Production of the active antifungal Pisum sativum defensin 1 (Psd1) in Pichia pastoris: overcoming the inefficiency of the STE13 protease

Plant defensins are small cysteine-rich proteins that present high activity against fungi and bacteria and inhibition of insect proteases and alpha-amylases. Here, we present the expression in Pichia pastoris, purification and characterization of the recombinant Pisum sativum defensin 1(rPsd1); a pea defensin which presents four disulfide bridges and high antifungal activity. For this, we had to overcome the inefficiency of the STE13 protease. Our strategy was to clone the corresponding cDNA directly in-frame with a variant of the widely used secretion signal from the Saccharomyces cerevisiae alpha-mating factor, devoid of the STE13 proteolytic signal cleavage sequence. Using an optimized expression protocol, which included a buffered basal salt media formulation, it was possible to obtain about 63.0mg/L of 15N-labeled and unlabeled rPsd1. The recombinants were purified to homogeneity by gel filtration chromatography, followed by reversed-phase HPLC. Mass spectrometry of native and recombinant Psd1 revealed that the protein expressed heterologously was post-translationally processed to the same mature protein as the native one. Circular dichroism and nuclear magnetic resonance spectroscopy analysis indicated that the recombinant protein had the same folding when compared to native Psd1. In addition, the rPsd1 was fully active against Aspergillus niger, if compared with native Psd1. To our knowledge, this is the first heterologous expression of a fully active plant defensin in a high-yield flask.     

Solution structure of Pisum sativum defensin 1 by high resolution NMR: plant defensins, identical backbone with different mechanisms of action.

Pisum sativum defensin 1 (Psd1) is a 46 amino acid residue plant defensin isolated from seeds of pea. The three-dimensional structure in solution of Psd1 was determined by two-dimensional NMR data recorded at 600 MHz. Experimental restraints were used for structure calculation using CNS and torsion-angle molecular dynamics. The 20 lowest energy structures were selected and further subjected to minimization, giving a root-mean-square deviation of 0.78(+/- 0.22) A in the backbone and 1.91(+/-0.60) A for over all atoms of the molecule. The protein has a globular fold with a triple-stranded antiparalell beta-sheet and an alpha-helix (from residue Asn17 to Leu27). Psd1 presents the so called "cysteine stabilized alpha/beta motif" and presents identical three-dimensional topology in the backbone with other defensins and neurotoxins. Comparison of the electrostatic surface potential among proteins with high three-dimensional (selected using the softwares TOP and DALI) topology gave insights into the mode of action of Psd1. The surface topologies between proteins that present antifungal activity or sodium channel inhibiting activity are different. On the other hand the surface topology presents several common features with potassium channel inhibitors, suggesting that Psd1 presents this activity. Other common features with potassium channel inhibitors were found including the presence of a lysine residue essential for inhibitory activity. The identity of Psd1 in primary sequence is not enough to infer a mechanism of action, in contrast with the strategy proposed here.     

Backbone dynamics of the antifungal Psd1 pea defensin and its correlation with membrane interaction by NMR spectroscopy

Plant defensins are cysteine-rich cationic peptides, components of the innate immune system. The antifungal sensitivity of certain exemplars was correlated to the level of complex glycosphingolipids in the membrane of fungi strains. Psd1 is a 46 amino acid residue defensin isolated from pea seeds which exhibit antifungal activity. Its structure is characterized by the so-called cysteine-stabilized α/β motif linked by three loops as determined by two-dimensional NMR. In the present work we explored the measurement of heteronuclear Nuclear Overhauser Effects, R1 and R2 ¹⁵N relaxation ratios, and chemical shift to probe the backbone dynamics of Psd1 and its interaction with membrane mimetic systems with phosphatidylcholine (PC) or dodecylphosphocholine (DPC) with glucosylceramide (CMH) isolated from Fusarium solani. The calculated R2 values predicted a slow motion around the highly conserved among Gly12 residue and also in the region of the Turn3 His36-Trp38. The results showed that Psd1 interacts with vesicles of PC or PC:CMH in slightly different forms. The interaction was monitored by chemical shift perturbation and relaxation properties. Using this approach we could map the loops as the binding site of Psd1 with the membrane. The major binding epitope showed conformation exchange properties in the μs-ms timescale supporting the conformation selection as the binding mechanism. Moreover, the peptide corresponding to part of Loop1 (pepLoop1: Gly12 to Ser19) is also able to interact with DPC micelles acquiring a stable structure and in the presence of DPC:CMH the peptide changes to an extended conformation, exhibiting NOE mainly with the carbohydrate and ceramide parts of CMH.     

