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International audience; A bioelectrode for electroenzymatic synthesis was prepared, combining a layer for NADH regeneration and a renewable layer for enzymatic substrate reduction. The covalent immobilization of a rhodium complex mediator ([Cp*Rh(bpy)Cl]+) on the surface of a bucky paper electrode was achieved by following an original protocol in two steps. A bipyridine ligand was first grafted on the electrode by electro‐reduction of bipyridyl diazonium cations generated from 4‐amino‐2,2′‐bipyridine, and the complex was then formed by reaction with [RhCp*Cl2]2. A turnover frequency of 1.3 s−1 was estimated for the electrocatalytic regeneration of NADH by this immobilized complex, with a Faraday efficiency of 83 %. The bucky paper electrode was then overcoated by a bio‐doped porous layer made of glassy fibers with immobilized D‐sorbitol dehydrogenase. This assembly allowed for the efficient separation of the enzyme and the rhodium catalyst, which is a prerequisite for effective bioelectrocatalysis with such bioelectrochemical system, while allowing effective mass transport of NAD+/NADH cofactor from one layer to the other. Thereby, it was possible to reuse the same mediator‐functionalized bucky paper with three different enzyme layers. The bioelectrode was applied to the electroenzymatic reduction of D‐fructose to D‐sorbitol. A turnover frequency of 0.19 s−1 for the rhodium complex was observed in the presence of 3 mM D‐fructose and a total turnover number higher than 12000 was estimated.

International audience; The functional and biological significance of the selected CASP12 targets are described by the authors of the structures. The crystallographers discuss the most interesting structural features of the target proteins and assess whether these features were correctly reproduced in the predictions submitted to the CASP12 experiment.

International audience; Covalent bonding of (2,2′-bipyridyl) (pentamethylcyclopentadienyl)-rhodium complex at the surface of a carbon-based porous electrode was achieved by combining diazonium electrografting, Huisgen cycloaddition, and metal complexation. The immobilized catalyst was applied to electrochemical regeneration of the reduced form of nicotinamide adenine dinucleotide (NADH). The different steps of surface functionalization were characterized by X-ray photoelectron spectroscopy and electrochemistry. The Faradaic efficiency of NADH regeneration was 87%. The chemical bonding provided good stability in solution under convection over 14 days, which is much better than the simple adsorption of the Rh complex on the electrode surface. Finally, the system was tested in the presence of NAD-dependent dehydrogenases that were immobilized in a sol–gel film on the top of the functionalized porous carbon electrode. A total turnover of 3790 and turnover frequency of 164 h–1 was observed. Two enzymatic reactions were considered: d-fructose reduction to d-sorbitol catalyzed by d-sorbitol dehydrogenase and hydroxyacetone reduction to 1,2-propanediol with galactitol dehydrogenase. The electrocatalytic regeneration of 1 mM NADH by the immobilized rhodium species was kept after 90 h electrolysis in the conditions of electroenzymatic synthesis.

International audience; Thiol–ene click chemistry can be exploited for the immobilization of cysteine-tagged dehydrogenases in an active form onto carbon electrodes (glassy carbon and carbon felt). The electrode surfaces have been first modified with vinylphenyl groups by electrochemical reduction of the corresponding diazonium salts generated in situ from 4-vinylaniline. The grafting process has been optimized in order to not hinder the electrochemical regeneration of NAD+/NADH cofactor and soluble mediators such as ferrocenedimethanol and [Cp*Rh(bpy)Cl]+. Having demonstrated the feasibility of thiol–ene click chemistry for attaching ferrocene moieties onto those carbon surfaces, the same approach was then applied to the immobilization of d-sorbitol dehydrogenases with cysteine tag. These proteins can be effectively immobilized (as pointed out by XPS), and the cysteine tag (either 1 or 2 cysteine moieties at the N terminus of the polypeptide chain) was proven to maintain the enzymatic activity of the dehydrogenase upon grafting. The bioelectrode was applied to electroenzymatic enantioselective reduction of d-fructose to d-sorbitol, as a case study.

