Extraction and Purification of Asparaginase enzyme from Pisum sativum plant and studying their cytotoxicity against L20B tumor cell line

 Plant samples of Pisum sativum were collected from crop fields in the Collage of Agriculture/ University of Baghdad and were classified as Pisum sativum subspp. Jof according to their morphological characteristics. Activity of asparaginase was detected in seeds, stems and leaves extracts. Results showed that maximum asparaginase activity was detected in seeds extracts which was 30.0 U/mg in comparison with 26.4 and 16.1 U/mg in extracts of leaves and stems respectively. According to theseresults plant seeds were used as a source for asparaginase production, characterization, and studying its antitumor activity.  Optimum conditions for the activity of crude asparaginase extracted from plants seeds were studied. Results showed maximum activity of asparaginase was achieved when the enzyme was incubated with 200mM of asparagines in a ratio of 1:3 (V/V) at 37°C for 30 minutes in presence of 0.05 M of potassium phosphate buffer solution at pH8.  Crude asparaginase extracted from plant seeds was purified in two steps, ion exchange chromatography by DEAE-Cellulose and gel filtration chromatography by Sephadex G-200. Specific activity of purified asparaginase was 228.8 U/mg.  Asparaginase purified from seeds extracts was then characterized. Results of characterization showed that the molecular weight of asparaginase was 66,464 Kelo dalton, and the optimum pH for enzyme activity and stability was pH 8.5, while the optimum temperature for enzyme activity and stability was 37°C and 40°C respectively On the other hand the enzyme activation energy was 6260 calories/mol, and the temperature coefficient (Q10) for asparaginase was 1.32.