JWSS - Journal of Water and Soil Science

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Proteolytic enzymes play important roles in food and drug industries. Actinidin, the most abundant protein of kiwifruit is a cystein protease (EC 3.4.22.14). In the present study, protein contents and levels of actinidin in kiwifruit cultivars were assayed and the enzyme was purified by a simple procedure. Actinidin was purified using two steps: (1)) precipitation by ammonium sulfate and (2) ion-exchange chromatography on diethylaminoethyl Sepharose. Molecular mass and proteolytic activity of the purified enzyme were determined by SDS-PAGE and casein digestion test, respectively. Protein content of the cultivars was compared by three different methods, namely, Bradford, UV and Lowry methods. The yield and degree of enzyme purity were 65 and 95 percents, respectively. Molecular mass of the purified enzymes were estimated 29 KDa in reducing SDS-PAGE. Results of the protein assays showed that protein and actinidin contents of kiwifruit were overestimated using UV or Lowry methods for assay. It was concluded that Bradford method is more accurate than UV or Lowry methods for measurement of protein level in kiwifruit.

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Plant pathogenic microorganisms produce a variety of enzymes capable of degrading different polysaccharides of the plant cell walls. Pathogens use these enzymes to penetrate and colonize host cells. Polygalacturonases are thought to be the first cell wall-degrading enzymes secreted by pathogens when they grow on plant cell walls. Oligogalacturonic acids with the polymerization degrees of 10 to 13 are intermediate products of pectin degradation by the action of polygalacturonases and are known to activate plant defense responses. PG- inhibiting proteins (PGIPs) present in the cell wall of many plants increase the stability of oligogalacturonic acids in the tissues by modulating fungal PG activities. These glycoproteins of the plant cell extracellular matrix retard the advancement of fungal hyphae, reduce tissue maceration, and prevent colonization of pathogen. In this study, Phaseolus vulgaris PGIPs were extracted from hypocotyle of Derakhshan and Naz bean cultivars. PvPGIPs were purified by afinity chromatography and analyzed by SDS-PAGE. Three major bands in the range of 47-55 kDa were detected. Average yield of The affinity-purified PGIPs was 1.68 mg per 100 gram of fresh bean hypocotyle. The inhibitory effect of PGIP was assayed on the PG activities of highly virulent isolates of Fusarium oxysporum (F15) and Ascochyta rabiei (IK04). The inhibitory activity of crude PGIP from Naz and Derakhshan cultivars on polygalacturonase activity of F. oxysporum was 18 and 28 units, respectively. These inhibitory activities increased to 40 units after purification. The inhibitory effect of crude PGIPs from both these two cultivars on PG activity of A. rabiei was 9 units, while purified PGIPs inhibited this PG activity to 18 and 29 units, respectively.