Genome Sequencing and Carbohydrate-Active Enzyme (CAZyme) Repertoire of the White Rot Fungus Flammulina elastica.

Next-generation sequencing (NGS) of the Flammulina elastica (wood-rotting basidiomycete) genome was performed to identify carbohydrate-active enzymes (CAZymes). The resulting assembly (31 kmer) revealed a total length of 35,045,521 bp (49.7% GC content). Using the AUGUSTUS tool, 12,536 total gene structures were predicted by ab initio gene prediction. An analysis of orthologs revealed that 6806 groups contained at least one F. elastica protein. Among the 12,536 predicted genes, F. elastica contained 24 species-specific genes, of which 17 genes were paralogous. CAZymes are divided into five classes: glycoside hydrolases (GHs), carbohydrate esterases (CEs), polysaccharide lyases (PLs), glycosyltransferases (GTs), and auxiliary activities (AA). In the present study, annotation of the predicted amino acid sequences from F. elastica genes using the dbCAN CAZyme database revealed 508 CAZymes, including 82 AAs, 218 GHs, 89 GTs, 18 PLs, 59 CEs, and 42 carbohydrate binding modules in the F. elastica genome. Although the CAZyme repertoire of F. elastica was similar to those of other fungal species, the total number of GTs in F. elastica was larger than those of other basidiomycetes. This genome information elucidates newly identified wood-degrading machinery in F. elastica, offers opportunities to better understand this fungus, and presents possibilities for more detailed studies on lignocellulosic biomass degradation that may lead to future biotechnological and industrial applications.

A protease/peptidase from culture medium of Flammulina velutipes that acts on arabinogalactan-protein.

Arabinogalactan-proteins (AGPs) are highly diverse plant proteoglycans found on the plant cell surface. AGPs have large arabinogalactan (AG) moieties attached to a core-protein rich in hydroxyproline (Hyp). The AG undergoes hydrolysis by various glycoside hydrolases, most of which have been identified, whereas the core-proteins is presumably degraded by unknown proteases/peptidases secreted from fungi and bacteria in nature. Although several enzymes hydrolyzing other Hyp-rich proteins are known, the enzymes acting on the core-proteins of AGPs remain to be identified. The present study describes the detection of protease/peptidase activity toward AGP core-proteins in the culture medium of winter mushroom (Flammulina velutipes) and partial purification of the enzyme by several conventional chromatography steps. The enzyme showed higher activity toward Hyp residues than toward proline and alanine residues and acted on core-proteins prepared from gum arabic. Since the activity was inhibited in the presence of Pefabloc SC, the enzyme is probably a serine protease.

Changes in trehalose content, enzyme activity and gene expression related to trehalose metabolism in Flammulina velutipes under heat shock.

Trehalose plays important roles in the protection of organisms against adverse environmental conditions. The growth and development of Flammulina velutipes is regulated and controlled under complex external conditions. This study investigated the effect of heat stress on trehalose metabolism in mycelia and fruiting bodies. The activities of enzymes involved in trehalose metabolism, the transcriptional levels of the corresponding genes and the trehalose content in the mycelia of Flammulina velutipes strain Dan3 under relatively high temperatures were investigated. The mycelia and fruiting bodies of a strain cultivated in a factory were collected at different stages to examine the trehalose content and expression levels of various genes. The results showed that intracellular trehalose significantly accumulated in the mycelia in response to 37 °C heat shock. Heat shock significantly stimulated the activities of trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase, thereby promoting the accumulation of trehalose for the first 2-6 h. The activity of neutral trehalase also decreased during this period. In addition, changes in the activities of trehalose-6-phosphate synthase, trehalose-6-phosphate phosphatase and neutral trehalase paralleled changes in the expression levels of the regulatory genes. As for the trehalose phosphorylase, the degradation of trehalose was stronger than its synthesis under heat stress. Heat shock can induce a stress response in the mycelia through the regulation of genes related to trehalose metabolism and the subsequent promotion and control of the transcription and translation of enzymes. The analysis of the trehalose and gene expression levels in the cultivated strain suggests that a substantial amount of trehalose had accumulated in the mycelia prior to induction of the primordia, and the fruiting bodies could possibly utilize degraded trehalose that translocated from the mycelia to maintain their growth.