International audience; The proof-of-concept for enzymatic electrosynthesis with simultaneous energy production has been demonstrated by using the regioselective conversion of D-sorbitol to D-fructose by D-sorbitol dehydrogenase as a model reaction. The bioreactor was composed of two electrodes, a carbon felt bioanode modified by multi-walled carbon nanotubes (MWCNT) containing D-sorbitol dehydrogenase immobilized in a silica matrix and an oxygen gas-flow cathode. The electrochemical regeneration of the NAD+ cofactor at the bioanode was achieved by using a poly(methylene green) mediator electrodeposited on the carbon felt/MWCNT electrode. The conversion rate was 1.18 mg day−1 cm−3 and an energy power output up to 14.6 μW cm−3 at 0.1 V was reached.

Bradyrhizobium japonicumsorbitol dehydrogenase is NADH-dependent and is active at elevated temperatures. The best substrate is D-glucitol (a synonym for D-sorbitol), although L-glucitol is also accepted, giving it particular potential in industrial applications. Crystallization led to a hexagonal crystal form, with crystals diffracting to 2.9 Å resolution. In attempts to phase the data, a molecular-replacement solution based upon PDB entry 4nbu (33% identical in sequence to the target) was found. The solution contained one molecule in the asymmetric unit, but a tetramer similar to that found in other short-chain dehydrogenases, including the search model, could be reconstructed by applying crystallographic symmetry operations. The active site contains electron density consistent with D-glucitol and phosphate, but there was not clear evidence for the binding of NADH. In a search for the features that determine the thermostability of the enzyme, theTmfor the orthologue fromRhodobacter sphaeroides, for which the structure was already known, was also determined, and this enzyme proved to be considerably less thermostable. A continuous β-sheet is formed between two monomers in the tetramer of theB. japonicumenzyme, a feature not generally shared by short-chain dehydrogenases, and which may contribute to thermostability, as may an increased Pro/Gly ratio.

International audience; Gold nanoparticles have been co-encapsulated with D-sorbitol dehydrogenase and diaphorase in electrodeposited thin sol-gel films. We demonstrate here that the nanoparticles allow improving significantly the catalytic efficiency of the bioelectrodes. The hybrid material was electrodeposited in the cavities of macroporous electrodes and characterized by high-resolution transmission electron microscopy and electrochemistry. Well-defined silica gel layers containing homogeneously dispersed nanoparticles were obtained, leading to high current densities in the presence of D-sorbitol. The access to the internal porosity of the macroporous electrode was not hindered by the presence of the electrodeposited sol-gel layer. In addition, a very significant enhancement factor, that describes the current increase with the increasing macroporous surface area was obtained due to the contribution of the tridimensional gold nanoparticle network.

Endogenous galactitol-1-phosphate 5-dehydrogenase (GPDH) (EC 1.1.1.251) from Escherichia coli spontaneously interacts with Ni 2+-NTA matrices becoming a potential contaminant for recombinant, target His-tagged proteins. Purified recombinant, untagged GPDH (rGPDH) converted galactitol into tagatose, and d-tagatose-6-phosphate into galactitol-1-phosphate, in a Zn 2+- and NAD(H)-dependent manner and readily crystallized what has permitted to solve its crystal structure. In contrast, N-terminally His-tagged GPDH was marginally stable and readily aggregated. The structure of rGPDH revealed metal-binding sites characteristic from the medium-chain dehydrogenase/reductase protein superfamily which may explain its ability to interact with immobilized metals. The structure also provides clues on the harmful effects of the N-terminal His-tag. Structured summary of protein interactions: GPDH and GPDH bind by molecular sieving (View interaction) GPDH and GPDH bind by x-ray crystallography (View interaction) GPDH and GPDH bind by cosedimentation in solution (View interaction). © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved., JMM and RM thank the Ministerio de Ciencia e Innovación for their research grantsBFU2010-17929, CSD2006-00015 and AGL2011-22745, CSD2007-00063 FUN-C-FOOD and Comunidad de Madrid S2009/AGR-1469, respectively.

