Phytases are phosphatases essential for initializing the sequential release of orthophosphates from phytic acid, i.e., the main seed storage form of phosphorous (P) in small grain cereals ( Boyd et al., 1972 Lott, 1984 Vohra and Satyanarayana, 2003). In addition to their extended biological roles, nepenthesins have recently been implicated for various industrial applications, for example, as a tool for Digestion in Hydrogen/Deuterium Exchange Mass Spectrometry ( Yang et al., 2015), and treatment for celiac disease ( Rey et al., 2016). They display a high diversity and widespread tissue expression, suggesting their participation in various physiological processes in plants. Several genes coding for protein homologs to nepenthesins have been identified in Arabidopsis thaliana and Oryza sativa ( Takahashi et al., 2008 Chen et al., 2009). Their biological function is mainly linked to the degradation of insect proteins as a nitrogen source. Later, they have been purified from various carnivorous plant species and characterized in vitro ( Bekalu et al., 2020a). They were initially described from the pitcher fluid of the carnivorous plant, Nepenthes ( Athauda et al., 2004). As described for the nepenthesin-like PAPs, nepenthesins are characterized by diverse N-terminal sequence and nepenthesin-type PAP insertion (NAP-I) sequence ( Simoes and Faro, 2004 Soares et al., 2019 Bekalu et al., 2020a). They represent only the extracellular protease of plant origin. Nepenthesins are the first group of proteases reported from the nepenthesin-like plant aspartic proteases (PAPs). graminearum (rFgPHY1) and Fusarium culmorum (FcPHY1) phytases induced substantial degradation of both Fusarium phytases, indicating that HvNEP-1-mediated proteolysis of the fungal phytases contributes to the HvNEP-1-based suppression of Fusarium. The co-incubation of rHvNEP-1 with recombinant F. The quantitative PCR analysis of trichothecene biosynthesis genes ( TRI) confirmed that rHvNEP-1 strongly repressed the expression of TRI4, TRI5, TRI6, and TRI12 in F. Moreover, rHvNEP-1 suppressed in vitro fungal growth and strongly reduced the production of mycotoxin, 15-acetyldeoxynivalenol (15-ADON), from Fusarium graminearum. Recombinant HvNEP-1 (rHvNEP-1) strongly inhibited the activity of Aspergillus and Fusarium phytases, which are enzymes that release inorganic phosphorous from phytic acid. Signal peptide lacking HvNEP-1 was expressed in Pichia pastoris and biochemically characterized. This study describes the effect of nepenthesin 1 ( HvNEP-1) protease from barley ( Hordeum vulgare L.) on fungal histidine acid phosphatase (HAP) phytase activity. Nepenthesins are categorized under the subfamily of the nepenthesin-like plant aspartic proteases (PAPs) that form a distinct group of atypical PAPs.
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