Functional Expression of Two Unusual Acidic Peroxygenases from Candolleomyces aberdarensis in Yeasts by Adopting Evolved Secretion Mutations.

Fungal unspecific peroxygenases (UPOs) are emergent biocatalysts that perform highly selective C-H oxyfunctionalizations of organic compounds, yet their heterologous production at high levels is required for their practical use in synthetic chemistry. Here, we achieved functional expression of two new unusual acidic peroxygenases from Candolleomyces (Psathyrella) aberdarensis (PabUPO) in yeasts and their production at a large scale in a bioreactor. Our strategy was based on adopting secretion mutations from an Agrocybe aegerita UPO mutant, the PaDa-I variant, designed by directed evolution for functional expression in yeast, which belongs to the same phylogenetic family as PabUPOs, long-type UPOs, and shares 65% sequence identity. After replacing the native signal peptides with the evolved leader sequence from PaDa-I, we constructed and screened site-directed recombination mutant libraries, yielding two recombinant PabUPOs with expression levels of 5.4 and 14.1 mg/liter in Saccharomyces cerevisiae. These variants were subsequently transferred to Pichia pastoris for overproduction in a fed-batch bioreactor, boosting expression levels up to 290 mg/liter, with the highest volumetric activity achieved to date for a recombinant peroxygenase (60,000 U/liter, with veratryl alcohol as the substrate). With a broad pH activity profile, ranging from pH 2.0 to 9.0, these highly secreted, active, and stable peroxygenases are promising tools for future engineering endeavors as well as for their direct application in different industrial and environmental settings. IMPORTANCE In this work, we incorporated several secretion mutations from an evolved fungal peroxygenase to enhance the production of active and stable forms of two unusual acidic peroxygenases. The tandem-yeast expression system based on S. cerevisiae for directed evolution and P. pastoris for overproduction on an ∼300-mg/liter scale is a versatile tool to generate UPO variants. By employing this approach, we foresee that acidic UPO variants will be more readily engineered in the near future and adapted to practical enzyme cascade reactions that can be performed over a broad pH range to oxyfunctionalize a variety of organic compounds.

Heme-thiolate ferryl of aromatic peroxygenase is basic and reactive.

A kinetic and spectroscopic characterization of the ferryl intermediate (APO-II) from APO, the heme-thiolate peroxygenase from Agrocybe aegerita, is described. APO-II was generated by reaction of the ferric enzyme with metachloroperoxybenzoic acid in the presence of nitroxyl radicals and detected with the use of rapid-mixing stopped-flow UV-visible (UV-vis) spectroscopy. The nitroxyl radicals served as selective reductants of APO-I, reacting only slowly with APO-II. APO-II displayed a split Soret UV-vis spectrum (370 nm and 428 nm) characteristic of thiolate ligation. Rapid-mixing, pH-jump spectrophotometry revealed a basic pKa of 10.0 for the Fe(IV)-O-H of APO-II, indicating that APO-II is protonated under typical turnover conditions. Kinetic characterization showed that APO-II is unusually reactive toward a panel of benzylic C-H and phenolic substrates, with second-order rate constants for C-H and O-H bond scission in the range of 10-10(7) M(-1)⋅s(-1). Our results demonstrate the important role of the axial cysteine ligand in increasing the proton affinity of the ferryl oxygen of APO intermediates, thus providing additional driving force for C-H and O-H bond scission.

A Peroxygenase from Chaetomium globosum Catalyzes the Selective Oxygenation of Testosterone.