Multilayer electrodes for highly enantioselective sugar conversion are obtained by using monoatomically high platinum islands as pattern for further electrode modification with pyranose-2-oxidase (P2Ox). We describe this kind of electrode for the glucose oxidation. The reaction is monitored via the H2O2 production by the enzyme as side product. As first layer, a self-assembled monolayer (SAM) is arranged on the Au(111) surface via dip coating in a solution of 4-mercaptopyridine. On top of the SAM, a controlled distribution of platinum islands is obtained by a combination of electroless and electrochemical deposition using a solution of K2PtCl4. The platinum islands have a diameter of about 6 nm. Thereon, poly(allylamine hydrochloride) (PAA) obtained by dip coating provides a linker for immobilization of the enzyme pyranose-2-oxidase. Our assumption is that only one enzyme molecule is bound to one platinum island. This well ordered surface structure is probably the reason for the very high conversion efficiency.

A supplemental cysteine-tag modification of the enzyme galactitol dehydrogenase (GatDH), expressed in Escherichia coli has enabled a directed and covalent immobilisation of the protein on gold surfaces without secondary linkers. The successful enzyme immobilisation was verified by SPR measurements, and the activity of the immobilized GatDH was demonstrated by cyclic voltammetry (CV) while NAD+ and the redox mediator 4-carboxy-2,5,7-trinitrofluorenyliden-malonnitrile (CTFM) were both kept in solution. The results represent a proof of concept for the development of electrochemical reactors for the generation of enantiopure fine chemicals as building blocks for pharmaceutical products.

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.

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 μM).

Sorbitol dehydrogenase (SDH) originating from recombinant Escherichia coli cells is immobilized on gold electrodes. First of all, (4-carboxy-2,5,7-trinitrofluorenyliden)malon-nitrile (CTFM) is adsorbed on the surface as mediator. In a second step, the cofactor β-nicotinamide adenine dinucleotide (NAD+) is immobilized on the gold electrode. Due to the formation of a complex between the mediator and the cofactor, the electron transfer rate can be enhanced by adding calcium ions to the buffer. The immobilization of NAD+ and SDH on the surface has been achieved by cross-linking with the glutaraldehyde/bovine serum albumin system. The successful biofunctionalization is monitored by cyclic voltammetry.

A pure culture of 2,4-dichlorophenol (2,4-DCP)-degrading bacteria was isolated from a natural enrichment that had been adapted to chlorophenols in the aeration pond of the Baikalsk pulp and paper mill (Russia). The bacteria were identified by 16S rDNA intergenic region analysis, using PCR with universal primers. Comparative analysis of the 16S rDNA sequence (1545 bp) in the GenBank database revealed that these bacteria are related to Bacillus cereus GN1. Degradation of 2,4-DCP was studied using this culture in liquid medium under aerobic conditions, at initial concentrations of 20–560mM 2,4-DCP. The 2,4-DCP degradation rates by B. cereus GN1 could be determined at concentrations up to 400mM. However, higher concentrations of 2,4-DCP (560mM) were inhibitory to cell growth. r 2006 Elsevier Ltd. All rights reserved.

International audience; A gene in Bradyrhizobium japonicum USDA 110, annotated as a ribitol dehydrogenase (RDH), had 87 % sequence identity (97 % positives) to the N-terminal 31 amino acids of an l-glucitol dehydrogenase from Stenotrophomonas maltophilia DSMZ 14322. The 729-bp long RDH gene coded for a protein consisting of 242 amino acids with a molecular mass of 26.1 kDa. The heterologously expressed protein not only exhibited the main enantio selective activity with d-glucitol oxidation to d-fructose but also converted l-glucitol to d-sorbose with enzymatic cofactor regeneration and a yield of 90 %. The temperature stability and the apparent K (m) value for l-glucitol oxidation let the enzyme appear as a promising subject for further improvement by enzyme evolution. We propose to rename the enzyme from the annotated RDH gene (locus tag bll6662) from B. japonicum USDA as a d-sorbitol dehydrogenase (EC 1.1.1.14).

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 (M r) 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 K m 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.

