Biocatalysts For Oxidation Research Articles

H2 Production from Biomass for H2/O2 Biofuel Cells

Fuel cells using renewable resources are attractive devices able to take up the energy challenges launched by fossil fuel exhaustion. Hydrogen can be produced from biomass and biocatalysts can replace noble metals such as platinum. Hydrogenase enzymes arise as particularly efficient biocatalysts for H2 oxidation. However, bottlenecks must be overcome before the development of these biofuel cells: 1- biohydrogen production is rate limited, 2- many hydrogenases are inhibited by oxygen and CO, 3- the size of the enzymes limits their electrical connection, hence the current densities. This work will discuss some solutions to get through these limitations, such as controlled bacterium consortia to determine the biophysical parameters able to enhance bioproduction of H2, and the efficient immobilization of an O2-tolerant hydrogenase which allows the development of a new generation of H2/O2 biofuel cell.A multidisciplinary approach including microbiology, microscopies and electrochemistry was used for the study of a synthetic microbial consortium in view of the optimization of biohydrogen production. Electrochemical tools, associated to the full characterization of carbon materials, were used to design a H2/O2 biofuel cell.Numerous microorganisms can produce hydrogen by “dark fermentation”. Literature essentially reports the use of pure microbial cultures to produce biohydrogen from simple sugars. In this work one bacterium from Clostridium (H2 producer) and one from Desulfovibrio (H2 producer or consumer depending on its environment) have been studied as pure and mixed cultures. We demonstrate in this work (i) a physical interaction between the strains via nanotubes, (ii) an exchange of cytoplasmic constituents, (iii) a higher hydrogen production by the consortium compared to isolated bacteria. A modification in the metabolic pathway is proposed that can explain such results.1 Besides, screening energetic pathways in exotic micro-organisms lead to the identification of a membrane-bound hydrogenase which oxidizes hydrogen into protons with high efficiency while presenting outstanding O2, CO and temperature resistances.2,3 When immobilized onto carbon nanotube networks and in combination with a multicopper oxidase for O2 reduction, this enzyme has been proved to be a valuable candidate in a preliminary H2/O2 biofuel cell.4 Fishbone carbon nanofibers (CNFs) are other promising carbon materials which are electrically conductive, and bear lots of reactive sites due to the stacking of graphene cones. They can be quite easily functionalized for an efficient entrapment of proteins. We report in this work the use of such synthesized CNFs for modification of electrodes and immobilization of hydrogenase. We discuss the physico-chemical parameters that limit the efficiency of the bioelectrode, and define their optimization for a great enhancement of the current densities for H2 oxidation.Combined with a thermophilic multicopper oxidase at the cathode, immobilization of hydrogenase on CNFs allow to design a new generation of a high power H2/O2 biofuel cell able to be fed in the future with H2 produced from biomass.5 References[1] Y. Rafrafi et al., Int. J. Hydrogen Energ. 38 (2013) 4975.[2] X. Luo et al., J. Biol. Inorg. Chem. 14(2009)1275.[3] A. Ciaccafava et al., Angew. Chem. Int. Ed., 51(2012)953.[4] A. Ciaccafava et al., Electrochem. Com. 23(2012)25.[5] A. de Poulpiquet et al., PCCP(2013)

Flavoprotein monooxygenases for oxidative biocatalysis: recombinant expression in microbial hosts and applications

External flavoprotein monooxygenases comprise a group of flavin-dependent oxidoreductases that catalyze the insertion of one atom of molecular oxygen into an organic substrate and the second atom is reduced to water. These enzymes are involved in a great number of metabolic pathways both in prokaryotes and eukaryotes. Flavoprotein monooxygenases have attracted the attention of researchers for several decades and the advent of recombinant DNA technology caused a great progress in the field. These enzymes are subjected to detailed biochemical and structural characterization and some of them are also regarded as appealing oxidative biocatalysts for the production of fine chemicals and valuable intermediates toward active pharmaceutical ingredients due to their high chemo-, stereo-, and regioselectivity. Here, we review the most representative reactions catalyzed both in vivo and in vitro by prototype flavoprotein monooxygenases, highlighting the strategies employed to produce them recombinantly, to enhance the yield of soluble proteins, and to improve cofactor regeneration in order to obtain versatile biocatalysts. Although we describe the most outstanding features of flavoprotein monooxygenases, we mainly focus on enzymes that were cloned, expressed and used for biocatalysis during the last years.