Cloning and characterization of a novel O-methyltransferase from Flammulina velutipes that catalyzes methylation of pyrocatechol and pyrogallol structures in polyphenols.

A novel O-methyltransferase gene was isolated from Flammulina velutipes. The isolated full-length cDNA was composed of a 690-nucleotide open reading frame encoding 230 amino acids. A database search revealed that the deduced amino acid sequence was similar to those of other O-methyltransferases; the highest identity was only 61.8% with Laccaria bicolor. The recombinant enzyme was expressed by Escherichia coli. BL21 (DE3) was assessed for its ability to methylate (-)-epigallocatechin-3-O-gallate (EGCG). LC-TOF-MS and NMR revealed that the enzyme produced five kinds of O-methylated EGCGs: (-)-epigallocatechin-3-O-(3-O-methyl)gallate, (-)-epigallocatechin-3-O-(4-O-methyl)gallate, (-)-epigallocatechin-3-O-(3,4-O-dimethyl)gallate, (-)-epigallocatechin-3-O-(3,5-O-dimethyl)gallate, and (-)-4'-O-methylepigallocatechin-3-O-(3,5-O-dimethyl)gallate. The substrate specificity of the enzyme for 20 kinds of polyphenols was assessed using the crude recombinant enzyme of O-methyltransferase. This enzyme introduced methyl group(s) into polyphenols with pyrocatechol and pyrogallol structures.

Homocitrate synthase expression and lysine content in fruiting body of different developmental stages in Flammulina velutipes.

Homocitrate synthase (EC 2.3.3.14) regulates the first step of fungal lysine biosynthesis. The gene encoding homocitrate synthase was identified in whole genomic sequencing of Flammulina velutipes and contains seven introns. The homocitrate synthase gene of F. velutipes strain W23 (Fvhcs) is 1780 bp in length and encodes a 464 amino acid protein with a predicted molecular weight 50.7 kDa. Phylogenetic analysis of Fvhcs and other homocitrate synthase proteins from diverse fungi produced a topology congruent with the current best estimate of organismal phylogeny. Analysis of protein domains by InterProScan and a motif search found that Fvhcs gene encodes homocitrate synthase protein conserved across Agaricomycotina. In addition, we sequenced the transcriptome of different developmental stages and structures of the fruiting body to analyze the expression levels of the Fvhcs gene. The data showed a correlation between Fvhcs gene expression and lysine values in different developmental stages and structures of F. velutipes.

Laccase production and metabolic diversity among Flammulina velutipes strains.

Twelve Flammulina velutipes strains originating from Poland were identified using internal transcribed spacer (ITS) region sequencing. Based on the sequences obtained, the genomic relationship of the analyzed strains was determined. All F. velutipes strains were also characterized using Biolog FF MicroPlates to obtain data on C-substrate utilization and mitochondrial activity. The ability to decompose various substrates differed among the F. velutipes strains up to five times. The highest catabolic activities were characteristic for only two strains with capabilities to decompose up to 22 carbon sources. The correlation between carbon repression and laccase production by F. velutipes was analyzed based on glucose assimilation by these strains. Moreover, the influence of metal ions (Cu(2+), Cd(2+)), veratric and ferulic acids, and temperature on laccase activities in the analyzed strains was determined. The results obtained proved that all the inducers influenced laccase expression in almost all the analyzed strains. However, the degree of induction depended not only on the strain used but also on the day of the induction.

Reduction of oxalate levels in tomato fruit and consequent metabolic remodeling following overexpression of a fungal oxalate decarboxylase.

The plant metabolite oxalic acid is increasingly recognized as a food toxin with negative effects on human nutrition. Decarboxylative degradation of oxalic acid is catalyzed, in a substrate-specific reaction, by oxalate decarboxylase (OXDC), forming formic acid and carbon dioxide. Attempts to date to reduce oxalic acid levels and to understand the biological significance of OXDC in crop plants have met with little success. To investigate the role of OXDC and the metabolic consequences of oxalate down-regulation in a heterotrophic, oxalic acid-accumulating fruit, we generated transgenic tomato (Solanum lycopersicum) plants expressing an OXDC (FvOXDC) from the fungus Flammulina velutipes specifically in the fruit. These E8.2-OXDC fruit showed up to a 90% reduction in oxalate content, which correlated with concomitant increases in calcium, iron, and citrate. Expression of OXDC affected neither carbon dioxide assimilation rates nor resulted in any detectable morphological differences in the transgenic plants. Comparative proteomic analysis suggested that metabolic remodeling was associated with the decrease in oxalate content in transgenic fruit. Examination of the E8.2-OXDC fruit proteome revealed that OXDC-responsive proteins involved in metabolism and stress responses represented the most substantially up- and down-regulated categories, respectively, in the transgenic fruit, compared with those of wild-type plants. Collectively, our study provides insights into OXDC-regulated metabolic networks and may provide a widely applicable strategy for enhancing crop nutritional value.