Evaluation of the membrane lipid selectivity of the pea defensin Psd1

Psd1, a 46 amino acid residues defensin isolated from the pea Pisum sativum seeds, exhibits anti-fungal activity by a poorly understood mechanism of action. In this work, the interaction of Psd1 with biomembrane model systems of different lipid compositions was assessed by fluorescence spectroscopy. Partition studies showed a marked lipid selectivity of this antimicrobial peptide (AMP) toward lipid membranes containing ergosterol (the main sterol in fungal membranes) or specific glycosphingolipid components, with partition coefficients (K(p)) reaching uncommonly high values of 10(6). By the opposite, Psd1 does not partition to cholesterol-enriched lipid bilayers, such as mammalian cell membranes. The Psd1 mutants His36Lys and Gly12Glu present a membrane affinity loss relative to the wild type. Fluorescence quenching data obtained using acrylamide and membrane probes further clarify the mechanism of action of this peptide at the molecular level, pointing out the potential therapeutic use of Psd1 as a natural antimycotic agent.     

cDNA cloning and heterologous expression of functional cysteine-rich antifungal protein Psd1 in the yeast Pichia pastoris.

In the present work, we describe the cDNA cloning, expression in Pichia pastoris, purification, and characterization of the recombinant Pisum sativum defensin 1 (rPsd1), a novel Cys-rich protein presenting four disulfide bridges and high antifungal activity. Several parameters that affect the level of protein expression were assayed. The best condition yielded 13.8 mg/L (1.50 microg/10(8) cells) of active rPsd1. The recombinant rPsd1 was purified to homogeneity by cation exchange, followed by reversed-phase HPLC, and subjected to automated amino acid sequencing, which revealed four additional amino acids (EAEA) at the N-terminal region. Circular dichroism, intrinsic fluorescence, and nuclear magnetic resonance spectroscopy analysis indicated that the recombinant protein has a very similar folding and a correct disulfide-bonding pattern when compared to native Psd1. Nevertheless, the rPsd1 presented a more species-specific antifungal activity. The importance of the N- and C-termini for Psd1 activity is pointed out.     

Antifungal Pisum sativum defensin 1 interacts with Neurospora crassa cyclin F related to the cell cycle

Plant defensins, components of the plant innate immune system, are cationic cysteine-rich antifungal peptides. Evidence from the literature [Thevissen, K., et al. (2003) Peptides 24, 1705-1712] has demonstrated that patches of fungi membrane containing mannosyldiinositolphosphorylceramide and glucosylceramides are selective binding sites for the plant defensins isolated from Dahlia merckii and Raphanus sativus, respectively. Whether plant defensins interact directly or indirectly with fungus intracellular targets is unknown. To identify physical protein-protein interactions, a GAL4-based yeast two-hybrid system was performed using the antifungal plant peptide Pisum sativum defensin 1 (Psd1) as the bait. Target proteins were screened within a Neurospora crassa cDNA library. Nine out of 11 two-hybrid candidates were nuclear proteins. One clone, detected with high frequency per screening, presented sequence similarity to a cyclin-like protein, with F-box and WD-repeat domains, related to the cell cycle control. GST pull-down assay corroborated in vitro this two-hybrid interaction. Fluorescence microscopy analysis of FITC-conjugated Psd1 and DAPI-stained fungal nuclei showed in vivo the colocalization of the plant peptide Psd1 and the nucleus. Analysis of the DNA content of N. crassa conidia using flow cytometry suggested that Psd1 directed cell cycle impairment and caused conidia to undergo endoreduplication. The developing retina of neonatal rats was used as a model to observe the interkinetic nuclear migration during proliferation of an organized tissue from the S toward the M phase of the cell cycle in the presence of Psd1. The results demonstrated that the plant defensin Psd1 regulates interkinetic nuclear migration in retinal neuroblasts.     