1,5-Anhydro-d-fructose (1,5-AF) is an interesting building block for enantioselective and stereoselective organic synthesis. Enzymes acting on this compound are potential targets for structure-based protein/enzyme design to extend the repertoire of catalytic modifications of this and related building blocks. Recombinant 1,5-anhydro-d-fructose reductase (AFR) from Sinorhizobium meliloti 1021 was produced in Escherichia coli, purified using a fused 6×His affinity tag and crystallized in complex with the cofactor NADP(H) using the hanging-drop technique. Its structure was determined to 1.93 A resolution using molecular replacement. The structure displays an empty substrate-binding site and can be interpreted as an open conformation reflecting the enzyme state shortly after the release of product, presumably with bound oxidized cofactor NADP+. Docking simulations indicated that amino-acid residues Lys94, His151, Trp162, Arg163, Asp176 and His180 are involved in substrate binding, catalysis or product release. The side chain of Lys94 seems to have the ability to function as a molecular switch. The crystal structure helps to characterize the interface relevant for dimer formation as observed in solution. The crystal structure is compared with the structure of the homologue from S. morelense, which was solved in a closed conformation and for which dimer formation in solution could not be verified but seems to be likely based on the presented studies of S. meliloti AFR.

International audience; A reagentless D-sorbitol biosensor based on NAD-dependent D-sorbitol dehydrogenase (DSDH) immobilized in a sol-gel carbon nanotubes-poly(methylene green) composite has been developed. It was prepared by durably immobilizing the NAD(+) cofactor with DSDH in a sol-gel thin film on the surface of carbon nanotubes functionalized with poly(methylene green). This device enables selective determination of D-sorbitol at 0.2 V with a sensitivity of 8.7 mu Ammol(-1)Lcm(-2) and a detection limit of 0.11 mmolL(-1). Moreover, this biosensor has excellent operational stability upon continuous use in hydrodynamic conditions.

This review describes the input of sol-gel chemistry to the immobilization of polyol dehydrogenases on electrodes, for applications in bioelectrocatalysis. The polyol dehydrogenases are described and their application for biosensing, biofuel cell and electrosynthesis are briefly discussed. The immobilization of proteins via sol-gel approaches is described, including a discussion on the difficulty to maintain the activity of proteins in a silica matrix and the strategies developed to offer a proper environment to the proteins by developing optimal organic-inorganic hybrid materials. Finally, the co-immobilization of the NAD + co-factor and of mediators for the elaboration of reagentless devices is presented, based on published and original data. All-in-all, sol-gel approaches appear to be a very promising for development of original electrochemical applications involving dehydrogenases in near future.

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.

International audience; 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 12 h 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.

International audience; Multiwalled carbon nanotubes (MWCNT) have been functionalized, for the electrocatalytic detection of NADH, by microwave treatment, electrochemical deposition of poly(methylene green) or wrapping with an Os‐complex modified polymer. Sol‐gel thin films have been then electrodeposited on the carbon nanotube layers for co‐immobilization of D‐sorbitol dehydrogenase and diaphorase when necessary and NAD+ via covalent linkage using glycidoxypropyltrimethoxysilane. The comparison of these systems shows that the electrodeposited sol‐gel matrix can significantly affect the operational behavior of functionalized MWCNT. Only MWCNT wrapped with the Os‐complex modified polymer and covered with a sol‐gel biocomposite allowed the electrochemical detection of D‐sorbitol in a reagentless configuration.

International audience; We report here that the electrochemically assisted deposition (EAD) of silica thin films can be a good strategy to co-encapsulate d-sorbitol dehydrogenase (DSDH) and diaphorase in an active form. This is achieved via the electrolysis of a hydrolyzed sol containing the biomolecules to initiate the poly-condensation of silica precursors upon electrochemically induced pH increase at the electrode/solution interface. DSDH was found to be very sensitive to the silica gel environment and the addition of a positively-charged polyelectrolyte was necessary to ensure effective operational behavior of the biomolecules. The composition of the sol and the conditions for electrolysis have been optimized with respect to the intensity of the electrochemical response to d-sorbitol oxidation. The Km of DSDH in the electrodeposited film was in the range of 3 mM, slightly better than the value determined biochemically in solution (6.5 mM). The co-immobilization of DSDH and diaphorase in this way led on the one hand to the possible reduction of NAD+ to NADH (simultaneously to d-sorbitol oxidation) and on the other hand to the safe re-oxidation of the co-factor using a mediator (ferrocenedimethanol) as electron relay. The bioelectrocatalytic response looks promising for electro-enzymatic applications. To support this idea, the EAD of sol–gel bio-composite has been extended to macroporous electrodes displaying a much bigger electroactive surface area.

