Isolation and purification of anticoagulant enzymes from plant latex
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Abstract
The plant latex is a complex mixture of organic, inorganic and hydrolytic enzymes such as proteases which acts as anticoagulant and finds its uses in surgeries, heart diseases and blood clot treatment. In the present study, latex was collected from Euphorbia milii, Jatropha malacophylla, Thevetia peruviana, Euphorbia pulcherimma and Artocarpus
altilis to evaluate the best source of anticoagulant enzyme though four other plants were originally screened as well but failed to show the enzyme. Among the five, A. altilis and J. malacophylla showed the highest anticoagulant activity by milk clotting, blood clotting and APTT assay. The samples were subjected to three-step purification, i.e., salt precipitation, dialysis, ion exchange and gel filtration. The fold purification of A. altilis was found to be 5.37 and 2.31 for J. malacophylla respectively after the gel filtration. The percentage of yield of A. altilis was found to be 63.23% and 26.4% for J. malacophylla. Molecular weight of J. malacophylla sample and A. altilis was found to be ~80kDa and ~105kDa respectively, determined by SDS PAGE. Both enzymes showed optimum activity at pH 7. A. altilis showed optimum activity at 35°C, incubation time of 40 minutes, substrate concentration of 60mM, with MgCl2 as activator the activity increased at 600?L and with EDTA as inhibitor the activity increased at 400?L. J. malacophylla showed optimum activity at 45°C, incubation time of 10 minutes, substrate concentration of 80mM and stable at 35 °C which is the human body temperature. A. altilis showed optimum conditions for human administration making it therapeutically viable.
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Anticoagulant, APTT, Gel filtration, Ion exchange, SDS PAGE
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