Martin Hofrichter

Mineralization of 14C-labelled synthetic lignin and extracellular enzyme activities of the wood-colonizing ascomycetes Xylaria hypoxylon and Xylaria polymorpha

Two wood-dwelling ascomycetes, Xylaria hypoxylon and Xylaria polymorpha, were isolated from rotting beech wood. Lignin degradation was studied following the mineralization of a synthetic

Oxidations catalyzed by fungal peroxygenases.

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Directed Evolution of Unspecific Peroxygenase from Agrocybe aegerita

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Life in leaf litter: novel insights into community dynamics of bacteria and fungi during litter decomposition.

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5‐hydroxymethylfurfural conversion by fungal aryl‐alcohol oxidase and unspecific peroxygenase

-labelled lignin in solid and liquid media. Approximately 9% of the synthetic lignin was mineralized by X. polymorpha during the growth on beech wood meal, and the major fraction (65.5%) was polymerized into water- and dioxan-insoluble material. Both fungi produced laccase (up to 1,200xa0U l−1) in an agitated complex medium based on tomato juice; peroxidase activity (<80xa0U l−1) was only detected for X. polymorpha in soybean meal suspension. The enzymatic attack of X. polymorpha on beech wood resulted in the formation of three fractions of water-soluble lignocellulose fragments with molecular masses of 200, 30 (major fraction) and 3xa0kDa, as demonstrated by high-performance size exclusion chromatography. This fragment pattern differs considerably from that of the white-rot fungus Bjerkandera adusta, which preferentially released smaller lignocellulose fragments (0.8xa0kDa). The finding that X. polymorpha produced large lignocellulose fragments, along with the fact that high levels of hydrolytic enzymes (esterase 630xa0U l−1, xylanase 120xa0U l−1) were detected, indicates the cleavage of bonds between the lignin and hemicellulose moieties.

Widespread occurrence of expressed fungal secretory peroxidases in forest soils

The degradation of the nitroaromatic pollutant 2,4,6-trinitrotoluene (TNT) by the manganese-dependent peroxidase (MnP) of the white-rot fungus Phlebia radiata and the main reduction products formed were investigated. In the presence of small amounts of reduced glutathione (10 mM), a concentrated cell-free preparation of MnP from P. radiata exhibiting an activity of 36 nkat/ml (36 nmol Mn(II) oxidized per sec and per ml) transformed 10 mg/l of TNT within three days. The same preparation was capable of completely transforming the reduced derivatives of TNT. When present at 10 mg/l, the aminodinitrotoluenes were transformed in less than two days and the diaminonitrotoluenes in less than three hours. Experiments with 14C-U-ring labeled TNT and 2-amino-4,6-dinitrotoluene showed that these compounds were mineralized by 22% and 76%, respectively, within 5 days. Higher concentrations of reduced glutathione (50 mM) led to a severe inhibition of the degradation process. It is concluded that Phlebia radiata is a good candidate for the biodegradation of TNT as well as its reduction metabolites.

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

The enzymatic oxyfunctionalization of organic molecules under physiological conditions has attracted keen interest from the chemical community. Unspecific peroxygenases (EC 1.11.2.1) secreted by fungi represent an intriguing enzyme type that selectively transfers peroxide-borne oxygen with high efficiency to diverse substrates including unactivated hydrocarbons. They are glycosylated heme-thiolate enzymes that form a separate superfamily of heme proteins. Among the catalyzed reactions are hydroxylations, epoxidations, dealkylations, oxidations of organic hetero atoms and inorganic halides as well as one-electron oxidations. The substrate spectrum of fungal peroxygenases and the product patterns show similarities both to cytochrome P450 monooxygenases and classic heme peroxidases. Given that selective oxyfunctionalizations are among the most difficult to realize chemical reactions and that respectively transformed molecules are of general importance in organic and pharmaceutical syntheses, it will be worth developing peroxygenase biocatalysts for industrial applications.

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

ABSTRACT 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 k cat/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.