Unspecific peroxygenases (UPO, EC 1.11.2.1) secreted by fungi open an efficient way to selectively oxyfunctionalize diverse organic substrates, including less-activated hydrocarbons, by transferring peroxide-borne oxygen. We investigated a cell-free approach to incorporate epoxy and hydroxyl functionalities directly into the bulky molecule testosterone by a novel unspecific peroxygenase (UPO) that is produced by the ascomycetous fungus Chaetomium globosum in a complex medium rich in carbon and nitrogen. Purification by fast protein liquid chromatography revealed two enzyme fractions with the same molecular mass (36 kDa) and with specific activity of 4.4 to 12 U mg-1 . Although the well-known UPOs of Agrocybe aegerita (AaeUPO) and Marasmius rotula (MroUPO) failed to convert testosterone in a comparative study, the UPO of C. globosum (CglUPO) accepted testosterone as substrate and converted it with total turnover number (TTN) of up to 7000 into two oxygenated products: the 4,5-epoxide of testosterone in ß-configuration and 16α-hydroxytestosterone. The reaction performed on a 100 mg scale resulted in the formation of about 90 % of the epoxide and 10 % of the hydroxylation product, both of which could be isolated with purities above 96 %. Thus, CglUPO is a promising biocatalyst for the oxyfunctionalization of bulky steroids and it will be a useful tool for the synthesis of pharmaceutically relevant steroidal molecules.

Fatty Acid Chain Shortening by a Fungal Peroxygenase.

A recently discovered peroxygenase from the fungus Marasmius rotula (MroUPO) is able to catalyze the progressive one-carbon shortening of medium and long-chain mono- and dicarboxylic acids by itself alone, in the presence of H2 O2 . The mechanism, analyzed using H218 O2 , starts with an α-oxidation catalyzed by MroUPO generating an α-hydroxy acid, which is further oxidized by the enzyme to a reactive α-keto intermediate whose decarboxylation yields the one-carbon shorter fatty acid. Compared with the previously characterized peroxygenase of Agrocybe aegerita, a wider heme access channel, enabling fatty acid positioning with the carboxylic end near the heme cofactor (as seen in one of the crystal structures available) could be at the origin of the unique ability of MroUPO shortening carboxylic acid chains.

Peroxygenase-Catalyzed Oxyfunctionalization Reactions Promoted by the Complete Oxidation of Methanol.

Peroxygenases catalyze a broad range of (stereo)selective oxyfunctionalization reactions. However, to access their full catalytic potential, peroxygenases need a balanced provision of hydrogen peroxide to achieve high catalytic activity while minimizing oxidative inactivation. Herein, we report an enzymatic cascade process that employs methanol as a sacrificial electron donor for the reductive activation of molecular oxygen. Full oxidation of methanol is achieved, generating three equivalents of hydrogen peroxide that can be used completely for the stereoselective hydroxylation of ethylbenzene as a model reaction. Overall we propose and demonstrate an atom-efficient and easily applicable alternative to established hydrogen peroxide generation methods, which enables the efficient use of peroxygenases for oxyfunctionalization reactions.

Steroid hydroxylation by basidiomycete peroxygenases: a combined experimental and computational study.

The goal of this study is the selective oxyfunctionalization of steroids under mild and environmentally friendly conditions using fungal enzymes. With this purpose, peroxygenases from three basidiomycete species were tested for the hydroxylation of a variety of steroidal compounds, using H2O2 as the only cosubstrate. Two of them are wild-type enzymes from Agrocybe aegerita and Marasmius rotula, and the third one is a recombinant enzyme from Coprinopsis cinerea. The enzymatic reactions on free and esterified sterols, steroid hydrocarbons, and ketones were monitored by gas chromatography, and the products were identified by mass spectrometry. Hydroxylation at the side chain over the steroidal rings was preferred, with the 25-hydroxyderivatives predominating. Interestingly, antiviral and other biological activities of 25-hydroxycholesterol have been reported recently (M. Blanc et al., Immunity 38:106-118, 2013, http://dx.doi.org/10.1016/j.immuni.2012.11.004). However, hydroxylation in the ring moiety and terminal hydroxylation at the side chain also was observed in some steroids, the former favored by the absence of oxygenated groups at C-3 and by the presence of conjugated double bonds in the rings. To understand the yield and selectivity differences between the different steroids, a computational study was performed using Protein Energy Landscape Exploration (PELE) software for dynamic ligand diffusion. These simulations showed that the active-site geometry and hydrophobicity favors the entrance of the steroid side chain, while the entrance of the ring is energetically penalized. Also, a direct correlation between the conversion rate and the side chain entrance ratio could be established that explains the various reaction yields observed.

