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  • Introduction With an annual world production of around


    Introduction With an annual world production of around 750 million tons, wheat is grown on more agricultural area than any other food crop and is the main source of plant protein in human nutrition [1,2]. Wheat is composed of 8–15% protein, from which 85–90% is gluten [3]. The unique viscoelastic and adhesive properties of gluten facilitate the production of porous bakery products. These are consumed as staple foods in many parts of the world and contribute to the “westernization” of the diet. Less obvious sources of gluten and gluten hydrolysates are convenience foods and cosmetics where they serve as stabilizing and binding agents [4]. Gluten, even if consumed only once a day, may trigger a lifelong autoimmune disorder, called celiac disease, from which one percent of the world’s population is supposed to suffer [3]. So far, celiac disease is treated by a strict gluten free diet, implying that those affected are strongly limited in their choice of food. The causative components of wheat gluten, the so-called gliadins, occur as a group of storage proteins (α-/β-, γ- and ω-forms distinguished by solubility) not only in wheat, but also in barley, rye, oats and spelt and show a high abundance of glutamyl and prolyl peptides [5]. Due to the particular cyclic structure of proline, most peptidases do not efficiently cleave the peptide bonds of proline-rich proteins, such as gluten, casein, collagen and gelatin [6]. As a result, the hydrolysis of food proteins remains not only incomplete, but may also lead to the release of stable, proline-rich bitter peptides [7]. To overcome these problems, 2×Taq PCR Master Mix(with dye) able to hydrolyze proline-rich peptides might offer a new strategy for celiac disease therapy and bitterness reduction. Prolyl-specific endopeptidases (EC belong to the serine peptidase family and exhibit the unique property of hydrolyzing peptides at the carboxy terminus of internal proline residues [8]. The endopeptidases occur in a number of organisms, including bacteria, fungi and insects [9]. Peptides causing an inflammatory response were detoxified by the treatment with a prolyl endopeptidase from Flavobacterium meningosepticum [10]. A prolyl-specific endopeptidase was applied in brewing and in the debittering of casein hydrolysates [11]. Due to the long tradition in food fermentation processes, e.g. soy sauce, sake, miso, and shochu production, some strains of Aspergillus are generally recognized as safe and are thus attractive candidates for generating new prolyl endopeptidases. A prolyl endopeptidase from Aspergillus niger (An-Pep) belonging to peptidase family S28.004 showed a debittering effect on hydrolysates when incubated with β-casein [11]. Several studies confirmed that An-Pep efficiently degraded gluten in vitro under conditions similar to those present in the gastrointestinal tract indicating that co-administration of An-Pep with a gluten-containing meal might help reducing gluten toxicity [12]. Aspergillus oryzae was noted to produce a prolyl endopeptidase with a high affinity to longer peptides compared to Ala-Pro-pNA and hydrolyzing intact casein [13]. Edible basidiomycetes (mushrooms) are another potent source of prolyl endopeptidases. Agaricus bisporus and Coprinopsis clastophylla secreted extracellular prolyl endopeptidases in liquid culture [14,15]. During the screening of prolyl-specific peptidase-producing organisms, Flammulina velutipes emerged as a promising candidate [16]. The aim of this study was to purify this novel extracellular prolyl-specific endopeptidase (FvpP) from a submerged culture, identify the gene sequence and verify the presumed catalytic activity by heterologous expression in A. oryzae. A further target, was to biochemically characterize and to compare properties and applicability with the commercially available standard enzyme An-Pep.
    Materials and methods
    Results and discussion
    Conflict of interest
    Acknowledgment We thank Ulrich Krings for nLC-ESI-QTOF-MS/MS sequencing. Furthermore, the authors gratefully acknowledge Elizabeth Skellam for providing the pTAex3 vector and the Aspergillus oryzae strain.