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Within the kingdom of fungi, the division Basidiomycota represents more than 30,000 species, some with huge genomes indicating great metabolic potential. The fruiting bodies of many basidiomycetes are appreciated as food ("mushrooms"). Solid-state and submerged cultivation processes have been established for many species. Specifically, xylophilic fungi secrete numerous enzymes but also form smaller metabolites along unique pathways; both groups of compounds may be of interest to the food processing industry. To stimulate further research and not aim at comprehensiveness in the broad field, this review describes some recent progress in fermentation processes and the knowledge of fungal genetics. Processes with potential for food applications based on lipases, esterases, glycosidases, peptidases and oxidoreductases are presented. The formation and degradation of colourants, the degradation of harmful food components, the formation of food ingredients and particularly of volatile and non-volatile flavours serve as examples. In summary, edible basidiomycetes are foods—and catalysts—for food applications and rich donors of genes to construct heterologous cell factories for fermentation processes. Options arise to support the worldwide trend toward greener, more eco-friendly and sustainable processes. [ABSTRACT FROM AUTHOR]

A combined system of a unique dye-decolorizing peroxidase (Ftr-DyP) and a laccase obtained from the basidiomycete Funalia trogii converted the precursor (+)-valencene completely to the high-value grapefruit flavour constituent (+)-nootkatone, reaching a concentration maximum of 1100 mg/L. In the presence of 1 mM Mn2+ and 2.5 mM p-coumaric acid, (+)-nootkatone was the predominating volatile product, and only traces of substrate and the nootkatols were detectable after 24 h. Hence, the two-enzyme-system reproduced the oxidizing activity observed before for the crude culture supernatant. The newly discovered Ftr-DyP was purified, sequenced and further characterized as a thermostable, non-glycosylated protein with a pH-optimum in the acidic range and a calculated mass of 52.3 kDa. Besides the typical activity of DyPs towards anthraquinone dyes, Ftr-DyP also oxidized Mn2+ and showed activity in the absence of hydrogen peroxide. Neither the DyP from Mycetinis scorodonius nor the manganese peroxidase from Nematoloma frowardii were able to replace Ftr-DyP in this reaction. A hypothetical reaction mechanism is presented. [ABSTRACT FROM AUTHOR]

The feruloyl esterase (FAE) gene EST1 from the basidiomycete Pleurotus sapidus was heterologously expressed in Escherichia coli and Pichia pastoris. Catalytically active recombinant Est1 was secreted using P. pastoris as a host. For expression in P. pastoris, the expression vector pPIC9K was applied. The EST1 gene was cloned with an N-terminal α-mating factor pre-pro sequence and expressed under the control of a methanol inducible alcohol oxidase 1 promotor. Est1 was purified to homogeneity using ion exchange and hydrophobic interaction chromatography. The recombinant Est1 showed optima at pH 5.0 and 50 °C, and released ferulic acid from saccharide esters and from the natural substrate destarched wheat bran. Substrate specificity profile and descriptor-based analysis demonstrated unique properties, showing that Est1 did not fit into the current FAE classification model. Transferuloylation synthesis of feruloyl-saccharide esters was proven for mono- and disaccharides. [ABSTRACT FROM AUTHOR]

Investigating the secretion of esterases by the basidiomycetous fungus Pleurotus sapidus in a Tween 80-rich nutrient medium, an enzyme was discovered that hydrolyzed the ester bond of feruloylated saccharides. The enzyme was purified by ion exchange and size exclusion chromatography. Polyacrylamide gel electrophoresis analysis showed a monomeric protein of about 55 kDa. The complete coding sequence with an open reading frame of 1,665 bp encoded a protein (Est1) consisting of 554 amino acids. The enzyme showed no significant homology to any published feruloyl esterase sequences, but possessed putative conserved domains of the lipase/esterase superfamily. Substrate specificity studies classified the new enzyme as type-A feruloyl esterase, hydrolyzing methyl ferulate, methyl sinapate, and methyl p-coumarate but no methyl caffeate. The enzyme had a pH optimum of 6 and a temperature optimum at 50 °C. Ferulic acid was efficiently released from ferulated saccharides, and the feruloyl esterase exhibited moderate stability in biphasic systems (50 % toluene or tert-butylmethyl ether). [ABSTRACT FROM AUTHOR]

