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A prolyl endopeptidase, FvpP, was identified in the culture supernatant of the basidiomycete Flammulina velutipes and functionally expressed for gene verification in Aspergillus oryzae. The wild-type FvpP with a molecular mass of 50 kDa under denaturing conditions, exhibited optima at pH 5, and 45 °C and an isoelectric point of 3.8. Five mM Fe2+ and Fe3+ activated the prolyl endopeptidase, while it was not affected by 10 mM CaCl2, EDTA, and Pepstatin A. Five mM Cu2+, Mn2+, Zn2+, Ni2+, Co2+ and low Z-Pro-prolinal concentrations decreased the enzyme activity. Concluded from comparative RP-HPLC and high-resolution QTOF-MS/MS analyses of the hydrolytic cleavage products of gliadin, casein and gelatin, FvpP possessed a P1-P1′-substrate specificity similar to the prolyl endopeptidase from Aspergillus niger (An-Pep), although the sequence similarity was only 39%. FvpP and An-Pep (S28.004; MEROPS) are unique in the serine peptidase family S28, because they share more sequence similarity with Pro-X carboxypeptidase (S28.001; MEROPS) and dipeptidyl peptidase II (S28.002; MEROPS) than with prolyl oligopeptidases of the serine peptidase family S09. As a potential degrader of the antigenic epitopes of α-gliadin, FvpP might provide an efficient tool for the processing of wheat and other gluten containing cereals to better digestible foods for celiac patients.

A glycosidase of the basidiomycete Bjerkandera adusta (BadGluc) was found in screenings to possess a strong decolorizing ability towards malvidin-3-galactoside, an anthocyanin abundant in various berry fruits. The BadGluc was purified from the culture supernatant via FPLC, and the corresponding gene was identified which showed low similarity to other characterized glucosidases. Scanning the primary sequence with PROSITE no active site motif was detected. Eventually, a specific 18 aa consensus pattern was identified manually. The active site motif possessed an undescribed sequence which was only found in a few hypothetical proteins. The corresponding gene was cloned and expressed in Pichia pastoris GS115 yielding activities up to 100 U/L using 4-nitrophenyl-β-d-glucopyranoside (pNPG) as substrate. The enzyme possessed a good temperature (70% after 1 h at 50°C) and pH stability (70% between pH 2 and 7.5), and preferably catalysed the hydrolysis of delphinidin-3-glucoside and cyanidin-3-glucoside, regardless of the position of the terminal Hexa-His tag. This novel glucosidase worked in aqueous solution as well as on pre-stained fabrics making it the first known candidate anthocyanase for applications in the detergent and food industries.

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.

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.

Some l-arginyl dipeptides were recently identified as salt taste enhancers, thus opening the possibility to reduce dietary sodium uptake without compromising palatability. A screening of 15 basidiomycete fungi resulted in the identification of 5 species secreting a high peptidolytic activity (>3 kAU/mL; azocasein assay). PFP-LC-MS/MS and HILIC-MS/MS confirmed that l-arginyl dipeptides were liberated when casein or lysozyme served as substrate. Much higher yields of dipeptides (42–75 μmol/g substrate) were released from lysozyme than from casein. The lysozyme hydrolysate generated by the complex set of peptidases of Trametes versicolor showed the highest l-arginyl dipeptide yields and a significant salt taste enhancing effect in a model cheese matrix and in a curd cheese. With a broad spectrum of novel specific and nonspecific peptidases active in the slightly acidic pH range, T. versicolor might be a suitable enzyme source for low-salt dairy products.