Purification and characterization of a novel O-methyltransferase from Flammulina velutipes.

An enzyme catalyzing the methylation of phenolic hydroxyl groups in polyphenols was identified from mycelial cultures of edible mushrooms to synthesize O-methylated polyphenols. Enzyme activity was measured to assess whether methyl groups were introduced into (-)-epigallocatechin-3-O-gallate (EGCG) using SAM as a methyl donor, and (-)-epigallocatechin-3-O-(3-O-methyl)-gallate (EGCG3″Me), (-)-epigallocatechin-3-O-(4-O-methyl)-gallate (EGCG4″Me), and (-)-epigallocatechin-3-O-(3,5-O-dimethyl)-gallate (EGCG3″,5″diMe) peaks were detected using crude enzyme preparations from mycelial cultures of Flammulina velutipes. The enzyme was purified using chromatographic and two-dimensional electrophoresis. The purified enzyme was subsequently analyzed on the basis of the partial amino acid sequence using LC-MS/MS. Partial amino acid sequencing identified the 17 and 12 amino acid sequences, VLEVGTLGGYSTTWLAR and TGGIIIVDNVVR. In database searches, these sequences showed high identity with O-methyltransferases from other mushroom species and completely matched 11 of 17 and 9 of 12 amino acids from five other mushroom O-methyltransferases.

Expression of an oxalate decarboxylase impairs the necrotic effect induced by Nep1-like protein (NLP) of Moniliophthora perniciosa in transgenic tobacco.

Oxalic acid (OA) and Nep1-like proteins (NLP) are recognized as elicitors of programmed cell death (PCD) in plants, which is crucial for the pathogenic success of necrotrophic plant pathogens and involves reactive oxygen species (ROS). To determine the importance of oxalate as a source of ROS for OA- and NLP-induced cell death, a full-length cDNA coding for an oxalate decarboxylase (FvOXDC) from the basidiomycete Flammulina velutipes, which converts OA into CO(2) and formate, was overexpressed in tobacco plants. The transgenic plants contained less OA and more formic acid compared with the control plants and showed enhanced resistance to cell death induced by exogenous OA and MpNEP2, an NLP of the hemibiotrophic fungus Moniliophthora perniciosa. This resistance was correlated with the inhibition of ROS formation in the transgenic plants inoculated with OA, MpNEP2, or a combination of both PCD elicitors. Taken together, these results have established a pivotal function for oxalate as a source of ROS required for the PCD-inducing activity of OA and NLP. The results also indicate that FvOXDC represents a potentially novel source of resistance against OA- and NLP-producing pathogens such as M. perniciosa, the causal agent of witches' broom disease of cacao (Theobroma cacao L.).

The Potential of Peroxidases Extracted from the Spent Mushroom (Flammulina velutipes) Substrate Significantly Degrade Mycotoxin Deoxynivalenol.

Little is known about the degradability of mycotoxin deoxynivalenol (DON) by the spent mushroom substrate (SMS)-derived manganese peroxidase (MnP) and lignin peroxidase (LiP) and its potential. The present study investigated the growth inhibition of Fusarium graminearum KR1 and the degradation of DON by MnP and LiP extracted from SMS. The results from the 7-day treatment period showed that mycelium inhibition of F. graminearum KR1 by MnP and LiP were 23.7% and 74.7%, respectively. Deoxynivalenol production in the mycelium of F. graminearum KR1 was undetectable after treatment with 50 U/mL of MnP or LiP for 7 days. N-acetyl-D-glucosamine (GlcNAc) content and chitinase activity both increased in the hyphae of F. graminearum KR1 after treatment with MnP and LiP for 1, 3, and 6 h, respectively. At 12 h, only the LiP-treated group had higher chitinase activity and GlcNAc content than those of the control group (p < 0.05). However, more than 60% of DON degradabilities (0.5 mg/kg, 1 h) were observed under various pH values (2.5, 4.5, and 6.5) in both MnP (50 U/g) and LiP (50 U/g) groups, while DON degradability at 1 mg/kg was 85.5% after 50 U/g of LiP treatment for 7 h in simulated pig gastrointestinal tracts. Similarly, DON degradability at 5 mg/kg was 67.1% after LiP treatment for 4.5 h in simulated poultry gastrointestinal tracts. The present study demonstrated that SMS-extracted peroxidases, particularly LiP, could effectively degrade DON and inhibit the mycelium growth of F. graminearum KR1.