Discovery and Characterization of a 5-Hydroxymethylfurfural Oxidase from Methylovorus sp. Strain MP688

In the search for useful and renewable chemical building blocks, 5-hydroxymethylfurfural (HMF) has emerged as a very promising candidate, as it can be prepared from sugars. HMF can be oxidized to 2,5-furandicarboxylic acid (FDCA), which is used as a substitute for petroleum-based terephthalate in polymer production. On the basis of a recently identified bacterial degradation pathway for HMF, candidate genes responsible for selective HMF oxidation have been identified. Heterologous expression of a protein from Methylovorus sp. strain MP688 in Escherichia coli and subsequent enzyme characterization showed that the respective gene indeed encodes an efficient HMF oxidase (HMFO). HMFO is a flavin adenine dinucleotide-containing oxidase and belongs to the glucose-methanol-choline-type flavoprotein oxidase family. Intriguingly, the activity of HMFO is not restricted to HMF, as it is active with a wide range of aromatic primary alcohols and aldehydes. The enzyme was shown to be relatively thermostable and active over a broad pH range. This makes HMFO a promising oxidative biocatalyst that can be used for the production of FDCA from HMF, a reaction involving both alcohol and aldehyde oxidations.

Recalcitrant polysaccharide degradation by novel oxidative biocatalysts

The classical hydrolytic mechanism for the degradation of plant polysaccharides by saprophytic microorganisms has been reconsidered after the recent landmark discovery of a new class of oxidases termed lytic polysaccharide monooxygenases (LPMOs). LPMOs are of increased biotechnological interest due to their implication in lignocellulosic biomass decomposition for the production of biofuels and high-value chemicals. They act on recalcitrant polysaccharides by a combination of hydrolytic and oxidative function, generating oxidized and non-oxidized chain ends. They are copper-dependent and require molecular oxygen and an external electron donor for their proper function. In this review, we present the recent findings concerning the mechanism of action of these oxidative enzymes and identify issues and questions to be addressed in the future.

Reconstitution of Active Mycobacterial Binuclear Iron Monooxygenase Complex in Escherichia coli

Bacterial binuclear iron monooxygenases play numerous physiological roles in oxidative metabolism. Monooxygenases of this type found in actinomycetes also catalyze various useful reactions and have attracted much attention as oxidation biocatalysts. However, difficulties in expressing these multicomponent monooxygenases in heterologous hosts, particularly in Escherichia coli, have hampered the development of engineered oxidation biocatalysts. Here, we describe a strategy to functionally express the mycobacterial binuclear iron monooxygenase MimABCD in Escherichia coli. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the mimABCD gene expression in E. coli revealed that the oxygenase components MimA and MimC were insoluble. Furthermore, although the reductase MimB was expressed at a low level in the soluble fraction of E. coli cells, a band corresponding to the coupling protein MimD was not evident. This situation rendered the transformed E. coli cells inactive. We found that the following factors are important for functional expression of MimABCD in E. coli: coexpression of the specific chaperonin MimG, which caused MimA and MimC to be soluble in E. coli cells, and the optimization of the mimD nucleotide sequence, which led to efficient expression of this gene product. These two remedies enabled this multicomponent monooxygenase to be actively expressed in E. coli. The strategy described here should be generally applicable to the E. coli expression of other actinomycetous binuclear iron monooxygenases and related enzymes and will accelerate the development of engineered oxidation biocatalysts for industrial processes.