Malvidin-3-O-galactoside, a widely occurring anthocyanin, was incubated with laccases (LCCs) from the basidiomycetes Pleurotus pulmonarius (Ppu) and Funalia trogii (Ftr), and with bilirubin oxidases (BODs) from Bacillus pumilus (Bpu) and Magnaporthe oryzae (Mor). LC-MS and LC-MS2 analyses identified 2,6-dimethoxy-1,4-benzoquinone (DMBQ) and tentatively identified the corresponding coumarin galactoside fragment (m/z 355) resulting from the cleavage of the substrate. The enzymes cleaved malvidin-3-O-galactoside within a few minutes under acidic and neutral conditions and in the absence of a mediator. The fungal LCCs were more active at pH 4 and 5.5, while the BODs worked best in the neutral range. The initial cleavage product coumarin galactoside was further oxidised, as indicated by a continuous decline of concentration and concurrent formation of a brown precipitate. Based on the structure of the products and the general LCC mechanism, a reaction sequence was proposed starting with a stable tertiary carbocation, attack of molecular oxygen and formation of a 1,2-dioxetane intermediate between B and C rings which is finally split into the two carbonyl products. Similar reactions may be catalysed by plant oxidases in anthocyanin-rich tissues resulting in the formation of the strong antibacterial and cytotoxic DMBQ.

Fungal laccases with their pH optima in the acidic range may be suitable to improve the physical properties of lactic acid fermented dairy foods, such as yoghurt, by cross-linking of the milk proteins. Ions naturally occurring in milk may inhibit the laccase catalysed reaction. Hence, laccase activity was induced and accumulated in cultures of 20 fungal species and compared to the reference enzyme Laccase C (Lcc C). Laccases of Funalia trogii , Pleurotus flabellatus , Pleurotus eryngii , Pleurotus lampas and Trametes versicolor showed comparable or higher activities than Lcc C in the presence of calcium, chloride, magnesium, potassium, phosphate and citrate. In a cross-linking experiment, P. eryngii laccase (Lcc Per) converted casein completely into protein aggregates with higher molecular masses. A combination of 0.1 U/g Lcc Per and 5 mmol/L vanillin as mediator enhanced all rheological parameters, storage modulus G′, loss modulus G″ and complex shear viscosity η* of a skimmed milk yoghurt by approximately 30%, while a higher dose (1 U/g) resulted in fragmentation of caseins and weakening of the gel. At least 1 U/g of the more strongly inhibited Lcc C was required for measurable cross-linking, but no fragmentation occurred.

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.

Lactic acid fermented foods, such as yoghurt, suffer from structural losses in post-processing steps due to their shear sensitivity. The acidic pH optimum of fungal laccases offers potential to compensate for these losses and enhance the textural characteristics by cross-linking of milk proteins. Physical properties of skimmed milk yoghurt, as measured by dynamic oscillation rheology, changed upon addition of laccase alone or in combination with food-grade mediators. Among seven mediators examined, vanillin, vanillic acid, and caffeic acid were found to be most efficient. Variation of enzyme activity, mediator type and concentration showed best viscoelastic properties for the combination of 3 U laccase per gram yoghurt and 5 mmol L−1 caffeic acid. Evaluation of the loss factor, tan δ, showed increased elastic properties. SDS-PAGE showed a changed pattern of protein bands after the treatments. Overall, the combination of laccase with common food components provides an alternative to improve yoghurt texture.

The potentially carcinogenic and neurotoxic compound acrylamide occurs in commonly consumed starch-based foods. Asparaginases hydrolyze the precursor asparagine to aspartate and ammonia and promise a potential way to reduce the amount of free asparagine in the starting materials of food production. Coeliac disease is an autoimmune disorder which is considered to be present in approximately 1% of population. Gluten-derived peptides were identified with important physiological relevance due to their resistance to gastric and pancreatic peptidases. Prolyl-specific endopeptidases cleave the toxic peptides and thus alleviate gluten intolerance. The smoke flavor compound vinylguaiacol was produced via microbial transformation of ferulic acid. The advantages of the cold flavor generation are obvious, as the risk of polycyclic aromatic hydrocarbon formation is completely eliminated.

