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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. K m and k cat / K m values were determined to 0.3 mM and 78.4 mM −1 s −1 for p -coumaric acid and 1.9 mM and 45.1 mM −1 s −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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Highlights: [•] An alcohol oxidase of Phanerochaete chrysosporium with a distinct glycerol oxidase side-activity. [•] Several AOX are known from fungi, but so far no AOX has been published which is able to oxidize glycerol. [•] The present AOX from P. chrysosporium is the first AOX accepting both, small monovalent alcohols and glycerol. [•] The green oxidant hydrogen peroxide could be generated from industrial side-streams of glycerol. [Copyright &y& Elsevier]

Highlights: [•] EstBC from Auricularia auricula-judae established a new class of esterases. [•] The unique substrate profile and partial sequences were determined. [•] EstBC acted on both, benzoates and cinnamates efficiently. [•] The enzyme may be useful for hydrolyzing/synthesizing flavor esters or other fine chemicals. [Copyright &y& Elsevier]

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]

Abstract: The submerged cultivation of Pleurotus ostreatus var. florida on wheat gluten led to an increased solubility of the substrate. The coding sequence of one of the extracellular peptidases was obtained by PCR with degenerated primers and RACE. The sequence amounted to 1161bp, translating into 386 aa with a molecular mass of 38.7kDa. By comparison with homologous proteins the new enzyme was classified as a subtilisin-like peptidase of the proteinase K subfamily. P. ostreatus peptidase 1 (POP1) was expressed as a preproenzyme, whose 19 aa signal sequence was cleaved off during export into the extracellular space. The prosequence acted as an intramolecular chaperone and temporary inhibitor and was degraded in an autocatalytic process. The heterologous expression in Escherichia coli resulted in the formation of inclusion bodies, from which the peptidase was refolded by using β-cyclodextrin and CTAB. POP1 showed optima at pH 7.5 and 37°C. It was most stable at pH 8 and 30°C. The peptidase was completely inhibited by PMSF, antipain and Cu2+, while Ca2+ significantly enhanced its performance. The hydrolysis of N-Suc-AAPF-pNA was almost tripled in the presence of 5M NaCl. [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: 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]

Graphical abstract Highlights • A prolyl endopeptidase was isolated from the basidiomycete Flammulina velutipes. • Cleavage of gliadin, casein, and gelatin was confirmed by HR-QTOF-MS/MS. • FvpP degraded the antigenic epitope of α -gliadin which triggers celiac disease. • The biochemical properties of FvpP favour application in food processing. Abstract 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 CaCl 2 , 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. [ABSTRACT FROM AUTHOR]

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 6000 U L −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. [ABSTRACT FROM AUTHOR]

Abstract: 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 Hg2+, 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. K m and k cat 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. [Copyright &y& Elsevier]

Abstract: 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 0.4mg/ml was found. The method has been proven to be effective for the enrichment of laccase C. [Copyright &y& Elsevier]

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]