The toolbox of Auricularia auricula-judae dye-decolorizing peroxidase - Identification of three new potential substrate-interaction sites.

Dye-decolorizing peroxidases (DyPs) such as AauDyPI from the fungus Auricularia auricula-judae are able to oxidize substrates of different kinds and sizes. A crystal structure of an AauDyPI-imidazole complex gives insight into the binding patterns of organic molecules within the heme cavity of a DyP. Several small N-containing heterocyclic aromatics are shown to bind in the AauDyPI heme cavity, hinting to susceptibility of DyPs to azole-based inhibitors similar to cytochromes P450. Imidazole is confirmed as a competitive inhibitor with regard to peroxide binding. In contrast, bulky substrates such as anthraquinone dyes are converted at the enzyme surface. In the crystal structure a substrate analog, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), binds to a tyrosine-rich hollow harboring Y25, Y147, and Y337. Spin trapping with a nitric oxide donor uncovers Y229 as an additional tyrosine-based radical center in AauDyPI. Multi-frequency EPR spectroscopy further reveals the presence of at least one intermediate tryptophanyl radical center in activated AauDyPI with W377 as the most likely candidate.

Oxidation and nitration of mononitrophenols by a DyP-type peroxidase.

Substantial conversion of nitrophenols, typical high-redox potential phenolic substrates, by heme peroxidases has only been reported for lignin peroxidase (LiP) so far. But also a dye-decolorizing peroxidase of Auricularia auricula-judae (AauDyP) was found to be capable of acting on (i) ortho-nitrophenol (oNP), (ii) meta-nitrophenol (mNP) and (iii) para-nitrophenol (pNP). The pH dependency for pNP oxidation showed an optimum at pH 4.5, which is typical for phenol conversion by DyPs and other heme peroxidases. In the case of oNP and pNP conversion, dinitrophenols (2,4-DNP and 2,6-DNP) were identified as products and for pNP additionally p-benzoquinone. Moreover, indications were found for the formation of random polymerization products originating from initially formed phenoxy radical intermediates. Nitration was examined using (15)N-labeled pNP and Na(14)NO2 as an additional source of nitro-groups. Products were identified by HPLC-MS, and mass-to-charge ratios were evaluated to clarify the origin of nitro-groups. The additional nitrogen in DNPs formed during enzymatic conversion was found to originate both from (15)N-pNP and (14)NO2Na. Based on these results, a hypothetical reaction scheme and a catalytically responsible confine of the enzyme's active site are postulated.

Fungal unspecific peroxygenases: heme-thiolate proteins that combine peroxidase and cytochrome p450 properties.

Eleven years ago, a secreted heme-thiolate peroxidase with promiscuity for oxygen transfer reactions was discovered in the basidiomycetous fungus, Agrocybe aegerita. The enzyme turned out to be a functional mono-peroxygenase that transferred an oxygen atom from hydrogen peroxide to diverse organic substrates (aromatics, heterocycles, linear and cyclic alkanes/alkenes, fatty acids, etc.). Later similar enzymes were found in other mushroom genera such as Coprinellus and Marasmius. Approximately one thousand putative peroxygenase sequences that form two large clusters can be found in genetic databases and fungal genomes, indicating the widespread occurrence of such enzymes in the whole fungal kingdom including all phyla of true fungi (Eumycota) and certain fungus-like heterokonts (Oomycota). This new enzyme type was classified as unspecific peroxygenase (UPO, EC 1.11.2.1) and placed in a separate peroxidase subclass. Furthermore, UPOs and related heme-thiolate peroxidases such as well-studied chloroperoxidase (CPO) represent a separate superfamily of heme proteins on the phylogenetic level. The reactions catalyzed by UPOs include hydroxylation, epoxidation, O- and N-dealkylation, aromatization, sulfoxidation, N-oxygenation, dechlorination and halide oxidation. In many cases, the product patterns of UPOs resemble those of human cytochrome P450 (P450) monooxygenases and, in fact, combine the catalytic cycle of heme peroxidases with the "peroxide shunt" of P450s. Here, an overview on UPOs is provided with focus on their molecular and catalytic properties.

