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چکیده
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Introduction: Growing seedlings produce a variety of proteolytic activities, and they might be found equally important in enzyme technology. However, due to the presence of phenolic compounds, isolation and purification of proteins and other macromolecules like DNA in plant extracts is often difficult. Growing sunflower seedlings produces an array of proteolytic activities which are active in acidic, neutral or alkaline pH ranges. However, these plants are rich in soluble phenolic compounds like chlorogenic acid which affect protease activity purification. Methods: Sunflower seeds (Helianthus annuus) were cultured at 25 °C in dark. Four day old seedlings were used for protease purification. For removal of phenolics, extraction was done in the presence of polyvinylpolypyrrolidone (PVPP) and the protein was precipitated with ammonium sulphate. Throughout the experiment, the efficiencies of protein and protease purification steps were evaluated by Arnon assay, zymographic analysis using gelatin SDSPAGE and reducing SDS-PAGE. Results and discussion: Neither polyvinylpyrrolidone (soluble PVP) nor insoluble PVPP at 5% concentration were effective in the complete removal of phenolic compounds from a sunflower seedling protease preparation, as the extracted activity of an alkaline protease was not significantly improved by them. Furthermore, phenolics removal was confirmed by their quantification. Ammonium sulphate (30%) precipitation of the protease extract prepared in the presence of 5% insoluble PVPP, however, led to complete fractionation of the phenolic compounds in the precipitate, while the alkaline protease activity which was subsequently recovered in 65% ammonium sulphate precipitate had two-fold more specific activity. PVP behaved like PVPP but with reduced effectiveness.
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