Enzyme-based biofuel cells

Enzyme-based biofuel cells possess several positive attributes for energy conversion, including renewable catalysts, flexibility of fuels (including renewables), and the ability to operate at room temperature. However, enzyme-based biofuel cells remain limited by short lifetimes, low power densities and inefficient oxidation of fuels. Recent advances in biofuel cell technology have addressed these deficiencies and include methods to increase lifetime and environmental stability.     

Electron transfer in quinoproteins

Soluble quinoprotein dehydrogenases oxidize a wide range of sugar, alcohol, amine, and aldehyde substrates. The physiological electron acceptors for these enzymes are not pyridine nucleotides but are other soluble redox proteins. This makes these enzymes and their electron acceptors excellent systems with which to study mechanisms of long-range interprotein electron transfer reactions. The tryptophan tryptophylquinone (TTQ)-dependent methylamine dehydrogenase (MADH) transfers electrons to a blue copper protein, amicyanin. It has been possible to alter the rate of electron transfer by using different redox forms of MADH, varying reaction conditions, and performing site-directed mutagenesis on these proteins. From kinetic and thermodynamic analyses of the reaction rates, it was possible to determine whether a change in rate is due a change in Delta G(0), electronic coupling, reorganization energy or kinetic mechanism. Examples of each of these cases are discussed in the context of the known crystal structures of the electron transfer protein complexes. The pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase transfers electrons to a c-type cytochrome. Kinetic and thermodynamic analyses of this reaction indicated that this electron transfer reaction was conformationally coupled. Quinohemoproteins possess a quinone cofactor as well as one or more c-type hemes within the same protein. The structures of a PQQ-dependent quinohemoprotein alcohol dehydrogenase and a TTQ-dependent quinohemoprotein amine dehydrogenase are described with respect to their roles in intramolecular and intermolecular protein electron transfer reactions.     

Development of nanostructured bioanodes containing dendrimers and dehydrogenases enzymes for application in ethanol biofuel cells

This paper describes the use of the electrostatic layer-by-layer (LbL) technique for the preparation of bioanodes with potential application in ethanol/O(2) biofuel cells. More specifically, the LbL technique was employed for immobilization of dehydrogenase enzymes and polyamidoamine (PAMAM) dendrimers onto carbon paper support. Both mono (anchoring only the enzyme alcohol dehydrogenase, ADH) and bi-enzymatic (anchoring both ADH and aldehyde dehydrogenase, AldDH) systems were tested. The amount of ADH deposited onto the Toray? paper was 95 ng cm(-2) per bilayer. Kinetic studies revealed that the LbL technique enables better control of enzyme disposition on the bioanode, as compared with the results obtained with the bioanodes prepared by the passive adsorption technique. The power density values achieved for the mono-enzymatic system as a function of the enzyme load ranged from 0.02 to 0.063 mW cm(-2) for the bioanode containing 36 ADH bilayers. The bioanodes containing a gas diffusion layer (GDL) displayed enhanced performance, but their mechanical stability must be improved. The bi-enzymatic system generated a power density of 0.12 mW cm(-2). In conclusion, the LbL technique is a very attractive approach for enzyme immobilization onto carbon platform, since it enables strict control of enzyme disposition on the bioanode surface with very low enzyme consumption.     