Quantitating intraparticle O2 gradients in solid supported enzyme immobilizates: Experimental determination of their role in limiting the catalytic effectiveness of immobilized glucose oxidase

Enzymatic O2 -dependent oxidations are receiving increased attention for use in fine chemicals synthesis. Solid supported oxidation catalysts often show poor efficiency due to pronounced O2 diffusion restriction. Internal O2 supply therefore constitutes a key parameter for optimizing the enzyme immobilization. We herein describe an optical sensing method for quantitation of space-averaged intraparticle O2 concentrations in porous Sepabeads carriers. The method applies phosphorescence lifetime measurements on Sepabeads labeled with an O2 sensitive indicator dye. Using glucose oxidase immobilized at different loadings (0.005-12 mg/g) on labeled Sepabeads, we analyzed in real time during the enzymatic reaction the formation of O2 concentration differences between bulk liquid and the intraparticle environment. We show that the O2 gradient at apparent steady state increased with increasing enzyme loading, so that O2 eventually became totally depleted from inside the highly loaded carriers. We also show that the residual intraparticle O2 concentration was correlated with the catalytic effectiveness factor (η) of the enzyme immobilizate used, thus providing a direct measure of the magnitude of O2 diffusion limitation. Once corrected for diffusional effect, η was no longer dependent on enzyme loading and its constant value now described the intrinsic activity of immobilized glucose oxidase. Three common procedures of enzyme immobilization, involving adsorption, cross-linking, and covalent attachment, are shown to differ widely concerning the obtained intrinsic activity. Therefore, intraparticle O2 concentration data enable distinction between diffusional restriction and activity loss as the two principal factors limiting the effectiveness of immobilized O2 dependent enzymes, and thus they inform rational design of an optimally active oxidation biocatalyst on solid support.

Application of heme-containing biocatalysts for oxidation and determination of organosulfur compounds

The conditions of the oxidation of dibenzothiophene and aliphatic phenothiazines (promazine and chlorpromazine) in the presence of heme-containing biocatalysts (such as horseradish peroxidase and hemoglobin) have been optimized. It has been demonstrated that hemoglobin is an efficient catalyst for the fast and selective conversion of dibenzothiophene to the corresponding sulfoxide (yield is 91 ± 2% in 5 min); horseradish peroxidase is a more promising catalyst for the oxidation of phenothiazines. The effect of polar organic solvent (dimethyl sulfoxide) on the kinetics parameters of the reaction of oxidation of promazine in the presence of heme-containing biocatalysts has been studied. Procedures of determining phenothiazines based on spectrophotometric control of the rate of their oxidation have been developed. The applicable concentration ranges for the determination of promazine and chlorpromazine using horseradish peroxidase in aqueous medium were 10–100 μM and 10–200 μM respectively, and 20–500 μM for the determination of promazine in the presence of 10 vol % of dimethyl sulfoxide.

Applications of Flavoprotein Oxidases in Organic Synthesis: Novel Reactivities that Go Beyond Amine and Alcohol Oxidations

Oxidases represent a distinct and interesting class of oxidative biocatalysts. A major portion of the known oxidases contain a flavin as cofactor, with glucose oxidase as best known example. While a number of oxidases are well known in the field of biocatalysis, the total number of available oxidases is rather limited. However, by analysis of literature data and genome sequences a clear picture emerges: nature harbors a large number of unexplored flavoprotein oxidases that can catalyze a breadth of oxidative reactions. In this review a summary is provided of the unique and intriguing catalytic potential of newly discovered flavoprotein oxidases. The reactions that have been shown to be catalyzed range from enantio- and/or regioselective alcohol and amine oxidations to oxidative C-O or C-C bond formations. This illustrates that nature harbors a large collection of oxidases that can be of use for catalyzing complex oxidative reactions. Keywords: Alcohol, Biocatalysis, C-C bond, Cyclization, Oxidases, Flavoprotein

