Browsing by Author "Santos, Bruno Fernandes dos"
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- Purification and characterization of a serine protease from Alternaria alternataPublication . Santos, Bruno Fernandes dos; Bicho, Diana Faria; Tomaz, Cândida Ascensão TeixeiraAlternaria alternata is a common environmental allergenic fungi. The prevalence of Alternaria allergies varies in different geographic regions. A multicenter study performed in a group of almost 900 patients with allergies found hypersensitivity to Alternaria in 3–20% of the individuals (3 % in Portugal to 20 % in Spain). To date, 17 allergens of A. alternata have been identified. Different studies demonstrated the existence of A. alternata serine proteases that can induce lung inflammation and airway epithelial cell activation via PAR2. In 2014, the Alt a 15 (recombinant A. alternata serine protease) was added to allergens list of the official Allergen nomenclature subcommittee. In the present project, we aimed to purify the native serine protease from the A. alternata extract since this form is very important for carrying out characterization studies and to provide a good foundation for improving the accuracy of the diagnosis and the understanding and management of IgE-mediated fungal diseases. The purification protocol involved a first step of affinity chromatography using a Concanavalin A Sepharose column and a second purification step of ion exchange chromatography using a DEAE-Sepharose column. A global 28.03-fold purification of serine protease was obtained with a specific activity of 144.33 U/mg. The molecular weight of the enzyme was determined as 40 kDa by using SDS-PAGE, and the optimum pH and temperature were 8.0 and 50º C, respectively. The kinetic parameters KM and Vmax, determined against substrate casein, were 0.51 mg/mL and 7.38 µmol/min/mL, respectively. The protease was strongly inhibited by PMSF (phenylmethyl sulfony fluorid) indicating it is a serine protease. The purification strategy applied in this work could be of great importance to open a new possibility for obtaining native A. alternata serine proteases for application in diagnosis or clinical purposes.