Production, purification, characterization, antioxidant and antiproliferative activities of extracellular L-asparaginase produced by Fusarium equiseti AHMF4

Mervat Morsy Abbas Ahmed El-Gendy; Mohamed F Awad; Fareed Shawky El-Shenawy; Ahmed Mohamed Ahmed El-Bondkly

doi:10.1016/j.sjbs.2021.01.058

Production, purification, characterization, antioxidant and antiproliferative activities of extracellular L-asparaginase produced by Fusarium equiseti AHMF4

Saudi J Biol Sci. 2021 Apr;28(4):2540-2548. doi: 10.1016/j.sjbs.2021.01.058. Epub 2021 Feb 10.

Authors

Mervat Morsy Abbas Ahmed El-Gendy¹, Mohamed F Awad^{2

3}, Fareed Shawky El-Shenawy³, Ahmed Mohamed Ahmed El-Bondkly⁴

Affiliations

¹ Chemistry of Natural and Microbial Products Department, National Research Centre, Dokki, Giza 12622, Egypt.
² Department of Biology, College of Science, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia.
³ Botany and Microbiology Department, Faculty of Science, Al-Azhar University, Assiut Branch, Assiut, Egypt.
⁴ Genetics and Cytology Department, National Research Centre, Dokki, Giza 12622, Egypt.

Abstract

L-Asparaginase is an antileukemic agent that depletes L-asparagine "an important nutrient for cancer cells" through the hydrolysis of L-asparagine into L-aspartic acid and ammonia leading to leukemia cell starvation and apoptosis in susceptible leukemic cell populations. Moreover currently, bacterial L-asparaginase has been limited by problems of lower productivity, stability, selectivity and a number of toxicities along with the resistance towards bacterial L-asparaginase. Then the current work aimed to provide pure L-asparaginase with in-vitro efficacy against various human carcinomas without adverse effects related to current L-asparaginase formulations. Submerged fermentation (SMF) bioprocess was applied and improved to maximize L-asparaginase production from Fusarium equiseti AHMF4 as alternative sources of bacteria. The enzyme production in SMF was maximized to reach 40.78 U mL^-1 at the 7th day of fermentation with initial pH 7.0, incubation temperature 30 °C, 1.0% glucose as carbon source, 0.2% asparagine as nitrogen source, 0.1% alanine as amino acid supplement and 0.1% KH₂PO₄. The purification of AHMF4 L-asparaginase yielded 2.67-fold purification and 48% recovery with final specific activity of 488.1 U mg^-1 of protein. Purified L-asparaginase was characterized as serine protease enzyme with molecular weight of 45.7 kDa beside stability at neutral pH and between 20 and 40 °C. Interestingly, purified L-asparaginase showed promising DPPH radical scavenging activity (IC₅₀ 69.12 μg mL^-1) and anti-proliferative activity against cervical epitheloid carcinoma (Hela), epidermoid larynx carcinoma (Hep-2), hepatocellular carcinoma (HepG-2), Colorectal carcinoma (HCT-116), and breast adenocarcinoma (MCF-7) with IC₅₀ equal to 2.0, 5.0, 12.40, 8.26 and 22.8 μg mL^-1, respectively. The enzyme showed higher activity, selectivity and anti-proliferative activity against cancerous cells along with tiny cytotoxicity toward normal cells (WI-38) which indicates that it has selective toxicity and it could be applied as a less toxic alternative to the current formulations.

Keywords: Anti-proliferative; Antioxidant; Characterization; Fusarium sp.; L-Asparaginase; Optimization; Purification.