New quinoproteins in oxidative fermentation

Several quinoproteins have been newly indicated in acetic acid bacteria, all of which can be applied to fermentative or enzymatic production of useful materials by means of oxidative fermentation. (1) D-Arabitol dehydrogenase from Gluconobacter suboxydans IFO 3257 was purified from the bacterial membrane and found to be a versatile enzyme for oxidation of various substrates to the corresponding oxidation products. It is worthy of notice that the enzyme catalyzes D-gluconate oxidation to 5-keto-D-gluconate, whereas 2-keto-D-gluconate is produced by a flavoprotein D-gluconate dehydrogenase. (2) Membrane-bound cyclic alcohol dehydrogenase was solubilized and purified for the first time from Gluconobacter frateurii CHM 9. When compared with the cytosolic NAD-dependent cyclic alcohol dehydrogenase crystallized from the same strain, the reaction rate in cyclic alcohol oxidation by the membrane enzyme was 100 times stronger than the cytosolic NAD-dependent enzyme. The NAD-dependent enzyme makes no contribution to cyclic alcohol oxidation but contributes to the reduction of cyclic ketones to cyclic alcohols. (3) Meso-erythritol dehydrogenase has been purified from the membrane fraction of G. frateurii CHM 43. The typical properties of quinoproteins were indicated in many respects with the enzyme. It was found that the enzyme, growing cells and also the resting cells of the organism are very effective in producing L-erythrulose. Dihydroxyacetone can be replaced by L-erythrulose for cosmetics for those who are sensitive to dihydroxyacetone. (4) Two different membrane-bound D-sorbitol dehydrogenases were indicated in acetic acid bacteria. One enzyme contributing to L-sorbose production has been identified to be a quinoprotein, while another FAD-containing D-sorbitol dehydrogenase catalyzes D-sorbitol oxidation to D-fructose. D-Fructose production by the oxidative fermentation would be possible by the latter enzyme and it is superior to the well-established D-glucose isomerase, because the oxidative fermentation catalyzes irreversible one-way oxidation of D-sorbitol to D-fructose without any reaction equilibrium, unlike D-glucose isomerase. (5) Quinate dehydrogenase was found in several Gluconobacter strains and other aerobic bacteria like Pseudomonas and Acinetobacter strains. It has become possible to produce dehydroquinate, dehydroshikimate, and shikimate by oxidative fermentation. Quinate dehydrogenase was readily solubilized from the membrane fraction by alkylglucoside in the presence of 0.1 M KCl. A simple purification by hydrophobic chromatography gave a highly purified quinate dehydrogenase that was monodispersed and showed sufficient purity. When quinate dehydrogenase purification was done with Acinetobacter calcoaceticus AC3, which is unable to synthesize PQQ, purified inactive apo-quinate dehydrogenase appeared to be a dimer and it was converted to the monomeric active holo-quinate dehydrogenase by the addition of PQQ.     

Challenges in biocatalysis for enzyme-based biofuel cells

Enzyme-based biofuel cells are attracting attention rapidly partially due to the promising advances reported recently. However, there are issues to be addressed before biofuel cells become competitive in practical applications. Two critical issues are short lifetime and poor power density, both of which are related to enzyme stability, electron transfer rate, and enzyme loading. Recent progress in nanobiocatalysis opens the possibility to improve in these aspects. Many nano-structured materials, such as mesoporous media, nanoparticles, nanofibers, and nanotubes, have been demonstrated as efficient hosts of enzyme immobilization. It is evident that, when nanostructure of conductive materials are used, the large surface area of these nanomaterials can increase the enzyme loading and facilitate reaction kinetics, and thus improving the power density of biofuel cells. In addition, research efforts have also been made to improve the activity and stability of immobilized enzymes by using nanostructures. It appears to be reasonable to us to expect that progress in nanostuctured biocatalysts will play a critical role in overcoming the major obstacles in the development of powerful biofuel cells.     