Heterologous expression and structural characterization of two low pH laccases from a biopulping white-rot fungus Physisporinus rivulosus

The lignin-degrading, biopulping white-rot fungus Physisporinus rivulosus secretes several laccases of distinct features such as thermostability, extremely low pH optima and thermal activation for oxidation of phenolic substrates. Here we describe the cloning, heterologous expression and structural and enzymatic characterisation of two previously undescribed P. rivulosus laccases. The laccase cDNAs were expressed in the methylotrophic yeast Pichia pastoris either with the native or with Saccharomyces cerevisiae α-factor signal peptide. The specific activity of rLac1 and rLac2 was 5 and 0.3μkat/μg, respectively. However, mutation of the last amino acid in the rLac2 increased the specific laccase activity by over 50-fold. The recombinant rLac1 and rLac2 enzymes demonstrated low pH optima with both 2,6-dimethoxyphenol (2,6-DMP) and 2,2'-azino-bis(3-ethylbenzathiazoline-6-sulfonate). Both recombinant laccases showed moderate thermotolerance and thermal activation at +60°C was detected with rLac1. By homology modelling, it was deduced that Lac1 and Lac2 enzymes demonstrate structural similarity with the Trametes versicolor and Trametes trogii laccase crystal structures. Comparison of the protein architecture at the reducing substrate-binding pocket near the T1-Cu site indicated the presence of five amino acid substitutions in the structural models of Lac1 and Lac2. These data add up to our previous reports on laccase production by P. rivulosus during biopulping and growth on Norway spruce. Heterologous expression of the novel Lac1 and Lac2 isoenzymes in P. pastoris enables the detailed study of their properties and the evaluation of their potential as oxidative biocatalysts for conversion of wood lignin, lignin-like compounds and soil-polluting xenobiotics.

Cytochrome P450 monooxygenases: an update on perspectives for synthetic application

Cytochrome P450 monooxygenases (P450s) are versatile biocatalysts that catalyze the regio- and stereospecific oxidation of non-activated hydrocarbons under mild conditions, which is a challenging task for chemical catalysts. Over the past decade impressive advances have been achieved via protein engineering with regard to activity, stability and specificity of P450s. In addition, a large pool of newly annotated P450s has attracted much attention as a source for novel biocatalysts for oxidation. In this review we give a short up-to-date overview of recent results on P450 engineering for technical applications including aspects of whole-cell biocatalysis with engineered recombinant enzymes. Furthermore, we focus on recently identified P450s with novel biotechnologically relevant properties.

ChemInform Abstract: Exploring the Biocatalytic Scope of a Bacterial Flavin‐Containing Monooxygenase.

AbstractA novel fused oxidative biocatalyst consisting of a phosphite dehydrogenase and bacterial FMO is successfully applied in the oxidation of various aromatic and aliphatic sulfides to the corresponding sulfoxides.

Novel multienzyme oxidative biocatalyst for lignin bioprocessing

A novel multienzyme biocatalyst, based on coimmobilization of the laccase and horseradish peroxidase by cross linking and layer-by-layer coating with polyelectrolyte, was designed, synthesized and applied at the development of an oxidative cascade process on lignin. The efficiency and specificity of the new LbL-multienzyme system, the occurrence of a synergy of the co-immobilized enzymes, the lignin oxidation pathway and the nature of the structural modifications occurred in treated lignins have been investigated in the present effort by means of GPC analysis and quantitative 31P NMR techniques.

New biotech applications from evolved D-amino acid oxidases

D-Amino acid oxidase (DAAO) is a well-known flavoenzyme that catalyzes the oxygen-dependent oxidative deamination of amino acid D-isomers with absolute stereospecificity, which results in α-keto acids, ammonia and hydrogen peroxide. Recently, the extraordinary functional plasticity of DAAO has become evident; in turn, boosting research on this flavoprotein. Protein engineering has allowed for a redesign of DAAO substrate specificity, oxygen affinity, cofactor binding, stability, and oligomeric state. We review recent developments in utilizing DAAO, including as a biocatalyst for resolving racemic amino acid mixtures, as a tool for biosensing, and as a new mechanism of herbicide resistance. Perspectives for future biotechnological applications of this oxidative biocatalyst are also outlined.