The entomopathogenic ascomycete Paecilomyces farinosus (alternative name Isaria farinosa) synthesized a hydrophobin, irrespective of being grown in submerged or surface culture. The protein was extracted using trifluoroacetic acid and purified using preparative HPLC and SDS-PAGE. Partial sequences were obtained using ESI-MS/MS. The peptides were used as a start to apply a 'template switching oligo' protocol to elucidate the complete open reading frame of P. farinosus hydrophobin 1 ( pfah1). The deduced protein sequence comprised 107 amino acids (10.7 kDa) including a 16 amino acid long hydrophobic signal peptide, showed a calculated pI of 4.56, and was interrupted by one intron. Phylogenetic analyses revealed relationships to hydrophobins of the ascomycetes Magnaporthe grisea and Metarhizium anisopliae. Based on solubility, hydropathy pattern and phylogeny PfaH1 was assigned to the class Ia hydrophobins. (© 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [ABSTRACT FROM AUTHOR]

Two extracellular esterases were produced in submerged cultures of the basidiomycete Pleurotus sapidus. A foam fractionation device was designed and employed for the isolation of the esterolytic enzymes. The recovery of enzyme activity in the liquefied foam strongly depended on the superficial gas velocity. High purification and enrichment factors ( Ea = 62.0, P = 15.5) were achieved using nitrogen at 1.87 cm min−1 within 100 min. Increasing the superficial gas velocity to 2.49 cm min−1 improved the recovery of total esterase activity in the foam to >95% at the expense of reduced enrichment and purification factors. Differences in their physicochemical characteristics resulted in differing foaming properties of the two esterases secreted by P. sapidus. By variation of the pH value of the culture medium and addition of Triton X-100, both esterases were successively and quantitatively transferred into the foam in a two-step fractionation process. [ABSTRACT FROM AUTHOR]

The lipase Lip2 of the edible basidiomycete, Pleurotus sapidus, is an extracellular enzyme capable of hydrolysing xanthophyll esters with high efficiency. The gene encoding Lip2 was expressed in Escherichia coli TOP10 using the gene III signal sequence to accumulate proteins in the periplasmatic space. The heterologous expression under control of the araBAD promoter led to the high level production of recombinant protein, mainly as inclusion bodies, but partially in a soluble and active form. A fusion with a C-terminal His tag was used for purification and immunochemical detection of the target protein. This is the first example of a heterologous expression and periplasmatic accumulation of a catalytically active lipase from a basidiomycete fungus. [ABSTRACT FROM AUTHOR]

Exposition to UV-A light stimulated the growth and synthesis of raspberry ketone in submerged cultures of the basidiomycete Nidula niveotomentosa. To investigate the fungus' response to UVA light differentially expressed proteins were identified by means of 2D-electrophoresis. Light induced proteins were de novo sequenced by ESI-MS/MS spectrometry, and the encoding nucleotide sequences were cloned from cDNA or genomic DNA. The spectrum of UV-A light-induced proteins comprised several stress-related proteins including a catalase, heat-shock proteins, glutathione s-transferases and proteasomes. In addition, growth-related enzymes of the citric cycle were found to be up-regulated as a response to irradiation with UV-A. [ABSTRACT FROM AUTHOR]

An extracellular enzyme capable of efficient hydrolysis of xanthophyll esters was purified from culture supernatants of the basidiomycete Pleurotus sapidus . Under native conditions, the enzyme exhibited a molecular mass of 430 kDa, and SDS-PAGE data suggested a composition of eight identical subunits. Biochemical characterisation of the purified protein showed an isoelectric point of 4.5, and ideal hydrolysis conditions were observed at pH 5.8 and 40°C. Partial amino acid sequences were derived from N-terminal Edman degradation and from mass spectrometric ab initio sequencing of internal peptides. An 1861-bp cDNA containing an open reading frame of 1641 bp was cloned from a cDNA library that showed ca. 40% homology to Candida rugosa lipases. The P. sapidus carboxylesterase represents the first enzyme of the lipase/esterase family from a basidiomycetous fungus that has been characterised at the molecular level. [ABSTRACT FROM AUTHOR]

Abstract: Little is known on basidiomycete sources of feruloyl esterases (FAEs), although many wood-rotting representatives of these fungi typically grow on feruloyl-rich substrates. A major reason is that the almost ubiquitous presence of laccases interferes with the detection of FAE activity. Laccases polymerize the liberated ferulic acid (FA) in situ, thus detracting the product of enzymatic hydrolysis from its detection. A rapid HPLC-UV method was developed to detect the loss of FA, but also to quantify the hydrolysis of FA esters. The method allows at the same time to evaluate the substrate specificity of a FAE. Forty one basidiomycetes were tested for their FAE activities, and 25 out of the set were positive. The basidiomycetes hydrolyzing cinnamates with the highest conversion rates were Auricularia auricula-judae and Marasmius scorodonius. Moreover, a new FAE inducer, the nonionic detergent Tween 80, was found. This is the first comprehensive study on basidiomycete sources of FAEs. [Copyright &y& Elsevier]