First crystal structure of a fungal high-redox potential dye-decolorizing peroxidase: substrate interaction sites and long-range electron transfer.

Dye-decolorizing peroxidases (DyPs) belong to the large group of heme peroxidases. They utilize hydrogen peroxide to catalyze oxidations of various organic compounds. AauDyPI from Auricularia auricula-judae (fungi) was crystallized, and its crystal structure was determined at 2.1 Å resolution. The mostly helical structure also shows a ß-sheet motif typical for DyPs and Cld (chlorite dismutase)-related structures and includes the complete polypeptide chain. At the distal side of the heme molecule, a flexible aspartate residue (Asp-168) plays a key role in catalysis. It guides incoming hydrogen peroxide toward the heme iron and mediates proton rearrangement in the process of Compound I formation. Afterward, its side chain changes its conformation, now pointing toward the protein backbone. We propose an extended functionality of Asp-168, which acts like a gatekeeper by altering the width of the heme cavity access channel. Chemical modifications of potentially redox-active amino acids show that a tyrosine is involved in substrate interaction. Using spin-trapping experiments, a transient radical on the surface-exposed Tyr-337 was identified as the oxidation site for bulky substrates. A possible long-range electron transfer pathway from the surface of the enzyme to the redox cofactor (heme) is discussed.

Structural basis of substrate conversion in a new aromatic peroxygenase: cytochrome P450 functionality with benefits.

Aromatic peroxygenases (APOs) represent a unique oxidoreductase sub-subclass of heme proteins with peroxygenase and peroxidase activity and were thus recently assigned a distinct EC classification (EC 1.11.2.1). They catalyze, inter alia, oxyfunctionalization reactions of aromatic and aliphatic hydrocarbons with remarkable regio- and stereoselectivities. When compared with cytochrome P450, APOs appear to be the choice enzymes for oxyfunctionalizations in organic synthesis due to their independence from a cellular environment and their greater chemical versatility. Here, the first two crystal structures of a heavily glycosylated fungal aromatic peroxygenase (AaeAPO) are described. They reveal different pH-dependent ligand binding modes. We model the fitting of various substrates in AaeAPO, illustrating the way the enzyme oxygenates polycyclic aromatic hydrocarbons. Spatial restrictions by a phenylalanine pentad in the active-site environment govern substrate specificity in AaeAPO.

Differences in the secretion pattern of oxidoreductases from Bjerkandera adusta induced by a phenolic olive mill extract.