Efficient production of Al(OH)3-immobilized laccase with aHeterobasidion annosumstrain selected by microplate screening

Wild-type white rot fungi are the most important production organisms for laccase, a promising oxidative biocatalyst with numerous applications. This study aimed at identifying novel highly productive strains, finding optimal cultivation conditions for laccase production and establishing a simple immobilization procedure. By using a newly developed 96-well microplate cultivation method, 23 species of white rot fungi, represented by 29 strains, were directly compared with regard to the amount of secreted laccase. Both, with glucose and spruce saw dust as growth substrate a Heterobasidion annosum strain and a Physisporinus vitreus strain were the most productive (730–2200 U l−1 of secreted laccase). Cultivation conditions for laccase production with H. annosum were optimized in larger-scale liquid cultures. Aeration with a sparger lead to a 3·8-fold increase in laccase activity when compared to nonaerated flask cultures. More than 3000 U l−1 laccase was produced in glucose medium supplemented with yeast extract and the inducer veratryl alcohol. Culture supernatant was incubated with short-range ordered Al(OH)3 particles to directly immobilize and concentrate laccase by adsorption. Active laccase was recovered in 40% yield and the Al(OH)3-adsorbed laccase was suitable for repeated decolourization of indigo carmine. Microplate cultivation allowed a large-scale comparison of the capacity of different fungal species for laccase production. Laccase secretion of a highly productive H. annosum strain was found to vary strongly with different cultivation conditions. Adsorption to Al(OH)3 proved to be suitable as direct immobilization technique.

ChemInform Abstract: Recent Developments in the Application of Baeyer—Villiger Monooxygenases as Biocatalysts

AbstractReview: ca. 200 refs.

Recent Developments in the Application of Baeyer–Villiger Monooxygenases as Biocatalysts

Baeyer-Villiger monooxygenases (BVMOs) represent a specific class of monooxygenases that are capable of catalyzing a variety of oxidation reactions, including Baeyer-Villiger oxidations. The recently elucidated BVMO crystal structures have provided a more detailed insight into the complex mechanism of these flavin-containing enzymes. Biocatalytic studies on a number of newly discovered BVMOs have shown that they are very potent oxidative biocatalysts. In addition to catalyzing the regio- and enantioselective Baeyer-Villiger oxidations of a wide range of carbonylic compounds, epoxidations, and enantioselective sulfoxidations have also been shown to be part of their catalytic repertoire. This review provides an overview on the recent developments in BVMO-mediated biocatalytic processes, identification of the catalytic role of these enzymes in metabolic routes and prodrug activation, as well as the efforts in developing effective biocatalytic methodologies to apply BVMOs for the synthesis of high added value compounds.

Genome mining approach for the discovery of novel cytochrome P450 biocatalysts

Cytochrome P450 enzymes (P450s) are able to regioselectively and stereoselectively introduce oxygen into organic compounds under mild reaction conditions. These monooxygenases in particular easily catalyze the insertion of oxygen into less reactive carbon-hydrogen bonds. Hence, P450s are of considerable interest as oxidation biocatalysts. To date, although several P450s have been discovered through screening of microorganisms and have been further genetically engineered, the substrate range of these biocatalysts is still limited to fulfill the requirements for a large number of oxidation processes. On the other hand, the recent rapid expansion in the number of reported microbial genome sequences has revealed the presence of an unexpectedly vast number of P450 genes. This large pool of naturally evolved P450s has attracted much attention as a resource for new oxidation biocatalysts. In this review, we focus on aspects of the genome mining approach that are relevant for the discovery of novel P450 biocatalysts. This approach opens up possibilities for exploitation of the catalytic potential of P450s for the preparation of a large choice of oxidation biocatalysts with a variety of substrate specificities.