Evaluation of the antifungal activity of the purified chitinase 1 from the filamentous fungus Aphanocladium album

Abstract Crude protein preparations from the culture filtrate of the filamentous fungus Aphanocladium album , a hyperparasite of rust fungi, strongly inhibited growth of a strain of the fungus Nectria haematococca pathogenic on pea. Crude protein from the filtrate of the variant E3 of A. album , hyperproducing chitinase, was less inhibitory than crude protein from the filtrate of the wild-type strain E1. The antifungal potential of a purified chitinase from A. album , called chitinase 1, was compared to that of a plant chitinase with known antifungal activity, obtained from pea ( Pisum sativum ). Although purified chitinase 1 of A. album degraded chitin more completely than did pea chitinase, it did not inhibit growth of N. haematococca , either alone or in the presence of a pea β-1,3-glucanase. Furthermore, chitinase 1 from A. album failed to enhance the antifungal activity of pea chitinase. These results indicate that the extracellular proteins of A. album inhibit growth of some fungi by other means than through their chitinase 1 activity.     

Biological activity of phenylpropionic acid isolated from a terrestrial Streptomycetes.

The strain ANU 6277 was isolated from laterite soil and identified as Streptomyces sp. closely related to Streptomyces albidoflavus cluster by 16S rRNA analysis. The cultural, morphological and physiological characters of the strain were recorded. The strain exhibited resistance to chloramphenicol, penicillin and streptomycin. It had the ability to produce enzymes such as amylase and chitinase. A bioactive compound was isolated from the strain at stationary phase of culture and identified as 3-phenylpropionic acid (3-PPA) by FT-IR, EI-MS, 1H NMR and 13C NMR spectral studies. It exhibited antimicrobial activity against different bacteria like Bacillus cereus, B. subtilis, Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris, Pseudomonas aeruginosa, P. flourescens, Staphylococcus aureus and some fungi including Aspergillus flavus, A. niger, Candida albicans, Fusarium oxysporum, F. udum and Penicillium citrinum. The antifungal activity of 3-PPA of the strain was evaluated in in vivo and in vitro conditions against Fusarium udum causing wilt disease in pigeon pea. The compound 3-PPA is an effective antifungal agent when compared to tricyclozole (fungicide) to control wilt caused by F. udum, but it exhibited less antifungal activity than carbendazim.     

Carboxylic acid and phosphate ester derivatives of fluconazole: synthesis and antifungal activities

Two classes of fluconazole derivatives, (a) carboxylic acid esters and (b) fatty alcohol and carbohydrate phosphate esters, were synthesized and evaluated in vitro against Cryptococcus neoformans, Candida albicans, and Aspergillus niger. All carboxylic acid ester derivatives of fluconazole (1a-l), such as O-2-bromooctanoylfluconazole (1g, MIC=111 microg/mL) and O-11-bromoundecanoylfluconazole (1j, MIC=198 microg/mL), exhibited higher antifungal activity than fluconazole (MIC > or = 4444 microg/mL) against C. albicans ATCC 14053 in SDB medium. Several fatty alcohol phosphate triester derivatives of fluconazole, such as 2a, 2b, 2f, 2g, and 2h, exhibited enhanced antifungal activities against C. albicans and/or A. niger compared to fluconazole in SDB medium. For example, 2-cyanoethyl-omega-undecylenyl fluconazole phosphate (2b) with MIC value of 122 microg/mL had at least 36 times greater antifungal activity than fluconazole against C. albicans in SDB medium. Methyl-undecanyl fluconazole phosphate (2f) with a MIC value of 190 microg/mL was at least 3-fold more potent than fluconazole against A. niger ATCC 16404. All compounds had higher estimated lipophilicity and dermal permeability than those for fluconazole. These results demonstrate the potential of these antifungal agents for further development as sustained-release topical antifungal chemotherapeutic agents.     

Identification and characterization of the mitochondrial membrane sorting signals in phosphatidylserine decarboxylase 1 from Saccharomyces cerevisiae

Phosphatidylserine decarboxylase 1 (Psd1p) catalyzes the formation of the majority of phosphatidylethanolamine (PE) in the yeast Saccharomyces cerevisiae. Psd1p is localized to mitochondria, anchored to the inner mitochondrial membrane (IMM) through membrane spanning domains and oriented towards the mitochondrial intermembrane space. We found that Psd1p harbors at least two inner membrane-associated domains, which we named IM1 and IM2. IM1 is important for proper orientation of Psd1p within the IMM (Horvath et al., J. Biol. Chem. 287 (2012) 36744–55), whereas it remained unclear whether IM2 is important for membrane-association of Psd1p. To discover the role of IM2 in Psd1p import, processing and assembly into the mitochondria, we constructed Psd1p variants with deletions in IM2. Removal of the complete IM2 led to an altered topology of the protein with the soluble domain exposed to the matrix and to decreased enzyme activity. Psd1p variants lacking portions of the N-terminal moiety of IM2 were inserted into IMM with an altered topology. Psd1p variants with deletions of C-terminal portions of IM2 accumulated at the outer mitochondrial membrane and lost their enzyme activity. In conclusion we showed that IM2 is essential for full enzymatic activity, maturation and correct integration of yeast Psd1p into the inner mitochondrial membrane.     

