Abstract
Purpose: To isolate, purify and characterize protease from the latex of the plant. Methods: Protease was isolated from the latex of Plumeria rubra Linn using acetone precipitation method and purified by a sequence of DEAE cellulose column chromatography, followed by two successive column purification in Sephadex G-50 and Sephadex G-200. The molecular weight of the purified protease was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE). The protease was given a trivial name, Plumerin-R. Results: Plumerin-R showed a single protein band on SDS-PAGE and molecular weight was approximately 81.85 kDa. It remained active over a broad range of temperature but had optimum activity at 55 °C and pH 7.0 when casein was used as substrate. Activation of the protease by a thiol-activating agent indicated the presence of sulfhydryl as an essential group for its activity. Conclusion: A protease from the latex of Plumeria rubra Linn was purified to homogeneity by a simple purification procedure and then characterized. Keywords: Protease, Plumerin-R, Sulfhydryl, Purification; Characterization
Highlights
Many proteases from plant latex have been isolated and their properties extensively investigated, e.g., ficin from Ficus carica, euphorbains from Euphorbia spp., papain and related proteases from Carica papaya [1, 2, 3] and calotropain from Calotropis gigantea [4].Proteases have been purified and characterized from oat, wheat flag, maize, Phaseolus vulgaris, Onopordum turcicum, Spinacia oleracea and Petroselinum crispum leaves [5]
Plumeria rubra Linn. (Apocynaceae) is a laticiferous tree that grows as a spreading shrub or small tree to a height of 7 - 8 m (20 25 ft)
Sephadex G-50 column elution profile (Figure 2) showed two protein peaks but enzyme activity was present in fractions 20 to 28
Summary
Many proteases from plant latex have been isolated and their properties extensively investigated, e.g., ficin from Ficus carica, euphorbains from Euphorbia spp., papain and related proteases from Carica papaya [1, 2, 3] and calotropain from Calotropis gigantea [4].Proteases have been purified and characterized from oat, wheat flag, maize, Phaseolus vulgaris, Onopordum turcicum, Spinacia oleracea and Petroselinum crispum leaves [5]. The present study was conducted to isolate, purify and characterize the protease from the stem latex of Plumeria rubra Linn. Protein estimation and enzyme assay were carried out for the acetone precipitated fraction. The acetone-precipitated fraction was purified with DEAE cellulose column chromatography.
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