Cross-linking in adhesive quinoproteins: studies with model decapeptides

Mytilus edulis foot protein-1 (mefp1) is a major component of the byssus, an adhesive holdfast in mussels. The recent report of 5, 5'-di(dihydroxyphenyl-L-alanine) (diDOPA) cross-links in byssus [McDowell et al. (1999) J. Biol. Chem. 274, 20293] has raised questions about the relationship of these to mefp1. About 80% of the primary structure of mefp1 consists of a tandemly repeated consensus sequence Ala(1)-Lys(2)-Pro(3)-Ser(4)-Tyr(5)-Pro(6)-Pro(7)-Thr(8)-Tyr(9)-Lys(10 ) with varying degrees of posttranslational hydroxylation to hydroxyprolines in positions 3, 6, and 7 and to DOPA in positions 5 and 9. Six natural or synthetic variants of this decapeptide were subjected to oxidation by tyrosinase or periodate. DOPA is the only residue to suffer losses in all oxidized peptides. Moreover, using MALDI TOF mass spectrometry, oxidized decapeptides all showed evidence of multimer formation and a mass loss of 6 Da per coupled pair of peptides. Multimer formation was inhibited by addition of DOPA-like o-diphenols, but addition of simple amines such as free Lys had no effect. The results are consistent with aryloxy coupling to diDOPA followed by reoxidation to diDOPA quinone. There are subtle but noteworthy variations, however, in multimer formation among the peptide congeners. Decapeptides with Pro(3) modified to trans-4-hydroxyproline do not form multimers beyond dimers; they also exhibit significant Lys losses following oxidation of DOPA. Moreover, in Ala-Lys-Hyp-Ser-Tyr-DiHyp-Hyp-Thr-DOPA-Lys, Tyr appears to be protected from oxidation by tyrosinase.     

The role of PQQ and quinoproteins in methylotrophic bacteria

Abstract Quinoprotein dehydrogenases play a non-exclusive role in the dissimilation of C₁ compounds. Methanol and methylamine oxidation occur by covalent catalysis while the reduction equivalents are transferred to the respiratory chain in one-electron steps. Cytochrome c _L is an excellent electron acceptor for methanol dehydrogenase at pH 7.0 and a bad one at pH 9.0. Efficient methanol oxidation (with NH₃ as activator) occurs at pH 9.0, but (due to the failure of NH₃) not at pH 7.0. Since stimulation occurred at the latter condition with a compound prepared from Hyphomicrobium X, most probably methanol oxidation in vivo requires the presence of a natural activator. The finding of pro-PQQ in methylamine dehydrogenase implicates that certain quinoproteins may have a modified tyrosine as cofactor. This type of quinoprotein is involved in assimilation routes which also occur in methylotrophs. l -Tyrosine and l -glutamate are the precursors of PQQ biosynthesis. Free intermediates in the route of biosynthesis have not been found. Most probably the whole process occurs on a protein matrix. In view of the significant amounts found in their culture fluid, methylotrophic bacteria seem particularly well suited for the fermentative production of PQQ.     

Effect of salts on luminescence of natural and recombinant luminescent bacterial biosensors

Effect of cations K+, Na+, Mg2+, and Ca2+ and anions SO4(2-), HCO3(-), and CO3(2-) on the luminescence intensity of the marine luminescent bacterium Photobacterium phorphoreum (Microbiosensor B-17 677f) and the recombinant strain Escherichia coli with cloned lux operon of P. leiognathi (Ekolyum-9). It is found that small concentrations of chlorides and sulfates of the cations studied had a concentration-dependent stimulatory effect on bacterial bioluminescence; as the concentration of agents increased, activation was succeeded by quenching. The strength of the inhibitory effect, which is characterized by EC50, decreased in the series Ca2+ > Na+ > Mg2+ > K+. Carbonates and hydrocarbonates had a pronounced inhibitory effect on the bioluminescence intensity, determined by an increase in pH. We showed that some types of highly mineralized water with a high hydrocarbonate content have a marked inhibitory effect on the luminescence intensity of microbial luminescent biosensors, mimicking the effect of chemical pollutants.     

Effect of salts on luminescence of natural and recombinant luminescent bacterial biosensors

Effect of cations K⁺, Na⁺, Mg²⁺, and Ca²⁺ and anions Cl^?, SO ₄ ^2? , HCO ₃ ^? , and CO ₃ ^2? on the luminescence intensity of the marine luminescent bacterium Photobacterium phorphoreum (Microbiosensor B-17 677f) and the recombinant strain Escherichia coli with cloned lux operon of P. leiognathi (Ecolum-9). It is found that small concentrations of chlorides and sulfates of the cations studied had a concentration-dependent stimulatory effect on bacterial bioluminescence; as the concentration of agents increased, activation was succeeded by quenching. The strength of the inhibitory effect, which is characterized by EC₅₀, decreased in the series Ca²⁺ > Na⁺ > Mg²⁺ > K⁺. Carbonates and hydrocarbonates had a pronounced inhibitory effect on the bioluminescence intensity, determined by an increase in pH. We showed that some types of highly mineralized water with a high hydrocarbonate content have a marked inhibitory effect on the luminescence intensity of microbial luminescent biosensors, mimicking the effect of chemical pollutants.     