Compartment-specific Synthesis of Phosphatidylethanolamine Is Required for Normal Heavy Metal Resistance

Control of lipid composition of membranes is crucial to ensure normal cellular functions. Saccharomyces cerevisiae has two different phosphatidylserine decarboxylase enzymes (Psd1 and Psd2) that catalyze formation of phosphatidylethanolamine. The mitochondrial Psd1 provides roughly 70% of the phosphatidylethanolamine (PE) biosynthesis in the cell with Psd2 carrying out the remainder. Here, we demonstrate that loss of Psd2 causes cells to acquire sensitivity to cadmium even though Psd1 remains intact. This cadmium sensitivity results from loss of normal activity of a vacuolar ATP-binding cassette transporter protein called Ycf1. Measurement of phospholipid levels indicates that loss of Psd2 causes a specific reduction in vacuolar membrane PE levels, whereas total PE levels are not significantly affected. The presence of a phosphatidylinositol transfer protein called Pdr17 is required for Psd2 function and normal cadmium tolerance. We demonstrate that Pdr17 and Psd2 form a complex in vivo that seems essential for maintenance of vacuolar PE levels. Finally, we refine the localization of Psd2 to the endosome arguing that this enzyme controls vacuolar membrane phospholipid content by regulating phospholipids in compartments that will eventually give rise to the vacuole. Disturbance of this regulation of intracellular phospholipid balance leads to selective loss of membrane protein function in the vacuole.     

Synthesis and evaluation of demethoxyviridin derivatives as potential antimicrobials

The in vitro antibacterial and antifungal activities of demethoxyviridin and some synthetic analogues were evaluated by the agar diffusion method. The minimum inhibitory concentrations (MIC) of the active compounds were also determined by the agar dilution method. Demethoxyviridin (1) showed moderate antibacterial activity against most of the strains tested. 1alpha-Hydroxydemethoxyviridin (3) showed antibacterial activity and the most potent in vitro antifungal activity with MIC of 20 microg/ml (0.062 mM) against Aspergillus niger, A. fumigatus, A. flavus, A. parasiticus, Fusarium solani, F. graminarum, Geotrichum candidum whereas 5'-methylfuro-(4',3',2'-4,5,6)androst-5-ene-3,17-dione (7) exhibited very weak antifungal activity against Candida albicans only.     

Stereoselective synthesis and antifungal activity of (Z)-trans-3-azolyl-2-methylchromanone oxime ethers

A series of (Z)-trans-3-azolyl-2-methylchromanone oxime ethers were stereoselectively synthesized and tested for in vitro antifungal activity. Many of these derivatives exhibit high activity against Candida albicans, Saccharomyces cerevisiae, Aspergillus niger, and Microsporum gypseum.     

Synthesis and antifungal properties of alpha-methoxy and alpha-hydroxyl substituted 4-thiatetradecanoic acids

4-thiatetradecanoic acid exhibited weak antifungal activities against Candida albicans (ATCC 60193), Cryptococcus neoformans (ATCC 66031), and Aspergillus niger (ATCC 16404) (MIC=4.8-12.7 mM). It has been demonstrated that alpha-methoxylation efficiently blocks beta-oxidation and significantly improve the antifungal activities of fatty acids. We examined whether antifungal activity of 4-thiatetradecanoic acid can be improved by alpha-substitution. The unprecedented (+/-)-2-hydroxy-4-thiatetradecanoic acid was synthesized in four steps (20% overall yield), while the (+/-)-2-methoxy-4-thiatetradecanoic acid was synthesized in five steps (14% overall yield) starting from 1-decanethiol. The key step in the synthesis was the hydrolysis of a trimethylsilyloxynitrile. In general, the novel (+/-)-2-methoxy-4-thiatetradecanoic acid displayed significantly higher antifungal activities against C. albicans (ATCC 60193), C. neoformans (ATCC 66031), and A. niger (ATCC 16404) (MIC=0.8-1.2 mM), when compared with 4-thiatetradecanoic acid. In the case of C. neoformans the (+/-)-2-hydroxy-4-thiatetradecanoic acid was more fungitoxic (MIC=0.17 mM) than the alpha-methoxylated analog, but not as effective against A. niger (MIC=5.5 mM). The enhanced fungitoxicity of the (+/-)-2-methoxy-4-thiatetradecanoic acid, as compared to decylthiopropionic acid, might be the result of a longer half-life in the cells due to a blocked beta-oxidation pathway which results in more time to exert its toxic effects. Thus, these novel fatty acids may have applications as probes to study fatty acid metabolic routes in human cells.     