Purification, characterization and molecular cloning of a novel endo-beta-N-acetylglucosaminidase from the basidiomycete, Flammulina velutipes.

Endo-beta-N-acetylglucosaminidases are thought to be key enzymes in the catabolism of asparagine-linked oligosaccharides. However, little is known about the enzymes of this type in basidiomycetes. We investigated endo-beta-N-acetylglucosaminidases in basidiomycetes using fluorescence-labeled glycoasparagines as substrates. Flammulina velutipes showed high activity and its enzyme was named endo-beta-N-acetylglucosaminidase FV (Endo FV). The enzyme purified from the fruiting bodies of F. velutipes was separated into two forms. Endo FV was specific for high mannose and hybrid-type oligosaccharides. The enzyme was remarkably less active against asparagine-linked oligosaccharides attached to glycoproteins. It transferred an asparagine-linked oligosaccharide to Glc, but not to Gal. cDNA of Endo FV was cloned. It was composed of a 996-bp open reading frame encoding 331 amino acid residues. A recombinant Endo FV expressed in Escherichia coli showed enzymatic activity. The Endo FV gene in the genome of F. velutipes had no introns. The gene encoding Endo FV showed little homology with genes of known endo-beta-N-acetylglucosaminidases. A chitinase active site motif existed in the deduced primary structure, indicating that Endo FV belongs to glycoside hydrolase family 18. The deduced amino acid sequence of Endo FV had regions conserved in class III chitinases from fungi though it showed little homology with the sequence of any other endo-beta-N-acetylglucosaminidases. A folding model of Endo FV indicated it to be homologous with the tertiary structure of Endo H which is quite similar in specificity for asparagine-linked oligosaccharides. This study suggests that Endo FV may become similar to Endo H in substrate specificity as a result of evolutionary convergence.

The Biosynthetic Pathway of Ergothioneine in Culinary-Medicinal Winter Mushroom, Flammulina velutipes (Agaricomycetes).

Ergothioneine is a natural 2-thiol-amidazole amino acid that plays an important role in inflammation, depression, and cardiovascular disease. Flammulina velutipes is a common basidiomycete mushroom rich in ergothioneine (EGT). However, the biosynthetic pathway of EGT in F. velutipes is still unclear. In this study, the F. velutipes ergothioneine biosynthetic gene 1 (Fvegtl), F. velutipes ergothioneine biosynthetic gene 2 (Fvegt2), and F. velutipes ergothioneine biosynthetic gene 3 (Fvegt3) were cloned and expressed, and the activities of the proteins encoded by these three genes (FvEgt1, F. velutipes ergothioneine biosynthase 1; FvEgt2, F. velutipes ergothioneine biosynthase 2; and FvEgt3, F. velutipes ergothioneine biosynthase 3) were identified. The results showed that FvEgtl not only has the function of methyltransferase, but also has the function of hercynlcysteineteine sulfoxide (Hersul) synthase, which can catalyze the production of Hersul from histidine and cysteine in F. velutipes. FvEgt2 and FvEgt3 are two functionally different cysteine desulfurase enzymes. Among them, FvEgt2 is a cysteine-cysteine desulfurase-which catalyzes the activation of the S-H bond on cysteine, while FvEgt3 is a pyridoxal phosphate (PLP)-dependent cysteine desulfurase responsible for catalyzing the production of ketimine complex. Our results show that FvEgt1/FvEgt2/FvEgt3 can simultaneously catalyze the production of EGT by histidine, cysteine, and pyridoxal phosphate. Collectively, the in vitro synthesis of EGT in the edible fungus F. velutipes was first achieved, which laid the foundation for the biological production of EGT.

