 days at 4 °C and 30 °C, and 213 days at 40 °C. The enzyme was unstable at temperatures above 40 °C in the range pH 2.0–4.0 and at a pH above 7.0.

Enhanced hydrogen production from aromatic acids by immobilized cells of Rhodopseudomonas palustris

Cells of the purple non-sulphur bacterium Rhodopseudomonas palustris DSM 131 were immobilized in agar, agarose, κ-carrageenan or sodium alginate gel. With alginate beads, prepared by an emulsion technique, and an optimal cell load of 10 mg dry weight/ml gel, the hydrogen production from aromatic acids was doubled as compared to that resulting from liquid cultures. Hydrogen yields of 60%, 57%, 86% or 88% of the maximal theoretical value were obtained from mandelate, benzoylformate, cinnamate or benzoate respectively. Benzoate concentrations above 16.5 mM were inhibitory. During a period of 55 days the process of hydrogen evolution with immobilized cells was repeated in five cycles with slowly decreasing efficiency.

Durable cofactor immobilization in sol-gel bio-composite thin films for reagentless biosensors and bioreactors using dehydrogenases.

A new strategy directed to the durable immobilization of NAD(+)/NADH cofactors has been tested, along with a suitable redox mediator (ferrocene), in biocompatible sol-gel matrices encapsulating a bi-enzymatic system (a dehydrogenase and a diaphorase, this latter being useful to the safe regeneration of the cofactor), which were deposited as thin films onto glassy carbon electrode surfaces. It involves the chemical attachment of NAD(+) to the silica matrix using glycidoxypropylsilane in the course of the sol-gel process (in smooth chemical conditions). This approach based on chemical bonding of the cofactor (which was checked by infrared spectroscopy) led to good performances in terms of long-term stability of the electrochemical response. The possibility to integrate all components (proteins, cofactor, mediator) in the sol-gel layer in an active and durable form gave rise to reagentless devices with extended operational stability (i.e. high amperometric response maintained for more than 12h of continuous use under constant potential, whereas the signal completely vanished within the first few minutes of working with non-covalently bonded NAD(+)). To confirm the wide applicability of the proposed approach, the same strategy has been applied to the elaboration of biosensors for D-sorbitol, D-glucose and L-lactate with using D-sorbitol dehydrogenase, D-glucose dehydrogenase and L-lactate dehydrogenase respectively. The analytical characteristics of the glucose sensors are given and compared to previous approaches described in the literature for the elaboration of reagentless biosensors.

Hydrogen production from aromatic acids byRhodopseudomonas palustris

The capability of five strains of the phototrophic bacteriumRhodopseudomonas palustris to produce molecular hydrogen (H2) from the aromatic acids benzoate,p-hydroxybenzoate, cinnamate and D- and L-mandelate was investigated. Optimal H2 production was achieved when the strains were grown anaerobically in the light at 10,000 lx under nitrogen (N) limitation using 1 mM L-glutamate as an N source. In the presence of 2 mM benzoate or L-mandelate as carbon and electron sources, strain DSM 131 produced 45% H2 of the maximal theoretical value and strain F2 32%, respectively. Increased H2 production correlated with increased nitrogenase activities, but H2 formation was not further stimulated by inhibition of the H2 uptake (hup) hydrogenase with ethylenediaminetetraacetic acid (EDTA).

A cold active (2R,3R)-(−)-di-O-benzoyl-tartrate hydrolyzing esterase from Rhodotorula mucilaginosa