A laccase of the basidiomycete Pleurotus pulmonarius (PpuLcc) possessed strong decolorizing abilities towards artificial and natural dyes. The PpuLcc was purified from the culture supernatant via FPLC, and the corresponding gene cloned and expressed in Pichia pastoris GS115. To examine the impact of the C-terminal tail region and the signal peptide on the recombinant expression of PpuLcc, a non-modified version or different truncations (−2, −5, −13 AA) of the target protein were combined with different secretion signals. Heterologous expression of codon optimized constructs resulted in extracellular activities of the PpuLcc variants of up to 7000 U L −1 (substrate ABTS) which was six times higher than non-codon optimized constructs. In contrast to previous works, altering the C-terminal end of the protein did not influence kinetic parameters or the rate of expression. The His -Tag purified enzymes showed high temperature optima (50–70 °C) and thermo stability. All of the recombinant variants degraded triarylmethane and azo dyes. Rapid bleaching of β -carotene (E 160a) and the polyene acid norbixin (E 160b) using a laccase was found for the first time. Thus, the enzyme may be useful in decolorizing unwanted polyene pigments, for example from the processing of cheese, bakery, desserts, ice cream or coloured casings. [ABSTRACT FROM AUTHOR]

Extracellular esterase activity was detected in submerged cultures of Rhizoctonia solani grown in the presence of sugar beet pectin or Tween 80. Putative type B feruloyl esterase (FAE) coding sequences found in the genome data of the basidiomycete were heterologously expressed in Pichia pastoris . Recombinant enzyme production on the 5-L bioreactor scale (Rs p CAE: 3245 U L −1 ) exceeded the productivity of the wild type strain by a factor of 800. Based on substrate specificity profiling, the purified recombinant Rs p CAE was classified as a p -coumaroyl esterase ( p CAE) with a pronounced chlorogenic acid esterase side activity. The Rs p CAE was also active on methyl cinnamate, caffeate and ferulate and on feruloylated saccharides. The unprecedented substrate profile of Rs p CAE together with the lack of sequence similarity to known FAEs or p CAEs suggested that the Rs p CAE represents a new type of enzyme. Hydroxycinnamic acids were released from agro-industrial side-streams, such as destarched wheat bran (DSWB), sugar beet pectin (SBP) and coffee pulp (CP). Overnight incubation of coffee pulp with the Rs p CAE resulted in the efficient release of p -coumaric (100%), caffeic (100%) and ferulic acid (85%) indicating possible applications for the valorization of food processing wastes and for the enhanced degradation of lignified biomass. [ABSTRACT FROM AUTHOR]

Ustilago maydis , an edible mushroom growing on maize ( Zea mays ), is consumed as the food delicacy huitlacoche in Mexico. A chlorogenic acid esterase from this basidiomycete was expressed in good yields cultivating the heterologous host Pichia pastoris on the 5 L bioreactor scale (reUmChlE; 45.9 U L −1 ). In contrast to previously described chlorogenic acid esterases, the reUmChlE was also active towards feruloylated saccharides. The enzyme preferred substrates with the ferulic acid esterified to the O-5 position of arabinose residues, typical of graminaceous monocots, over the O-2 position of arabinose or the O-6 position of galactose residues. Determination of k cat / K m showed that the reUmChlE hydrolyzed chlorogenic acid 18-fold more efficiently than methyl ferulate, p -coumarate or caffeate. Phenolic acids were released by reUmChlE from natural substrates, such as destarched wheat bran, sugar beet pectin and coffee pulp. Treatment of wheat dough using reUmChlE resulted in a noticeable softening indicating a potential application of the enzyme in bakery and confectionery. [ABSTRACT FROM AUTHOR]

Highlights: [•] Cultivation of shear stress sensitive E. ramulus and two basidiomycetes in a wave reactor. Comparison of the cultivations to STR cultivations. [•] Even cultivations that depend on strict anaerobic conditions were performed successfully in batch and fed-batch mode in the wave system. [•] Huge impact on morphology and resulting enzymatic activity was observed in the fungal cultures when comparing wave reactor and STR cultivations. [ABSTRACT FROM AUTHOR]