The secretome of the white-rot fungus Bjerkandera adusta produced in synthetic Kirk medium was compared to that supplemented with an aqueous phenol-rich extract of dry olive mill residues (ADOR). Distinct changes in the protein composition of oxidoreductases, namely diverse class-II peroxidases and aryl alcohol oxidases were found. In the ADOR-supplemented medium (ASC), 157 distinct proteins were identified by the secretome analysis, whereas only 59 of them were identified without ADOR supplementation (Kirk medium culture; KM). Proteome analysis indicated that the number of peroxidases produced in ASC was more than doubled (from 4 to 11) compared to KM. Two short manganese peroxidases (MnP1 and MnP6) and one versatile peroxidase (VP1) represented 29% of the relative abundance (NSAF) in ASC. Two of them (MnP1 and VP1) were also detected in KM at a relative abundance (NSAF) of only 3%. Further peroxidases present in ASC were one lignin peroxidase (LiP2), one generic peroxidase (GP) and three dye-decolorizing peroxidases (DyPs). The relative abundance of DyPs and aryl alcohol oxidases (AAO) were lower in ASC in comparison to KM. In addition to peptide sequence analysis, the secretion of Mn(2+)-oxidizing peroxidases as well as AAOs were followed by enzyme measurement. The Mn(2+)-oxidizing activity increased nearly 30-fold (from 10 to 281Ul(-1)) after ADOR addition. Two enzymes responsible for that activity were successfully purified (BadVPI and BadVPII). To prove a potential involvement of these enzymes in the degradation of aromatic compounds, BadVPI was tested for its ability to degrade the recalcitrant dehydrogenated polymer (DHP, synthetic lignin). These results show that natural phenol-rich materials act as secretome-stimulating additives. Applying these substances enables us to investigate fungal degradation and detoxification processes and gives more insight into the complexity of fungal secretomes, e.g. of white-rot fungi.

Directed evolution of unspecific peroxygenase from Agrocybe aegerita.

Unspecific peroxygenase (UPO) represents a new type of heme-thiolate enzyme with self-sufficient mono(per)oxygenase activity and many potential applications in organic synthesis. With a view to taking advantage of these properties, we subjected the Agrocybe aegerita UPO1-encoding gene to directed evolution in Saccharomyces cerevisiae. To promote functional expression, several different signal peptides were fused to the mature protein, and the resulting products were tested. Over 9,000 clones were screened using an ad hoc dual-colorimetric assay that assessed both peroxidative and oxygen transfer activities. After 5 generations of directed evolution combined with hybrid approaches, 9 mutations were introduced that resulted in a 3,250-fold total activity improvement with no alteration in protein stability. A breakdown between secretion and catalytic activity was performed by replacing the native signal peptide of the original parental type with that of the evolved mutant; the evolved leader increased functional expression 27-fold, whereas an 18-fold improvement in the kcat/Km value for oxygen transfer activity was obtained. The evolved UPO1 was active and highly stable in the presence of organic cosolvents. Mutations in the hydrophobic core of the signal peptide contributed to enhance functional expression up to 8 mg/liter, while catalytic efficiencies for peroxidative and oxygen transfer reactions were increased by several mutations in the vicinity of the heme access channel. Overall, the directed-evolution platform described is a valuable point of departure for the development of customized UPOs with improved features and for the study of structure-function relationships.

Degradation of atrazine by Frankia alni ACN14a: gene regulation, dealkylation, and dechlorination.

Atrazine is transformed to N-isopropylammelide through hydroxyatrazine as an intermediate as indicated by high-performance liquid chromatography/mass spectroscopy in culture filtrates of Frankia alni ACN14a and Frankia sp. EuI1c. Both Frankia strains have the ability to degrade atrazine via dechlorination and dealkylation and, subsequently, may be using it as a nitrogen and carbon source as detected here by increasing their growth patterns. Bioinformatic analysis of the Frankia genomes revealed that a potential gene cluster involved in atrazine decomposition contains three genes, namely, trzN (FRAAL1474 and FraEuI1c_5874), atzB (FRAAL1473 and FraEuI1c_5875), and atzR (FRAAL1471). The relative messenger RNA gene expression of the former genes was examined by qRT-PCR. The LysR-type transcriptional regulator atzR (FRAAL1471), which is expected to control the cluster expression, showed a 13-fold increase in the expression level under atrazine stress. Moreover, the putative adenosine aminohydrolase 3 atzB (FRAAL1473), which is expected to dealkylate the N-ethyl group of atrazine, showed also an increased expression by factor 16 with increased exposure. Eventually, the trzN (FRAAL1474) gene, which is predicted to encode a putative amidohydrolase catalyzing atrazine dechlorination, exhibited 31-fold increased expression. To our best knowledge, this is the first report about adenosine aminohydrolase 3 function in the dealkylation of the N-ethyl group from atrazine.