Coencapsulation of Oxygen Carriers and Glucose Oxidase in Polyelectrolyte Complex Capsules for the Enhancement of D-Gluconic Acid and δ-Gluconolactone Production

A novel encapsulated oxidative biocatalyst comprising glucose oxidase (GOD) coencapsulated with oxygen carriers within polyelectrolyte complex capsules was developed for the production of D-gluconic acid and delta-gluconolactone. The capsules containing immobilized GOD were produced by polyelectrolyte complexation with sodium alginate (SA) and cellulose sulfate (CS) as polyanions, poly(methylene-co-guanidine) (PMCG) as the polycation, CaCl(2) as the gelling agent and NaCl as the antigelling agent (GOD-SA-CS/PMCG capsules). Poly(dimethylsiloxane) (PDMS) and an emulsion of n-dodecane (DOD) or perfluorodecaline (PFD) with PDMS were used as the oxygen carriers and MnO(2) was used as a hydrogen peroxide decomposition catalyst. Water-soluble PDMS was found to act as both an oxygen carrier and an emulsifier of water-insoluble DOD and PFD. Stable microcapsules could be produced with concentrations of up to 4% (w/w) of PDMS, 10% (w/w) of DOD and PFD, and 25% (w/w) of MnO(2) in the polyanion solution of SA and CS. Roughly a two-fold increase in the GOD activity from 21.0+/-1.1 to 38.4+/-2.0 U*g(-1) and product space-time yields (STY) from 44.3+/-2.0 to 83.4+/-3.4 g*H*day(-1) could be achieved utilizing coencapsulated oxygen carriers compared to GOD encapsulated in the absence of oxygen carriers. This enhanced production does not significantly depend on the selected oxygen carrier under the conditions used in this study.

Very stable silica-gel-bound laccase biocatalysts for the selective oxidation in continuous systems

Cerrena unicolor laccase was immobilized by adsorption and covalent bonds formation on silica-gel carriers, functionalized with different organosilanes and surface densities. The effects of protein concentration, pH value of the coupling mixture and the enzyme purity on immobilization efficiency of the best carrier, moderately modified (0.75 mmol/g carrier) with 3-aminopropyltriethoxysilane were investigated. Activity of the best biocatalysts, expressed in ABTS oxidation, was 4028 U/mL of the carrier or 3530 U/mg of bound protein. Properties of immobilized laccase were determined to find excellent thermal stability improvement; t 1/2 for freely suspended enzyme was 2–3 min at 80 °C, whereas after immobilization over 100 min. Kinetic experiments in both batch and packed-bed reactors gave only four times lower k cat/ K m value than for the native enzyme. A packed-bed reactor with silica-gel-bound laccase beads appeared to be very efficient in ABTS oxidation and its exceptional potentials were shown in the continuous decolorization of indigo carmine for 18 days without loss in activity. This system offers perfect ability to degrade recalcitrant dyes, but we can also envisage its use, with ABTS acting as a mediator, in regeneration of nicotinamide cofactors.

Biocatalytic Synthesis of Dihydroxynaphthoic Acids by Cytochrome P450 CYP199A2

CYP199A2, a bacterial P450 monooxygenase from Rhodopseudomonas palustris, was found to exhibit oxidation activity towards three hydroxynaphthoic acids. Whole cells of the recombinant Escherichia coli strain expressing CYP199A2 efficiently catalyzed the regioselective oxidation of 1-, 3-, and 6-hydroxy-2-naphthoic acids to produce 1,7-, 3,7-, and 6,7-dihydroxynaphthoic acid respectively. These results suggest that CYP199A2 might be a useful oxidation biocatalyst for the synthesis of dihydroxynaphthoic acids.