Isolation and characterization of a gene coding for chitin deacetylase specifically expressed during fruiting body development in the basidiomycete Flammulina velutipes and its expression in the yeast Pichia pastoris.

Fv-pda, a gene coding for chitin deacetylase (CDA), was isolated from the basidiomycete Flammulina velutipes by differential display targeted for genes specifically expressed during fruiting body development. The fv-pda ORF comprises 250 amino acid residues and is interrupted by 10 introns. The fv-pda cDNA was expressed in the yeast Pichia pastoris, and the resulting recombinant FV-PDA was used for enzymatic characterization. The recombinant FV-PDA catalyses deacetylation of N-acetyl-chitooligomers, from dimer to pentamer, glycol chitin and colloidal chitin. The fv-pda was specifically expressed through the entire stage of fruiting body development, and the transcript was abundant in stipes of mature fruiting bodies. These results suggest that CDA plays an important role in the process of fruiting of F. velutipes.

Purification and characterization of a novel exo-beta-1,3-1,6-glucanase from the fruiting body of the edible mushroom Enoki (Flammulina velutipes).

To elucidate the role of beta-glucanases in the cell-wall degradation involved in morphogenesis, an exo-beta-1,3-1,6-glucanase (FvBGL1) was purified from fruiting bodies of the edible mushroom Enoki (Flammulina velutipes), and its enzymatic properties were studied. At least three beta-glucanases were detected in the crude extract by zymogram assay when 1% laminarin was used as substrate. The molecular mass of FvBGL1 was estimated by SDS-PAGE to be 80 kDa. The optimum pH and temperature for the action of FvBGL1 were 6.1 and 60 degrees C respectively. FvBGL1 was completely inactivated by 1 mM mercuric ions. FvBGL1 hydrolyzed F. velutipes cell-wall beta-glucan as well as beta-1,3- and beta-1,6-glucans from various sources with glucose as the only reaction product. Transglucosylation was observed when the enzyme acted on laminarinonaose. FvBGL1 can be assumed to degrade F. velutipes cell-wall beta-1,3-glucan, but most probably acts more efficiently in concert with other endogenous beta-glucan degrading enzymes.

Highly-efficient liposome-mediated transformation system for the basidiomycetous fungus Flammulina velutipes.

Flammulina velutipes is a well-known edible mushroom cultivated all over the world. However, because of the low transformation frequency, the expensive instruments required, and the complicated, time-consuming procedures necessary, there is insufficient genetic research on F. velutipes. In this study, we report a liposome-mediated transformation (LMT) system for the genetic transformation of F. velutipes. Using the LMT system, we obtained 82 ± 4 stable F. velutipes transformants per 105 protoplasts, which is a clear increase in transformation frequency compared to the other methods used. We were able to detect the expression of an EGFP reporter gene in the F. velutipes transformants using fluorescence imaging assays. Furthermore, we used this method to transfer the laccase gene into F. velutipes and found that the transcriptional level and enzymatic activity increased in these transformants. Mitotic stability analysis showed that all of the selected transformants remained mitotically stable, even after five successive rounds of sub-culturing. These results demonstrate a new transgenic approach that will facilitate F. velutipes research.

Expression of C-5 sterol desaturase from an edible mushroom in fisson yeast enhances its ethanol and thermotolerance.

Bioethanol is an environment friendly and renewable source of energy produced by the fermentation of agricultural raw material by a variety of microorganisms including yeast. Obtaining yeast strains that are tolerant to stresses like high levels of ethanol and high temperature is highly desirable as it reduces cost and increases yield during bioethanol production. Here, we report that heterologous expression of C-5 Sterol desaturase (FvC5SD)-an ergosterol biosynthesis enzyme from an edible mushroom Flammulina velutipes in fission yeast, not only imparts increased thermotolerance but also tolerance towards high ethanol concentration and low pH. This tolerance could be attributed to an increase of ≈1.5 fold in the level of ergosterol and oleic acid (C-18 unsaturated fatty acid) as analysed by gas chromatography- mass spectrometry. FvC5SD is a membrane localized iron binding enzyme that introduces double bond at C-5 position into the Δ7-sterol substrates to yield Δ5, 7- sterols as products. In F. velutipes, FvC5SD transcript was observed to be upregulated by ≈5 fold under low pH condition and by ≈ 9 folds and ≈5 fold at 40°C and 4°C respectively when compared to normal growth temperature of 23°C. Besides, susceptibility to cell wall inhibiting drugs like Congo red and Calcoflour white was also found to increase in FvC5SD expressing S. pombe strain. Alteration in membrane sterol and fatty acid composition could also lead to increase in susceptibility to cell wall inhibiting drugs. Thus, this study has immense industrial application and can be employed to ensure competitiveness of fermentation process.