International audience; We report the electrochemical cofactor regeneration for electroenzymatic reduction as a promising alternative to the enzymatic recycling due to the avoidance of couple products. In the mediated electron transfer reaction appropriate redox mediators for the cofactor reduction are necessary. We have synthesized different redox mediators-based on rhodium complexes-and have screened their electrochemical performance at different pH values and concentrations. Suitable mediators have been tested in the presence of the cofactor NAD+ and then additionally in the presence of D-sorbitol dehydrogenase (DSDH) from Rhodobacter sphaeroides. The electroenzymatically generated product was analyzed by HPLC. As result of the screening some mediator species were identified which enable an efficient electrochemical cofactor reduction.

International audience; The influence of polyelectrolyte (PE) additives on the sol‐gel encapsulation of dehydrogenases has been evaluated using D‐sorbitol dehydrogenase (DSDH) as a model enzyme. Thin sol‐gel films were prepared on glassy carbon electrodes (GCE), which were then evaluated with respect to the electrochemical detection of D ‐sorbitol. DSDH encapsulation in pure silica thin films resulted in undetectable electrochemical signal. Adding positively‐charged PE that contributes to stabilize the negatively‐charged DSDH in the biocomposite film led to much faster and higher response of DSDH to its substrate, as shown by the significant improvement in bioelectrochemical detection of NADH produced during the oxidation of D‐sorbitol.

Rapid immobilization with the one-pot purification of galactitol dehydrogenase (GatDH) and formate dehydrogenase (FDH) is achieved by using iminodiacetic acid (IDA) with chelated Co2+ modified magnetic nanoparticles as a carrier. Lactate dehydrogenase (LDH) from recombinant Escherichia coli and FDH commencing Candida methylica were used as an auxiliary enzyme for the regeneration of NADH/NAD(+) with a representative synthesis of (S)-1,2-propanediol and L-tagatose starting from hydroxyacetone and galactitol. The affinity magnetic nanoparticles were characterized by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR), while the purity of GatDH and FDH was assayed by SDS-PAGE analysis. The immobilized two-enzyme system, reflecting the pH dependence of its constituent enzymes, showed optimal activity at pH 7 and 8 for (S)-1,2-propanediol and L-tagatose production, respectively. The immobilized enzyme system retained up to 70% of its activity after one week of repeated use. The use of affinity magnetic nanoparticles offers the advantage of a one-pot purification of His(6)-tagged GatDH and FDH followed by the production of rare sugar and chiral diol. (C) 2011 Elsevier B.V. All rights reserved.

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 Asn(116), Ser(144), Tyr(159), and Lys(163). 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 Ser(144), Ser(146), and Asn(151) 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.

Dehydrogenases represent a valuable tool in biotechnology for the production of fine chemicals and reactive compounds and as the building blocks of pharmaceutical and agrochemical active substances. Their activity in regiospecific and enantioselective hydroxyl group oxidation and ketone reduction has attracted increasing interest with respect to production processes, design of biosensors, and construction of biofuel cells. However, the electron transfer cofactors necessary for their enzymatic activity are usually high priced and, therefore, have to be regenerated in the corresponding processes. In the last few decades, multiple regeneration procedures have been developed for different groups of dehydrogenases with application potential; for example, whole cell systems, cofactor regeneration with a second substrate and/or a second enzyme, and photochemical or electrochemical methods. Here, we discuss the different systems with focus on electrochemical restoration of redox equivalents. Keywords: dehydrogenase; stereoselectivity; regioselectivity; redox reaction; cofactor regeneration; NAD(P)+; NAD(P)H2; electrochemistry

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A new method is described for immobilisation of enzymes on polymer-coated Pt islands. These islands are deposited on top of a SAM-covered Au(111) electrode by a combination of electroless and electrochemical deposition, which allows for a variation of island size and distance between the islands. Here we describe the immobilisation of pyranose-2-oxidase (P2Ox) and the catalytic response to D-glucose on such a nanopatterned surface, which provides optimum access to the active centres of the enzyme.