Radical formation on a conserved tyrosine residue is crucial for DyP activity.

Dye-decolorizing peroxidases (DyPs) are able to cleave bulky anthraquinone dyes. The recently published crystal structure of AauDyPI reveals that a direct oxidation in the distal heme cavity can be excluded for most DyP substrates. It is shown that a surface-exposed tyrosine residue acts as a substrate interaction site for bulky substrates. This amino acid is conserved in eucaryotic DyPs but is missing in the structurally related chlorite dismutases (Clds). Dye-decolorizing peroxidases of procaryotic origin equally possess a conserved tyrosine in the same region of the polypeptide albeit not at the homologous position.

Substrate oxidation by dye-decolorizing peroxidases (DyPs) from wood- and litter-degrading agaricomycetes compared to other fungal and plant heme-peroxidases.

Catalytic and physicochemical properties of representative fungal dye-decolorizing peroxidases (DyPs) of wood- (WRF) and litter-decomposing white-rot fungi (LDF) are summarized and compared, including one recombinant Mycetinis scorodonius DyP (rMscDyP; LDF), the wild-type Auricularia auricula-judae DyP (AauDyP; WRF), and two new DyPs secreted by the jelly fungi Exidia glandulosa (EglDyP; WRF) and Mycena epipterygia (MepDyP; LDF). Homogeneous preparations of these DyPs were obtained after different steps of fast protein liquid chromatography, and they increase the total number of characterized fungal DyP proteins to eight. The peptide sequences of AauDyP, MepDyP, and EglDyP showed highest homologies (52-56%) to the DyPs of M. scorodonius. Five out of the eight characterized fungal DyPs were used to evaluate their catalytic properties compared to classic fungal and plant heme peroxidases, namely lignin peroxidase of Phanerochaete chrysosporium (PchLiP; WRF), versatile peroxidase of Bjerkandera adusta (BadVP; WRF), and generic peroxidases of Coprinopsis cinerea (CiP) and Glycine max (soybean peroxidase=SBP). All DyPs tested possess unique properties regarding the stability at low pH values: 50-90% enzymatic activity remained after 4-h exposition at pH 2.5, and the oxidation of nonphenolic aromatic substrates (lignin model compounds) was optimal below pH 3. Furthermore, all DyPs efficiently oxidized recalcitrant dyes (e.g., Azure B) as well as the phenolic substrate 2,6-dimethoxyphenol. Thus, DyPs combine features of different peroxidases on the functional level and may be part of the biocatalytic system secreted by fungi for the oxidation of lignin and/or toxic aromatic compounds.

The wood rot ascomycete Xylaria polymorpha produces a novel GH78 glycoside hydrolase that exhibits α-L-rhamnosidase and feruloyl esterase activities and releases hydroxycinnamic acids from lignocelluloses.

Soft rot (type II) fungi belonging to the family Xylariaceae are known to substantially degrade hardwood by means of their poorly understood lignocellulolytic system, which comprises various hydrolases, including feruloyl esterases and laccase. In the present study, several members of the Xylariaceae were found to exhibit high feruloyl esterase activity during growth on lignocellulosic materials such as wheat straw (up to 1,675 mU g(-1)) or beech wood (up to 80 mU g(-1)). Following the ester-cleaving activity toward methyl ferulate, a hydrolase of Xylaria polymorpha was produced in solid-state culture on wheat straw and purified by different steps of anion-exchange and size-exclusion chromatography to apparent homogeneity (specific activity, 2.2 U mg(-1)). The peptide sequence of the purified protein deduced from the gene sequence and verified by de novo peptide sequencing shows high similarity to putative α-L-rhamnosidase sequences belonging to the glycoside hydrolase family 78 (GH78; classified under EC 3.2.1.40). The purified enzyme (98 kDa by SDS-PAGE, 103 kDa by size-exclusion chromatography; pI 3.7) converted diverse glycosides (e.g., α-L-rhamnopyranoside and α-L-arabinofuranoside) but also natural and synthetic esters (e.g., chlorogenic acid, hydroxycinnamic acid glycoside esters, veratric acid esters, or p-nitrophenyl acetate) and released free hydroxycinnamic acids (ferulic and coumaric acid) from arabinoxylan and milled wheat straw. These catalytic properties strongly suggest that X. polymorpha GH78 is a multifunctional enzyme. It is the first fungal enzyme that combines glycosyl hydrolase with esterase activities and may help this soft rot fungus to degrade lignocelluloses.