Laccase Production Among Medicinal Mushrooms from the Genus Flammulina (Agaricomycetes) Under Different Treatments in Submerged Fermentation.

The laccase activities of 13 strains of medicinal and edible mushrooms from the Flammulina genus (F. velutipes, F. rossica, and F. fennae) were studied. The effects of both fungal isolates and culture media were investigated. The laccase activities indicated significant differences among Flammulina strains (P < 0.001), and the cultural media significantly affected the laccase activities in Flammulina spp. (P < 0.001). The morphological characteristics of the 3 Flammulina species were similar, but differences in phylogenetic analysis and laccase activity existed among different species. Although isolates of each Flammulina species differed, the laccase variables among different Flammulina species were greater than those within the same species. The presence of simple carbon and nitrogen sources increased the maximum laccase enzyme activity, but the occurrence of both laccase activity and maximum laccase enzyme activity was delayed compared with lignocellulosic material. The laccase activities of most Flammulina isolates were increased when exposed to copper ions. Our findings offer insights into laccase productivity in response to different Flammulina species or strains and different submerged fermentation treatments.

Characterization of ß-glucosidase produced by the white rot fungus Flammulina velutipes.

ß-Glucosidase production by the white rot fungus Flammulina velutipes CFK 3111 was evaluated using different carbon and nitrogen sources under submerged fermentation. Maximal extracellular enzyme production was 1.6 U/ml, corresponding to a culture grown in sucrose 40 g/l and asparagine 10 g/l. High production yield was also obtained with glucose 10 g/l and asparagine 4 g/l medium (0.5 U/ml). Parameters affecting the enzyme activity were studied using p-nitrophenyl-ß-D-glucopyranoside as the substrate. Optimal activity was found at 50°C and pHs 5.0 to 6.0. Under these conditions, ß-glucosidase retained 25% of its initial activity after 12 h of incubation and exhibited a half-life of 5 h. The addition of MgCl2, urea, and ethanol enhanced the ß-glucosidase activity up to 47%, whereas FeCl2, CuSO4, Cd(NO3)2, and cetyltrimethylammonium bromide inflicted a strong inhibitory effect. Glucose and cellobiose also showed an inhibitory effect on the ß-glucosidase activity in a concentration-dependent manner. The enzyme had an estimated molecular mass of 75 kDa. To the best of our knowledge, F. velutipes CFK 3111 ß-glucosidase production is amongst the highest reported to date, in a basidiomycetous fungus.

[Cloning of a laccase gene from Flammulina velutipes and study on its expression in Pichia pastoris].

Laccase(EC1.10.3.2) can be used for enzymatic detoxification of lignocellulosic hydrolysates. By using molecular techniques such as RACE (rapid amplification of cDNA ends) and Genome-Walking, a laccase gene and its corresponding full-length cDNA were cloned from Flammulina velutipes and designated as glccFv and IccFv. The sequences were submitted to GenBank, and the accession numbers obtained were AY485826 and AY450406, respectively. Analysis of amino acids sequence suggested that one laccase from Polyporus ciliatus possessed the highest homology with the protein encoded by lccFv showing for 72%. The ORF (open reading frame) of lccFv was transformed into Pichia pastoris strain GS115 through the P. pastoris expression vector pHBM906, which contains both the promoter and transcription terminator of the AOX1 gene. The recombinant laccase LCCFv was detected from the engineering strain GS115 (pHBM557) which was fermented with BMMY liquid medium and induced by 1.0% (V/V) methanol at 20 degrees C with the highest expression level (0.1070 U/mL). The optimal reaction temperature of LCCFv that secreted from P. pastoris GS115(pHBM557) was 45 degrees C, the optimal reaction pH value was pH3.9 and the thermostability and pH stability were very well under the optimal conditions.

Factors limiting the enzymatic hydrolysis of wheat gluten.

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.