Four extracellular enzymes, a versatile peroxidase, a manganese peroxidase, a dye-decolorizing peroxidase and a lignin peroxidase were discovered in liquid cultures of the basidiomycete Bjerkandera adusta. All of them cleaved β-carotene effectively. Expression was enhanced in the presence of β-carotene or Coomassie Brilliant Blue and peaked after 7-9 days. The monomeric proteins were purified by ion exchange and size exclusion chromatography and exhibited molecular masses of 41, 43, 51 and 43 kDa, respectively. The coding sequences showed homologies from 61 to 89 % to peroxidases from other basidiomycetes. The novel enzymes retained strong activity even in the absence of hydrogen peroxide and at alkaline pH. De-staining of fabrics using detergent-tolerant enzymes may help to save the most important bio-resources, energy and water, in washing processes and led to green processes in textile cleaning.

An extracellular laccase (Lacc10) was discovered in submerged cultures of Pleurotus ostreatus var. florida bleaching s-carotene effectively without the addition of a mediator (650 mU/L, pH 4). Heterologous expression in P. pastoris confirmed the activity and structural analyses revealed a carotenoid-binding domain, which formed the substrate-binding pocket and is reported here for the first time. In order to increase activity, 106 basidiospore-derived monokaryons and crosses of compatible progenies were generated. These showed high intraspecific variability in growth rate and enzyme formation. Seventy-two homokaryons exhibited a higher activity-to-growth-rate-relation than the parental dikaryon, and one isolate produced a very high activity (1800 mU/L), while most of the dikaryotic hybrids showed lower activity. The analysis of the laccase gene of the monokaryons revealed two sequences differing in three amino acids, but the primary sequences gave no clue for the diversity of activity. The enzyme production in submerged cultures of monokaryons was stable over seven sub-cultivation cycles.

An intracellular alcohol oxidase (AOX) was isolated from the white-rot basidiomycete Phanerochaete chrysosporium (Pch), grown on l-lactate induction medium, and purified to electrophoretic homogeneity. The dimeric protein consisted of two identical 75kDa subunits. The open reading frame of 1,956bp resulted in a monomer consisting of 651 amino acids. The enzyme showed a pI at 5.4, a pH optimum of 9, a temperature optimum at 50°C, possessed putative conserved domains of the GMC superfamily, a FAD binding domain, and showed up to 86% homology to alcohol oxidase sequences of Gloeophyllum trabeum and Coprinopsis cinerea. As was shown for the first time for an AOX from a basidiomycete, not only methanol, but also lower primary alcohols and glycerol were accepted as substrates. An assay based on aldehyde dehydrogenase confirmed d-glyceraldehyde as the product of the reaction. A bioprocess based on this enzyme could alleviate the problems associated with the huge side-stream of glycerol occurring during the manufacture of biodiesel, yielding the green oxidant hydrogen peroxide.

The first heterologous expression of an iron-containing lipoxygenase from a basidiomycete in Pichia pastoris is reported. Five different expression constructs of the lipoxygenase gene LOX1 from Pleurotus sapidus were cloned and successfully transferred into P. pastoris SMD1168, but only one pPIC9K vector construct was functionally expressed. In this construct the vector-provided α-factor signal sequence was replaced by insertion of a second Kozak sequence between the signal sequence and the LOX1 gene. His(+) transformants were screened for their level of resistance to geneticin (G418). Lox1 was expressed under different culture conditions and purified using the N-terminal His-tag. Relative enzyme activity increased significantly 48h after methanol induction and was highest with 2mll(-1) inducer. The recombinant enzyme showed an optimal lipoxygenase activity at pH 7 and 30-35°C and a vmax like the wild-type enzyme.