A spectrophotometric assay for the detection of fungal peroxygenases.

Rapid and simple spectrophotometric methods are required for the unambiguous detection of recently discovered fungal peroxygenases in vivo and in vitro. This paper describes a peroxygenase-specific assay using 5-nitro-1,3-benzodioxole as substrate. The product, 4-nitrocatechol, produces a yellow color at pH 7, which can be followed over time at 425 nm (ε(425)=9,700 M(-1) cm(-1)), and a red color when adjusted to pH >12, which can be measured in form of an end-point determination at 514 nm (ε(514)=11,400 M(-1) cm(-1)). The assay is suitable for detecting peroxygenase activities in complex growth media and environmental samples as well as for high-throughput screenings.

The aromatic peroxygenase from Marasmius rutola--a new enzyme for biosensor applications.

The aromatic peroxygenase (APO; EC 1.11.2.1) from the agraric basidomycete Marasmius rotula (MroAPO) immobilized at the chitosan-capped gold-nanoparticle-modified glassy carbon electrode displayed a pair of redox peaks with a midpoint potential of -278.5 mV vs. AgCl/AgCl (1 M KCl) for the Fe(2+)/Fe(3+) redox couple of the heme-thiolate-containing protein. MroAPO oxidizes aromatic substrates such as aniline, p-aminophenol, hydroquinone, resorcinol, catechol, and paracetamol by means of hydrogen peroxide. The substrate spectrum overlaps with those of cytochrome P450s and plant peroxidases which are relevant in environmental analysis and drug monitoring. In M. rotula peroxygenase-based enzyme electrodes, the signal is generated by the reduction of electrode-active reaction products (e.g., p-benzoquinone and p-quinoneimine) with electro-enzymatic recycling of the analyte. In these enzyme electrodes, the signal reflects the conversion of all substrates thus representing an overall parameter in complex media. The performance of these sensors and their further development are discussed.

Novel Fatty Acid Chain-Shortening by Fungal Peroxygenases Yielding 2C-Shorter Dicarboxylic Acids.

Unspecific peroxygenases (UPOs), the extracellular enzymes capable of oxygenating a potpourri of aliphatic and aromatic substrates with a peroxide as co-substrate, come out with a new reaction: carbon-chain shortening during the conversion of fatty acids with the well-known UPOs from Coprinopsis cinerea (rCciUPO) and Cyclocybe (Agrocybe) aegerita (AaeUPO). Although a pathway (Cα-oxidation) for shortening the hydrocarbon chain of saturated fatty acids has already been reported for the UPO from Marasmius rotula (MroUPO), it turned out that rCciUPO and AaeUPO shorten the chain length of both saturated and unsaturated fatty acids in a different way. Thus, the reaction sequence does not necessarily start at the Cα-carbon (adjacent to the carboxyl group), as in the case of MroUPO, but proceeds through the subterminal (ω-1 and ω-2) carbons of the chain via several oxygenations. This new type of shortening leads to the formation of a dicarboxylic fatty acid reduced in size by two carbon atoms in the first step, which can subsequently be further shortened, carbon by carbon, by the UPO Cα-oxidation mechanism.