Abstract: A dioxygenase from the edible basidiomycete Pleurotus sapidus, originally researched because of its distinct ability to convert the sequiterpene (+)-valencene to the valuable grapefruit aroma (+)-nootkatone, was identified as a potent lipoxygenase (LOXPsa1). Kinetic parameters, pH and temperature optima of the pure recombinant enzyme were determined using linoleic acid as the substrate. K m, v max, and k cat were 40.3μM, 130.3U mg−1, and 157s−1, respectively. The maximal enzymatic activity was found at pH 7.0 and 35°C. Showing high specificity toward free linoleic acid, the enzyme was classified as lipoxygenase type 1. Conversion of linoleic acid yielded mainly (S)-13-hydroperoxy-9Z,11E-octadecadienoic acid (94% ee), as was confirmed by chiral HPLC analysis of the hydroperoxides. The amino acid sequence showed homology to lipoxygenases catalyzing S stereospecific oxygenation, and thus the enzyme was characterized as a 13S-lipoxygenase. This is the first lipoxygenase described to accept terpene hydrocarbons as substrates. [Copyright &y& Elsevier]

Abstract: Mycelium of the basidiomycete Pleurotus sapidus known to contain a novel dioxygenase was used for the bioconversion of car-3-ene [I]. After 4h of incubation 25.3mgL−1 car-3-en-5-one [V], 5.4mgL−1 car-3-en-2-one [VII], and 7.3mgL−1 car-2-en-4-one [XV] accumulated as major oxidation products. The identity of the respective carenones and their corresponding alcohols was confirmed by comparison with MS and NMR spectral data obtained for synthesized authentic compounds. The peak areas of oxidation products were at least five times higher as compared with autoxidation. A radical mechanism similar to lipoxygenase catalysis was proposed and substantiated with detailed product analyses. The reduction of assumed car-3-ene hydroperoxides to the corresponding alcohols evidenced the radical initiated formation of hydroperoxides and confirmed the regio- and stereo-selectivity of the dioxygenase. The introduction of molecular oxygen into the bicyclic car-3-ene [I] molecule occurred at allylic positions of a cyclic isopentenyl moiety with a pronounced preference for the position adjacent to the non-substituted carbon atom of the C–C-double bond. This co-factor independent selective oxygenation presents an alternative to P450 mono-oxygenase based approaches for the production of terpene derived flavor compounds, pharmaceuticals and other fine chemicals. [Copyright &y& Elsevier]

Abstract: Flammulina velutipes enjoys high popularity as an edible mushroom in Asian cuisines. Investigating the secretion of peptidases in nutrient media enriched with gluten, an enzyme was noticed that catalyzed the deamidation of l-asparagine and l-glutamine. The enzyme was purified to electrophoretic homogeneity by foaming and SEC. PAGE analysis revealed a protein of about 85kDa with 13kDa subunits indicating a hexameric protein. Degenerated primers were deduced from peptide fragments and the complete coding sequence of 372bp was determined. The gene of Flammulina velutipes asparaginase (FvNase) over expressed in E. coli achieved an l-asparagine-hydrolyzing activity of 16U/mL in crude extract, which was five times higher than its l-glutamine-hydrolyzing ability. The enzyme showed a pH-optimum at pH 7, remarkable tolerance towards elevated temperature and sodium chloride concentration in both the native and recombinant form, and no significant homology to any conserved domains of published asparaginases or glutaminases. [ABSTRACT FROM AUTHOR]

Abstract: The 1641bp cDNA encoding an extra-cellular lipase of the basidiomycete Pleurotus sapidus (Lip2) was cloned from a cDNA library. Expression of the cDNA in Escherichia coli, with and without signal sequence, led to the production of recombinant Lip2, mainly as inclusion bodies with low catalytic activity. Refolding yielded catalytically active protein. A C-terminal His tag was used for purification and immunochemical detection. The recombinant lipase hydrolysed xanthophyll esters with high efficiency, and omitting the signal sequence did not alter the catalytic properties. The P. sapidus lipase represents the first enzyme of the lipase/esterase family from a basidiomycetous fungus characterised on the molecular level and expressed in a manageable host. [Copyright &y& Elsevier]

Abstract: A laccase (E.C. 1.10.3.2) from Trametes spec. was isolated from aqueous media using foam fractionation. The pH value, superficial velocity, foaming period, and temperature were varied to optimise the transport of the active enzyme into the foam phase. Several detergents were added in varying concentrations to form and stabilize the foam, and the cationic detergent cetyltrimethylammonium bromide (CTAB) proved to be the most appropriate. From water as a model system, maximum recovery rates of 94% of laccase activity were achieved at pH 6.0 in 6min. For separation of the enzyme from protein rich culture media, the operation conditions had to be adjusted. At pH 5.4, 89% of laccase activity was transported into the foam phase after 15min. The method established was successfully applied to the isolation of an active laccase isoenzyme from submerged cultures of the basidiomycete Pleurotus sapidus. [Copyright &y& Elsevier]