Fibre-optic bacterial biosensors and their application for the analysis of bioavailable Hg and As in soils and sediments from Aznalcollar mining area in Spain

Fibre-optic biosensors for Hg and As were developed by attaching alginate-immobilised recombinant luminescent Hg- and As-sensor bacteria onto optical fibres. The optimised biosensors (consisting of seven layers of fibre-attached bacteria pre-grown till mid-logarithmic growth phase) enabled quantification of environmentally relevant concentrations of the target analytes: 2.6 microg l-1 of Hg(II) and 141 microg l-1 of As(V) or 18 microg l-1 of As(III). The highest viability and sensitivity for target analyte was obtained when fibre tips were stored in CaCl2 solution at -80 degrees C. Applicability of the fibre-optic biosensors in parallel to the respective non-immobilised sensors was assessed on 10 natural soil and sediment samples from Aznalcollar mining area (Spain). On the average 0.2% of the total Hg and 0.87% of the total As proved bioavailable to fibre-attached bacteria. Interestingly, about 20-fold more Hg and 4-fold more As was available to non-immobilised sensor bacteria indicating the importance of direct cell contact (possible only for non-immobilised cells) for enhanced bioavailability of these metals in solid samples.     

Two novel bacterial biosensors for detection of nitrate availability in the rhizosphere

The nitrate-regulated promoter of narG in Escherichia coli was fused to promoterless ice nucleation (inaZ) and green fluorescent protein (GFP) reporter genes to yield the nitrate-responsive gene fusions in plasmids pNice and pNgfp, respectively. While the promoter of narG is normally nitrate responsive only under anaerobic conditions, the L28H-fnr gene was provided in trans to enable nitrate-dependent expression of these reporter gene fusions even under aerobic conditions in both E. coli DH5alpha and Enterobacter cloacae EcCT501R. E. cloacae and E. coli cells containing the fusion plasmid pNice exhibited more than 100-fold-higher ice nucleation activity in cultures amended with 10 mM sodium nitrate than in nitrate-free media. The GFP fluorescence of E. cloacae cells harboring pNgfp was uniform at a given concentration of nitrate and increased about 1,000-fold when nitrate increased from 0 to 1 mM. Measurable induction of ice nucleation in E. cloacae EcCT501R harboring pNice occurred at nitrate concentrations of as low as 0.1 microM, while GFP fluorescence was detected in cells harboring pNgfp at about 10 microM. In the rhizosphere of wild oat (Avena fatua), the whole-cell bioreporter E.cloacae(pNgfp) or E. cloacae(pNice) expressed significantly higher GFP fluorescence or ice nucleation activity when the plants were grown in natural soils amended with nitrate than in unamended natural soils. Significantly lower nitrate abundance was detected by the E. cloacae(pNgfp) reporter in the A. fatua rhizosphere compared to in bulk soil, indicating plant competition for nitrate. Ice- and GFP-based bacterial sensors thus are useful for estimating nitrate availability in relevant microbial niches in natural environments.     

The application of methacrylate-based polymers to enzyme biosensors

Enzyme electrodes based on methacrylates have received significant attention in the development of biosensors. This article reviews the use and application of methacrylate and its derivatives as an immobilization system for the preparation of enzyme electrodes. Resent examples, extracted from the literature, illustrate the superior performance of such materials in the fabrication of biosensors and bioreactors.     

Detection of quorum-sensing N-acyl homoserine lactone signal molecules by bacterial biosensors

Many Gram-negative bacteria use N-acyl homoserine lactones (AHLs) as quorum-sensing (QS) signal molecules. AHL QS has been the subject of extensive investigation in the last decade and has become a paradigm for bacterial intercellular signaling. Research in AHL QS has been considerably aided by simple methods devised to detect AHLs using bacterial biosensors that phenotypically respond when exposed to exogenous AHLs. This article reviews and discusses the currently available bacterial biosensors which can be used in detecting and studying the different AHLs.     