A selection of Pleurotus spp. was screened for intracellular lipoxygenase activity, and strains with distinguished activities were chosen for a screening on the molecular level. Lipoxygenase genes from five different Pleurotus spp. were amplified from the corresponding cDNA, functionally expressed using a cold shock expression system in Escherichia coli BL21DE3 Star cells and characterized for specific activity and reaction optima. All lipoxygenase sequences coded for proteins of 643 amino acids, sharing similarities >95% among each other and to a previously characterized LOXPsa1 from Pleurotus sapidus. Lipoxygenase activities were quantified using linoleic acid as substrate and reached similar values ranging from 95 U/mg to 118 U/mg, with Km values between 58 and 106 μM. Optimum reaction conditions were pH 7 and 30–35 C. However, the uncommon trait of accepting (+)-valencene, a sesquiterpene hydrocarbon substrate, differed strongly between two clusters of highly homologous sequences. No exchange of amino acids adjacent to the active site was found. Cloning and expression of a truncated LOXPsa1 sequence missing 144 amino acid residues of the N-terminal barrel domain yielded soluble protein but no measurable activity.

Benzoic acid esterases and ferulic acid esterases (FAE) are enzymes with different profiles of substrate specificity. An extracellular esterase (EstBC) from culture supernatants of the edible basidiomycete fungus Auricularia auricula-judae was purified by anion exchange chromatography, followed by preparative isoelectric focusing and hydrophobic interaction chromatography. EstBC showed a molecular mass of 36 kDa and an isoelectric point of 3.2 along with broad pH and temperature windows similar to fungal FAEs. However, EstBC exhibited also characteristics of a benzoic acid esterase acting on both benzoates and cinnamates, and most efficiently on methyl and ethyl benzoate, methyl 3-hydroxybenzoate and methyl salicylate. Feruloyl saccharides as well as lipase substrates, such as long chain fatty acids esterified with glycerol, polyethoxylated sorbitan and p -nitrophenol were not hydrolyzed. Protein database analyses with tryptic peptides of EstBC solely yielded hits regarding hypothetical proteins belonging to the alpha/beta hydrolase family. The uncommon substrate specificity of EstBC concomitant with a lack of sequence homology to known enzymes suggests a new type of enzyme.

A decarboxylase (IfPAD) from the ascomycete Isaria farinosa converted ferulic acid to 4-vinylguaiacol (4-VG), a volatile which imparts the distinct "smoke flavor" of pyrolized wood. The activity was enhanced by adding (E)-ferulic acid to the culture medium and peaked with 3.6 U g-1 mycelium (1 μmol 4-VG min-1). The coding sequence of 543 bp was translated into a 25 kDa protein with a homology of 91 % to putative phenolic acid decarboxylases of its teleomorph, Cordyceps militaris, and Beauveria bassiana, the anamorph of Cordyceps bassiana. Cold shock expression in Escherichia coli yielded 411 U g-1 wet mass. Substrate conversion required a hydroxyl substituent para to a trans-unsaturated C3-side chain of the aromatic ring. Km and kcat/Km values were determined to 0.3 mM and 78.4 mM-1s-1 for p-coumaric acid and 1.9 mM and 45.1 mM-1s-1 for (E)-ferulic acid, respectively. The native enzyme and its recombinant counterpart showed pH-optima at pH 6.0 and pH 5.5, and low temperature optima of 19 °C and 14 °C, respectively. IfPAD produced 4-VG from destarched wheat bran and sugar beet fiber, confirming activity on complex plant biomass. This is the first report on the biochemical characterization of a phenolic acid decarboxylase from a filamentous ascomycete.

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 pCAE: 3245UL-1) exceeded the productivity of the wild type strain by a factor of 800. Based on substrate specificity profiling, the purified recombinant Rs pCAE was classified as a p-coumaroyl esterase (pCAE) with a pronounced chlorogenic acid esterase side activity. The Rs pCAE was also active on methyl cinnamate, caffeate and ferulate and on feruloylated saccharides. The unprecedented substrate profile of Rs pCAE together with the lack of sequence similarity to known FAEs or pCAEs suggested that the Rs pCAE 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 pCAE 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.

