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

Doaa B. Darwish; Salma S. Alrdahe; Yahya S. Al-Awthan; Imadeldin Elfaki; Tarig M. Alnour; Ahmed B. Darwish; Entsar A. Saad; Basmah M. Alharbi; Salem A. Habeb; Magdy M. Youssef

doi:10.53730/ijhs.v6nS8.11477

Authors

Doaa B. Darwish Department of Biology, Faculty of Science, Tabuk University, 71491Tabuk, Saudi Arabia & Botany Department, Faculty of Science, Mansoura University,35516 Mansoura, Egypt
Salma S. Alrdahe Department of Biology, Faculty of Science, Tabuk University, 71491Tabuk, Saudi Arabia
Yahya S. Al-Awthan Department of Biology, Faculty of Science, Tabuk University, 71491Tabuk, Saudi Arabia & Department of Biology, Faculty of Science, Ibb University, 70270, Ibb, Yemen
Imadeldin Elfaki Biochemistry Department, Faculty of Science, Tabuk University, 71491Tabuk, Saudi Arabia
Tarig M. Alnour Medical laboratory technology Department, Faculty of Applied Medical Sciences, Tabuk University, 71491Tabuk, Saudi Arabia
Ahmed B. Darwish Zoology Department, Faculty of Science, Suez University, El Salam-1, 43533, Suez, Egypt
Entsar A. Saad Biochemistry Division, Chemistry Department, Faculty of Science, Damietta University, New Damietta, 34511, Egypt
Basmah M. Alharbi Department of Biology, Faculty of Science, Tabuk University, 71491Tabuk, Saudi Arabia
Salem A. Habeb Biochemistry Department, Faculty of Science, Tabuk University, 71491Tabuk, Saudi Arabia
Magdy M. Youssef
mmm_youssef@mans.edu.eg
Biochemistry Division, Chemistry Department, Faculty of Science, Mansoura University, 35516 Mansoura, Egypt

Keywords:

Bacillus subtilis, L asparaginase, overexpression, purification, immobilization, characterization

Abstract

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.

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