Analysis of two novel classes of plant antifungal proteins from radish (Raphanus sativus L.) seeds.

Two novel classes of antifungal proteins were isolated from radish seeds. The first class consists of two homologous proteins (Rs-AFP1 and Rs-AFP2) that were purified to homogeneity. They are highly basic oligomeric proteins composed of small (5-kDa) polypeptides that are rich in cysteine. Both Rs-AFPs have a broad antifungal spectrum and are among the most potent antifungal proteins hitherto characterized. In comparison with many other plant antifungal proteins, the activity of the Rs-AFPs is less sensitive to the presence of cations. Moreover, their antibiotic activity shows a high degree of specificity to filamentous fungi. The amino-terminal regions of the Rs-AFPs show homology with the derived amino acid sequences of two pea genes specifically induced upon fungal attack, to gamma-thionins and to sorghum alpha-amylase inhibitors. The radish 2S storage albumins were identified as the second novel class of antifungal proteins. All isoforms inhibit growth of different plant pathogenic fungi and some bacteria. However, their antimicrobial activities are strongly antagonized by cations.     

Enhancement in antimicrobial activity of 2-(phenyl)-3-(2-butyl-4-chloro-1H-imidazolyl)-5-butylate isoxazolidine

The trans rich isomer, 2-(phenyl)-3-(2-butyl-4-chloro-1H-imidazolyl)-5-butylate isoxazolidine A (>96% ee) was synthesized by the condensation of E isomer rich nitrone 4 (>98% ee) with butyl acrylate in an inert solvent. Obtained isoxazolidine was screened for its antifungal activity against Aspergillus niger, Cephalosporium acremonium, Fusarium moniliforme by using Nystatin as positive control. It was also tested for its antibacterial activity against Bacillus subtilis, Escherichia coli, and Staphylococcus aureus by using Streptomycin as positive control. Enhanced antifungal activity was observed in isoxazolidine of >96% ee compared to the isoxazolidine of >69% ee (B), and enhancement was not observed in antibacterial activity.     

Processing and Topology of the Yeast Mitochondrial Phosphatidylserine Decarboxylase 1

The inner mitochondrial membrane plays a crucial role in cellular lipid homeostasis through biosynthesis of the non-bilayer-forming lipids phosphatidylethanolamine and cardiolipin. In the yeast Saccharomyces cerevisiae, the majority of cellular phosphatidylethanolamine is synthesized by the mitochondrial phosphatidylserine decarboxylase 1 (Psd1). The biogenesis of Psd1 involves several processing steps. It was speculated that the Psd1 precursor is sorted into the inner membrane and is subsequently released into the intermembrane space by proteolytic removal of a hydrophobic sorting signal. However, components involved in the maturation of the Psd1 precursor have not been identified. We show that processing of Psd1 involves the action of the mitochondrial processing peptidase and Oct1 and an autocatalytic cleavage at a highly conserved LGST motif yielding the α- and β-subunit of the enzyme. The Psd1 β-subunit (Psd1β) forms the membrane anchor, which binds the intermembrane space-localized α-subunit (Psd1α). Deletion of a transmembrane segment in the β-subunit results in mislocalization of Psd1 and reduced enzymatic activity. Surprisingly, autocatalytic cleavage does not depend on proper localization to the inner mitochondrial membrane. In summary, membrane integration of Psd1 is crucial for its functionality and for maintenance of mitochondrial lipid homeostasis.     