Immobilization as a technical possibility for long-term storage of bacterial biosensors

For applications in field experiments, the recombinant strain Salmonella typhimurium TA1535 was immobilized to permit its immediate utilization after long storage periods. Salmonella typhimurium TA1535 cells contain the plasmid that has an inducible SOS promoter fused to a promoterless luxCDABFE operon from Photobacterium leiognathi. The induction of bioluminescence occurs in the presence of the DNA-damaging agent mitomycin C which stimulates the bacterial SOS response. Early stationary phase cells were immobilized at a cell concentration of 10(10) CFU/ml in microtiter plates and stored up to 6 weeks at 4 degrees C in a sealed container. Even after 4 weeks of storage, the bioluminescence kinetics and yield in response to different concentrations of mitomycin C were not significantly different from those of freshly prepared samples.     

Agarose-gel-immobilized recombinant bacterial biosensors for simple and disposable on-site detection of phenolic compounds

In this study, recombinant bacterial biosensors were immobilized in an agarose matrix and used for the simple and disposable field monitoring of phenolic compounds. In brief, Escherichia coli cells harboring the pLZCapR plasmid, which was previously designed to express the β-galactosidase reporter gene in the presence of phenolic compounds, were immobilized in agarose gel with or without a substrate [chlorophenol red β-galactopyranoside (CPRG)] and dispensed to the wells of a 96-well plate. Analytes were added to the wells, and color development was monitored either directly from wells containing intact cells co-immobilized with CPRG (SYS I), or using cells that were lysed prior to the addition of CPRG (SYS L). SYS L showed relatively higher intensities and faster color development than SYS I. However, both systems developed a red color (representing hydrolysis of CPRG) in the presence of 10 μM to 10~100 mM phenol, with maximum responses seen at 1~5 and 50 mM phenol for SYS I and SYS L, respectively. Other phenolic compounds (2-chlorophenol, 2-methylphenol, 3-methylphenol, 4-chlorophenol, 2-nitrophenol, resorcinol, catechol, and 2,5-dimethylphenol) were also detected by the systems, with varied detection ranges and responses. The agarose-immobilized biosensors were stable for 28 days, retaining 39~69% of their activities when stored at 4°C without nutrients or additives. The immobilized biosensors described herein do not require the on-site addition of a substrate (in the case of SYS I), the pretreatment of samples, or the use of unwieldy instruments for the on-site monitoring of phenolic compounds from environmental samples.     

Design and application of highly responsive fluorescence resonance energy transfer biosensors for detection of sugar in living Saccharomyces cerevisiae cells

A protein sensor with a highly responsive fluorescence resonance energy transfer (FRET) signal for sensing sugars in living Saccharomyces cerevisiae cells was developed by combinatorial engineering of the domain linker and the binding protein moiety. Although FRET sensors based on microbial binding proteins have previously been created for visualizing various sugars in vivo, such sensors are limited due to a weak signal intensity and a narrow dynamic range. In the present study, the length and composition of the linker moiety of a FRET-based sensor consisting of CFP-linker(1)-maltose-binding protein-linker(2)-YFP were redesigned, which resulted in a 10-fold-higher signal intensity. Molecular modeling of the composite linker moieties, including the connecting peptide and terminal regions of the flanking proteins, suggested that an ordered helical structure was preferable for tighter coupling of the conformational change of the binding proteins to the FRET response. When the binding site residue Trp62 of the maltose-binding protein was diversified by saturation mutagenesis, the Leu mutant exhibited an increased binding constant (82 microM) accompanied by further improvement in the signal intensity. Finally, the maltose sensor with optimized linkers was redesigned to create a sugar sensor with a new specificity and a wide dynamic range. When the optimized maltose sensors were employed as in vivo sensors, highly responsive FRET images were generated from real-time analysis of maltose uptake of Saccharomyces cerevisiae (baker's yeast).