Overexpression, biochemical characterization, and anticancer activates of L-asparaginase from Bacillus subtilis

• Published in: International journal of health sciences pp. 1731 - 1752
• Main Authors: Darwish, Doaa B., Alrdahe, Salma S., Al-Awthan, Yahya S., Elfaki, Imadeldin, Alnour, Tarig M., Darwish, Ahmed B., Saad, Instar A., Alharbi, Basmah M., Habeb, Salem A., Youssef, Magdy M.
• Format: Journal Article
• Language: English
• Published: 02.08.2022
• ISSN: 2550-6978; 2550-696X
• DOI: 10.53730/ijhs.v6nS8.11477

L-asparaginases convert L-asparagine into L-aspartate and ammonia. The L-asparaginase from Bacillus subtilis was cloned and expressed in the E. coli strain BL21(DE3)pLysS in the current study. Using glutathione sepharose 4B column chromatography, the L-asparaginase enzyme was uniformly purified 173.34 times, with a final specific activity of 1769.13 IU/mg protein and a yield of 56.14%. The isolated enzyme was identified as a 36 kDa polypeptide chain by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The immobilized enzyme was placed on top of the Ca alginate beads. The immobilized enzyme is quite stable and retains the majority of its activity at 4 °C (74 percent). The enzymatic and structural characteristics of free and immobilized recombinant L-asparaginase were studied. The activity of the free enzyme peaked after 30 min of incubation at pH 8.0 and 45 °C. After 30 minutes at 50 °C, the immobilized enzyme showed its peak activity at a pH of 8.5. The refined enzyme's amino acid makeup was identified. An enzyme that heals leukemia, Bacillus subtilis L-asparaginase, can be successfully mass-produced using this technique.