Phosphatidylserine Decarboxylase 1 Autocatalysis and Function Does Not Require a Mitochondrial-specific Factor

Phosphatidylethanolamine (PE) is a major cellular phospholipid that can be made by four separate pathways, one of which resides in the mitochondrion. The mitochondrial enzyme that generates PE is phosphatidylserine decarboxylase 1 (Psd1p). The pool of PE produced by Psd1p, which cannot be compensated for by the other cellular PE metabolic pathways, is important for numerous mitochondrial functions, including oxidative phosphorylation and mitochondrial dynamics and morphology, and is essential for murine development. To become catalytically active, Psd1p undergoes an autocatalytic processing step involving a conserved LGST motif that separates the enzyme into α and β subunits that remain non-covalently attached and are anchored to the inner membrane by virtue of the membrane-embedded β subunit. It was speculated that Psd1p autocatalysis requires a mitochondrial-specific factor and that for Psd1p to function in vivo, it had to be embedded with the correct topology in the mitochondrial inner membrane. However, the identity of the mitochondrial factor required for Psd1p autocatalysis has not been identified. With the goal of defining molecular requirements for Psd1p autocatalysis, we demonstrate that: 1) despite the conservation of the LGST motif from bacteria to humans, only the serine residue is absolutely required for Psd1p autocatalysis and function; 2) yeast Psd1p does not require its substrate phosphatidylserine for autocatalysis; and 3) contrary to a prior report, yeast Psd1p autocatalysis does not require mitochondrial-specific phospholipids, proteins, or co-factors, because Psd1p re-directed to the secretory pathway undergoes autocatalysis normally and is fully functional in vivo.     

Chitin-supplemented formulations improve biocontrol and plant growth promoting efficiency of Bacillus subtilis AF 1

Formulations of a chitinolytic biocontrol and a plant growth promoting Bacillus subtilis AF 1 were prepared in peat, in peat supplemented with either 0.5% chitin or Aspergillus niger mycelium, or in spent compost obtained from Agaricus bisporus cultivation and were evaluated for biocontrol of two fungal pathogens and plant growth promoting activities on pigeon pea and groundnut. A steady increase in cell numbers of introduced B. subtilis AF 1 was observed in all the formulations at 30 degrees C. The increase in cell numbers was about 5.0 log units. Peat or spent compost inoculated with physiologically active and dormant states of B. subtilis AF 1 showed different time period requirements to attain maximum cell numbers. The presence of chitin or A. niger (in peat) or A. bisporus (in spent compost) as supplement in the carrier material improved the multiplication of B. subtilis AF 1. When used as seed treatments, formulations of AF 1 in peat supplemented with chitin or chitin-containing materials showed better control of A. niger (causing crown rot of groundnut) and Fusarium udum (causing wilt of pigeon pea) than AF 1 culture alone, in both groundnut and pigeon pea. Bacillus subtilis AF 1 formulations promoted seed germination and biomass of both groundnut and pigeon pea even under pathogen pressure. Survival of AF 1 on fresh culture-treated and formulation product-treated plants was similar in pathogen-infested soil.     

Long-Term Fungal Inhibition by Pisum sativum Flour Hydrolysate during Storage of Wheat Flour Bread

In order to identify antifungal compounds from natural sources to be used as ingredients in the bakery industry, water/salt-soluble extracts (WSE) from different legume flour hydrolysates obtained by the use of a fungal protease were assayed against Penicillium roqueforti DPPMAF1. The agar diffusion assays allowed the selection of the pea (Pisum sativum) hydrolysate as the most active. As shown by the hyphal radial growth rate, the WSE had inhibitory activity towards several fungi isolated from bakeries. The MIC of the WSE was 9.0 mg/ml. Fungal inhibition was slightly affected by heating and variations in pH. The antifungal activity was attributed to three native proteins (pea defensins 1 and 2 and a nonspecific lipid transfer protein [nsLTP]) and a mixture of peptides released during hydrolysis. The three proteins have been reported previously as components of the defense system of the plant. Five peptides were purified from WSE and were identified as sequences encrypted in leginsulin A, vicilin, provicilin, and the nsLTP. To confirm antifungal activity, the peptides were chemically synthesized and tested. Freeze-dried WSE were used as ingredients in leavened baked goods. In particular, breads made by the addition of 1.6% (wt/wt) of the extract and fermented by baker''s yeast or sourdough were characterized for their main chemical, structural, and sensory features, packed in polyethylene bags, stored at room temperature, and compared to controls prepared without pea hydrolysate. Artificially inoculated slices of a bread containing the WSE did not show contamination by fungi until at least 21 days of storage and behaved like the bread prepared with calcium propionate (0.3%, wt/wt).