Agro-industrial side-streams are abundant and renewable resources of hydroxycinnamic acids with potential applications as antioxidants and preservatives in the food, health, cosmetic, and pharmaceutical industries. Feruloyl esterases (FAEs) from Schizophyllum commune were functionally expressed in Pichia pastoris with extracellular activities of 6000UL(-1). The recombinant enzymes, ScFaeD1 and ScFaeD2, released ferulic acid from destarched wheat bran and sugar beet pectin. Overnight incubation of coffee pulp released caffeic (>60%), ferulic (>80%) and p-coumaric acid (100%) indicating applicability for the valorization of food processing wastes and enhanced biomass degradation. Based on substrate specificity profiling and the release of diferulates from destarched wheat bran, the recombinant FAEs were characterized as type D FAEs. ScFaeD1 and ScFaeD2 preferably hydrolyzed feruloylated saccharides with ferulic acid esterified to the O-5 position of arabinose residues and showed an unprecedented ability to hydrolyze benzoic acid esters.

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).

The first enzyme of the basidiomycete Piptoporus soloniensis, a peptidase (PsoP1), was characterized after isolation from submerged cultures, purification by fractional precipitation, and preparative native-polyarylamide gel electrophoresis (PAGE). The native molecular mass of PsoP1 was 38 kDa with an isoelectric point of 3.9. Similar to chymosin from milk calves, PsoP1 showed a maximum milk-clotting activity (MCA) at 35-40 °C and was most stable at pH 6 and below 40 °C. The complete inhibition by pepstatin A identified this enzyme as an aspartic peptidase. Electrospray ionization-tandem MS showed an amino acid partial sequence that was more homologous to mammalian milk clotting peptidases than to the chymosin substitute from a fungal species, such as the Zygomycete Mucor miehei. According to sodium dodecyl sulfate-PAGE patterns, the peptidase cleaved κ-casein in a way similar to chymosin and hydrolyzed β-casein slowly, as it would be expected from an efficient chymosin substitute.

The basidiomycete Laetiporus sulphureus (the edible 'chicken mushroom') imparted a strong seasoning odour when grown on a substrate containing wheat gluten as the sole carbon and nitrogen source. Secreted fungal peptidases hydrolysed the gluten substrate, and numerous potent heterocyclic flavours, among them sotolon (3-hydroxy-4,5-dimethyl-5H-furan-2-one) and other furanones, emerged in the liquefying hydrolysate. The fungal cells were supplemented with presumed precursors of sotolon, such as acetaldehyde, a-ketobutanoic acid, a-oxopentandioic acid, L -glutamic acid, L -isoleucine, and L-(+)-ascorbic acid. Upon addition of isoleucine and ferrous iron the formation of sotolon became measurable within 2 days. Small amounts of 4-hydroxyisoleucine (HIL) were identified in the culture supernatant of L. sulphureus by HPLC-FD and LC-SIM-MS/MS. An enzymatic formation of sotolon via a presumed isoleucine hydroxylase could not be excluded; however, balancing biochemical vs. chemical pathways, a slow chemical hydroxylation of the isoleucine released during the enzymatic hydrolysis of gluten seemed much more likely because ferric iron was present in the culture medium.

A total of 28 basidiomycetes and 16 substrates were investigated to detect extracellular peptidases for the hydrolysis of wheat gluten as a complex Koji substrate. In a first screening, basidiomycetes were grown in submerged cultures containing gluten as only carbon and nitrogen source. Growth rate, protein concentration and peptidase activity were monitored. Flammulina velutipes, Armillaria mellea, Trametes versicolor, Meripilus giganteus, Fomitopsis pinicola, Phanerochaete chrysosporium and Hericium erinaceus showed high peptidase activity. To evaluate the effects of complex substrates on peptidase generation, a set of experiments was performed using surface cultures of Flammulina velutipes. This fungus grew well on many of the substrates and yielded a maximum of over 160.000 arbitrary U mL−1 on the surface liquid released from gluten pellets. Gelatin zymography visualized a complex mixture of secreted peptidases. Highest peptidase yield was obtained from a koji-type culture: A maximum of 63% of the gluten was converted to free amino acids in 14 days by peptidase activities in the range of 80,000–90,000 aU mL−1. The dark brown hydrolysate smelled and tasted like meat broth with no detectable bitterness.