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Summary The life cycle of a new microsporidian, Pleistophora tanzaniae n. sp. parasitizing natural population of rhinoceros beetle, Oryctes monoceros OLIV. in Tanzania is described by means of lightand electron microscopy. The midgut epithelium was the usual site of infection of the parasite. Some data on the ultrastructure of spore are given. The membraneous structure of the polaroplast in ultrathin sections is discussed.

Gliding motility, ultrastructure and nutrition of two newly isolated filamentous sulfate-reducing bacteria, strains 5ac10 and 4be13, were investigated. The filaments were always attached to surfaces. Growth was supported by addition of insoluble aluminium phosphate or agar as substrata for gliding movement. Electron microscopy of ultrathin sections revealed cell walls characteristic of Gramnegative bacteria; the undulated structure of the outer membrane may pertain to the translocation mechanism. Intracytoplasmic membranes were present. Acetate, higher fatty acids, succinate or fumarate served as electron donors and carbon sources. Strain 5ac10 grew also with lactate, but not with benzoate that was used only by strain 4be13. Strain 5ac10 was able to grow slowly on H2 plus CO2 or formate in the presence of sulfate without additional organic carbon source. The capacity of complete oxidation was shown by stoichiometric measurements with acetate plus sulfate. Both strains contained b- and c-type cytochromes. Desulfoviridin was detected only in strain 5ac10. The two filamentous gliding sulfate reducers are described as new species of a new genus, Desulfonema limicola and Desulfonema magnum.

A novel temperate bacteriophage, designated ΦRsG1, was isolated from Rhodobacter sphaeroides Y (previously designated Rhodopseudomonas sphaeroides) following exposure to mitomycin C. The phage morphology, as revealed from electron microscopy, showed a hexagonal head (90 by 46.5 nm) connected with a tail (116 by 9.4 nm), to which a collar was proximally attached. A morphologically similar phage was also produced by spontaneous lysis of the cells. While ΦRsG1 did not grow on any other bacterial strain tested, spontaneously produced phage particles propagated (and formed plaques) on R. sphaeroides Y still carrying ΦRsG1 in the prophage state. The genome of ΦRsG1 consisted of double stranded linear DNA with cohesive ends and a GC-content of 71.8 mol%. The DNA molecules formed circles in vitro with a mean contour length of 17.18±0.4 μm, which corresponds to a size of 49 kbase pairs (kb). On the other hand, DNA extracted from the virulent phage particles was heterogeneous and consisted of two DNA species of different size, occurring in a ratio of about 1:1. These molecules also circularized having contour lengths of 17.18±0.4 μm and 14.02±0.41 μm corresponding to 49 and 40 kb, respectively. Restriction digest analysis of the two DNA species and DNA from ΦRsG1 indicated that they are similar, and allowed the indentification of an 11.5 kb EcoRI fragment that carries the cohesive ends. Because DNA from ΦRsG1 and the 49 kb DNA of the virulent phage particles were indistinguishable with the criteria applied, it is suggested that phage particles containing the 40 kb DNA represent the virulent type of phage, termed ΦRsG1.1.

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.

Guanine nucleotides are intimately involved in a variety of regulatory functions, in particular in protein synthesis (1) and signal transduction (2, 3). Common to these functions is that activation of the target proteins occurs by binding of GTP, while deactivation is achieved by GTP hydrolysis. Following GDP dissociation from the guanine nucleotide binding protein reactivation is initiated by GTP binding. Interference with this cycle of events leads to a major disturbance of the metabolic reactions involved.

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.