In a screening procedure a pink-colored yeast was isolated from enrichment cultures with (2R,3R)-(−)-di-O-benzoyl-tartrate (benzoyl-tartrate) as the sole carbon source. The organism saar1 was identified by morphological, physiological, and 18S ribosomal DNA/internal transcribed spacer analysis as Rhodotorula mucilaginosa, a basidiomycetous yeast. During growth the yeast hydrolyzed the dibenzoyl ester stoichiometrically to the monoester using the separated benzoate as the growth substrate, before the monoester was further cleaved into benzoate and tartrate, which were both metabolized. The corresponding benzoyl esterase was purified from the culture supernatant and characterized as a monomeric glycosylated 86-kDa protein with an optimum pH of 7.5 and an optimum temperature of 45 °C. At 0 °C the esterase still exhibited 20% of the corresponding activity at 30 °C, which correlates it to psychrophilic enzymes. The esterase could hydrolyze short chain p-nitrophenyl-alkyl esters and several benzoyl esters like benzoyl-methyl ester, ethylene-glycol-dibenzoyl ester, phenyl-benzoyl ester, cocaine, and 1,5-anhydro-d-fructose-tribenzoyl ester. However feruloyl-ethyl ester was not hydrolyzed. The activity characteristics let the enzyme appear as a promising tool for synthesis of benzoylated compounds for pharmaceutical, cosmetic, or fine chemical applications, even at low temperatures.

Structural Insight into Substrate Differentiation of the Sugar-metabolizing Enzyme Galactitol Dehydrogenase from Rhodobacter sphaeroides D

Galactitol 2-dehydrogenase (GatDH) belongs to the protein superfamily of short-chain dehydrogenases. As an enzyme capable of the stereo- and regioselective modification of carbohydrates, it exhibits a high potential for application in biotechnology as a biocatalyst. We have determined the crystal structure of the binary form of GatDH in complex with its cofactor NAD(H) and of the ternary form in complex with NAD(H) and three different substrates. The active form of GatDH constitutes a homo-tetramer with two magnesium-ion binding sites each formed by two opposing C termini. The catalytic tetrad is formed by Asn116, Ser144, Tyr159, and Lys163. GatDH structurally aligns well with related members of the short-chain dehydrogenase family. The substrate binding pocket can be divided into two parts of different size and polarity. In the smaller part, the side chains of amino acids Ser144, Ser146, and Asn151 are important determinants for the binding specificity and the orientation of (pro-) chiral compounds. The larger part of the pocket is elongated and flanked by polar and non-polar residues, enabling a rather broad substrate spectrum. The presented structures provide valuable information for a rational design of this enzyme to improve its stability against pH, temperature, or solvent concentration and to optimize product yield in bioreactors.

Multiscale-tailored bioelectrode surfaces for optimized catalytic conversion efficiency.

We describe the elaboration of a multiscale-tailored bioelectrocatalytic system. The combination of two enzymes, D-sorbitol dehydrogenase and diaphorase, is studied with respect to the oxidation of D-sorbitol as a model system. The biomolecules are immobilized in an electrodeposited paint (EDP) layer. Reproducible and efficient catalysis of D-sorbitol oxidation is recorded when this system is immobilized on a gold electrode modified by a self-assembled monolayer of 4-carboxy-(2,5,7-trinitro-9-fluorenylidene)malonitrile used as a mediator. The insertion of mediator-modified gold nanoparticles into the EDP film increases significantly the active surface area for the catalytic reaction, which can be further enhanced when the whole system is immobilized in macroporous gold electrodes. This multiscale architecture finally leads to a catalytic device with optimized efficiency for potential use in biosensors, bioelectrosynthesis, and biofuel cells.

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.

8 Immunoelectron Microscopic Localization of Bacterial Enzymes: Pre- and Post-embedding Labelling Techniques on Resin-embedded Samples

Publisher Summary The pre-embedding labeling procedure and the post-embedding labeling method are useful means for the electron microscopic localization of bacterial antigens. The post-embedding procedure is a particularly reliable method for the quantitation of the amount of antigen in a cell. Another advantage is that the antibody has direct access to the antigen exposed by sectioning of the cells; therefore this method does not require permeabilization of membranes and does not encounter problems of restricted diffusion in the cytoplasm of a cell, as does the pre-embedding labelling procedure. The protein A–gold method provides a highly specific and sensitive labeling. Also antiserum raised in a wide variety of mammals can be used. In addition, the preparation of the colloidal gold and the protein A–gold complexes is a simple procedure. The non-covalent nature of the binding of proteins to colloidal gold, without diminishing the activity of the bound proteins, allows for the use of different protein–gold complexes for detecting an antigen or protein in bacterial cells. The gold particles are easily detectable on ultra-thin sections and therefore allow a quantitative estimation of the immunolabel.