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.

A novel stain solving subtilisin-like peptidase (PPP1) was identified from the culture supernatant of the agaricomycete Pleurotus pulmonarius. It was purified to homogeneity using a sequence of preparative isoelectric focusing, anion exchange and size exclusion chromatography. Peptides were identified by ab initio sequencing (nLC-ESI-QTOF-MS/MS), characterizing the enzyme as a member of the subtilase family (EC 3.4.21.X). An expression system was established featuring the pPIC9K vector, an alternative Kozak sequence, the codon optimized gene ppp1 gene without the native signal sequence with C-terminal hexa-histidine tag, and Pichia pastoris GS115 as expression host. Intracellular active enzyme was obtained from cultivations in shake flasks and in a five liter bioreactor. With reaction optima of 40 °C and a pH > 8.5, considerable bleaching of pre-stained fabrics (blood, milk and India ink), and the possibility of larger-scale production, the heterologous enzyme is well suitable for detergent applications, especially at lower temperatures as part of a more energy- and cost-efficient washing process. Showing little sequence similarity to other subtilases, this unique peptidase is the first subtilisin-like peptidase from Basidiomycota, which has been functionally produced in Pichia pastoris.

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 6 min. 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 15 min. The method established was successfully applied to the isolation of an active laccase isoenzyme from submerged cultures of the basidiomycete Pleurotus sapidus.

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.

An extracellular chlorogenic acid esterase from Ustilago maydis (UmChlE) was purified to homogeneity by using three separation steps, including anion-exchange chromatography on a Q Sepharose FF column, preparative isoelectric focusing (IEF), and, finally, a combination of affinity chromatography and hydrophobic interaction chromatography on polyamide. SDS-PAGE analysis suggested a monomeric protein of ∼71 kDa. The purified enzyme showed maximal activity at pH 7.5 and at 37°C and was active over a wide pH range (3.5 to 9.5). Previously described chlorogenic acid esterases exhibited a comparable affinity for chlorogenic acid, but the enzyme from Ustilago was also active on typical feruloyl esterase substrates. Kinetic constants for chlorogenic acid, methyl p -coumarate, methyl caffeate, and methyl ferulate were as follows: K m values of 19.6 μM, 64.1 μM, 72.5 μM, and 101.8 μM, respectively, and k cat / K m values of 25.83 mM −1 s −1 , 7.63 mM −1 s −1 , 3.83 mM −1 s −1 and 3.75 mM −1 s −1 , respectively. UmChlE released ferulic, p -coumaric, and caffeic acids from natural substrates such as destarched wheat bran (DSWB) and coffee pulp (CP), confirming activity on complex plant biomass. The full-length gene encoding UmChlE consisted of 1,758 bp, corresponding to a protein of 585 amino acids, and was functionally produced in Pichia pastoris GS115. Sequence alignments with annotated chlorogenic acid and feruloyl esterases underlined the uniqueness of this enzyme.

An increasing demand of lignin-degrading enzymes makes an effective enrichment of laccase C from culture broth necessary. In this work, the continuous foam fractionation of laccase C was investigated as an alternative to other common applied methods, such as salting-out, dialyses or ultrafiltration, which are usually accompanied by losses of catalytic activity or low operational capacity. After optimization of different process parameters (feed position, gas flow rate and surfactant concentration) an enrichment ratio of 11.7 was achieved for laccase C along with 70.2% recovery. Feed position at the top of the foaming column increased the recovery. Increasing the gas flow rate led to a lower enrichment but higher recovery, and an increased outlet flow rate had only negative effect on the recovery. With increasing concentration of cetyl trimethyl ammonium bromide (CTAB), which was applied as a surfactant to enable foaming, the enrichment ratio decreased (from 11.7 to 1.8) and recovery increased (from 70.2 up to 80.2%). A for the enrichment ratio and recovery optimum CTAB-concentration of 0.4 mg/ml was found. The method has been proven to be effective for the enrichment of laccase C.

Peanut shells, a major waste stream of food processing, served as a renewable substrate for inducing the production of laccases by basidiomycetes. Of 46 surface cultures examined, 29 showed laccase activity under the experimental conditions. The edible fungus Pleurotus sapidus was selected as the most active producer, immobilized on the shells, and cultivated in the fed-batch mode. A continuous rise in laccase activity was found, indicating the inducibility of laccase secretion by the peanut shells and the reusability of the mycelium. Two laccase isoenzymes were purified by decoupled 2-D electrophoresis, and amino acid sequence information was obtained by electrospray ionization tandem mass spectrometry. cDNAs of the corresponding gene and another laccase were cloned and sequenced using a PCR-based screening of a synthesized P. sapidus cDNA library. Data bank searches against public databases returned laccases of P. ostreatus and P. sajor-caju as the best hits. The potential use of laccases by the food industry is discussed.

A β-carotene-degrading enzyme activity was observed in liquid cultures of the basidiomycete Ganoderma applanatum. Supplementing the cultures with β-carotene induced the bleaching activity. Purification via hydrophobic interaction, ion exchange and size exclusion chromatography followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) resulted in a single protein band. LC-ion-trap-MS analyses and gene amplification identified two manganese peroxidase isoenzymes with 97.8 % identity on the amino acid level. These showed an estimated molecular mass of 48 kDa and an isoelectric point of 2.6. Properties not yet described for other manganese peroxidases were hydrogen-peroxide-independent catalysis and two maxima of the bleaching activity, a distinct one at pH 5 and a lower one at pH 8. During simulated washing studies, the applicability of the isoenzymes for the brightening of carotenoids under alkaline conditions was proven. The new enzymes may replace common bleaching agents to produce environmentally more compatible detergent formulations.

The enzymatic hydrolysis of wheat gluten for the production of seasonings using mixtures of endo- and exopeptidases results in yields typically below 40%. Possible limiting parameters, such as an increasing product inhibition, autopeptidolysis of the enzymes, and lack of cleavage sites, were studied using novel peptidases from Flammulina velutipes or the commercial Flavourzyme preparation. Seven intermittent electrodialysis steps (10 g/L gluten and 10 kaU/mL) for the in situ removal of amino acids minimized the product inhibition. During 16 h, hydrolysis progressed nearly linearly. Compared to the batch control, a 3-fold yield of amino acids released was obtained indicating that an integrated product removal alleviates the problem of product inhibition. Autopeptidolysis, as shown using sodium dodecyl sulfate polyacrylamide gel electrophoresis and enzyme activity assays, was suppressed with increasing concentrations of competing gluten substrate. Peptidases of F. velutipes showed product inhibition only, whereas a combined effect of product inhibition and lack of cleavage sites was observed for Flavourzyme.

An extracellular feruloyl esterase (PeFaeA) from the culture supernatant of Pleurotus eryngii was purified to homogeneity using cation exchange, hydrophobic interaction, and size exclusion chromatography. The length of the complete coding sequence of PeFaeA was determined to 1668 bp corresponding to a protein of 555 amino acids. The catalytic triad of Ser-Glu-His demonstrated the uniqueness of the enzyme compared to previously published FAEs. The purified PeFaeA was a monomer with an estimated molecular mass of 67 kDa. Maximum feruloyl esterase (FAE) activity was observed at pH 5.0 and 50 °C, respectively. Metal ions (5 mM), except Hg(2+), had no significant influence on the enzyme activity. Substrate specificity profiling characterized the enzyme as a type A FAE preferring bulky natural substrates, such as feruloylated saccharides, rather than small synthetic ones. Km and kcat of the purified enzyme for methyl ferulate were 0.15 mM and 0.85 s(-1). In the presence of 3 M NaCl activity of the enzyme increased by 28 %. PeFaeA alone released only little ferulic acid from destarched wheat bran (DSWB), whereas after addition of Trichoderma viride xylanase the concentration increased more than 20 fold.

Wheat gluten hydrolysis, used to generate seasonings, was studied using peptidases from Flammulina velutipes or commercial Flavourzyme. L-amino acids were added in a range from 0.5 to 75.0 mM, and L-isoleucine, L-leucine, L-valine, and L-phenylalanine were identified as the strongest inhibitors for both enzyme mixtures. L-serine inhibited Flammulina velutipes peptidases only, while L-histidine and L-glutamine inhibited Flavourzyme peptidases only. To reduce product inhibition by released L-amino acids, electrodialysis was explored. An increase of the degree of hydrolysis of up to 60% for Flammulina velutipes peptidases and 31% for Flavourzyme compared to that for the best control batch was observed after applying an electrodialysis unit equipped with an ultrafiltration membrane for two times 1 h during the 20 h of hydrolysis. The total transfer of free L-amino acids into the concentrate reached 25-30% per hour. Peptides passed the membrane less easily, although the nominal cutoff was 4 kDa.

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.

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 85 kDa with 13 kDa subunits indicating a hexameric protein. Degenerated primers were deduced from peptide fragments and the complete coding sequence of 372 bp was determined. The gene of Flammulina velutipes asparaginase (FvNase) over expressed in E. coli achieved an L-asparagine-hydrolyzing activity of 16 U/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.

Efficient techniques for the isolation of enzymes from a microbial production culture are required to meet the growing needs of the "White Biotechnologies" for novel catalysts. Traditional protein purification procedures typically comprise multistep operations, which inevitably come along with significant losses of enzyme activity. Foaming offers an alternative minimizing the processing steps, preserving the purification efficiency and decreasing the activity losses all at the same time. This review provides an insight into the foaming process itself and its application in separating enzymes from model systems and from complex media, such as microbial cultures. Examples demonstrate fractionated foaming and the tweezer technique.

A novelly developed tweezing-adsorptive bubble separation (ABS) method for the enrichment of metalloenzymes (laccase C and horseradish peroxidase) is introduced. The method is based on the chelation of the enzymes' active center and can also be applied for analysis. N-(2-acetamido)iminodiacetic acid served as a chelator and was synthesized with an octyl unit to become ADA-C8. Laccase was enriched 13.3-fold (66.31% recovery) and HPOX 17.8-fold (85.34%) without a significant loss of enzymatic activity. To prove that the entire enzyme is tweezed at the active center, ABS trials were done using ADA-C8 already complexed with Cu2+ and Fe3+. As only marginal enrichment occurred (ER laccase, 0.17; ER HPOX, 0.44), no chelating effect was concluded. It was determined how the chelation toward the active center was directed by applying other chelators such as EDTA, NTA, N,N-dimethylaminoglycine, oxalic acid, malonic acid, adipinic acid, and tripropylamine, which are similar in structure to ADA-C8. The results concluded that the chelation is 3-fold coordinated on the type 1 copper center of laccase, whereas that of HPOX only 1-fold at Fe3+ and additionally at the cationic amino acid arginine, which is also located at the active center. Tweezing-ABS has been proven to selectively and effectively enrich metalloenzymes.

Active lipases were isolated from the culture supernatant of the basidiomycetous fungus Pleurotus sapidus by foam fractionation. The pH value, the gas flow, and the foaming period were systematically varied to optimize the transport of the enzymes into the foam phase. On a 70-mL scale, a maximum recovery of hydrolytic activity of 95% was obtained at pH 7.0 and 60 mL N2 min(-1) after 50 min. The procedure, with minor modifications, was also applicable to native pellet cultures of P. sapidus. The same recovery of 95% was achieved, with purification and enrichment factors of 11.6 and 28.0, respectively.