Bioprocess development for L-asparaginase production by Streptomyces rochei, purification and in-vitro efficacy against various human carcinoma cell lines

Abstract

Bioprocess development for L-asparaginase production by Streptomyces rochei, purification and in-vitro efficacy against various human carcinoma cell lines noura el-Ahmady el-naggar ✉ & nancy M. el-Shweihy in the near future, the demand for L-asparaginase is expected to rise several times due to an increase in its clinical and industrial applications in various industrial sectors, such as food processing. Streptomyces sp. strain NEAE-K is potent L-asparaginase producer, isolated and identified as new subsp. Streptomyces rochei subsp. chromatogenes neAe-K and the sequence data has been deposited under accession number KJ200343 at the GenBank database. Sixteen different independent factors were examined for their effects on L-asparaginase production by Streptomyces rochei subsp. chromatogenes NEAE-K under solid state fermentation conditions using Plackett-Burman design. pH, dextrose and yeast extract were the most significant factors affecting L-asparaginase production. Thus, using central composite design, the optimum levels of these variables were determined. L-asparaginase purification was carried out by ammonium sulfate followed by DEAE-Sepharose CL-6B ion exchange column with a final purification fold of 16.18. The monomeric molecular weight of the purified L-asparaginase was 64 kD as determined by SDS-PAGE method. The in vitro effects of L-asparaginase were evaluated on five human tumor cell lines and found to have a strong anti-proliferative effects. The results showed that the strongest cytotoxic effect of L-asparaginase was exerted on the HeLa and HepG-2 cell lines (ic 50 = 2.16 ± 0.2 and 2.54 ± 0.3 U/mL; respectively). In addition, the selectivity index of L-asparaginase against HeLa and HepG-2 cell lines was 3.94 and 3.35; respectively. L-asparaginase is one of the amidase group, that catalyzes the hydrolysis of L-asparagine and releases L-aspartic acid and ammonia 1. L-asparaginase is an effective therapeutic enzyme used in combination with other drugs for treating melanosarcoma, reticulosarbom, lymphocytic leukemia, Hodgkin disease, chronic lymphosarcoma, acute myelomonocytic leukemia, acute myelocytic leukemia and acute lymphoblastic leukemia in children and adults 2,3. In pediatric oncology, L-asparaginase is the recommended drug used for acute lymphoblastic leukemia therapy, resulting in a total recovery of more than 90 percent of kids within a period of four weeks 3. The anti-leukemic impact of L-asparaginase based on the fact that the tumor cells need a huge quantity of L-asparagine to survive. The tumor cells cannot synthesize L-asparagine for their needs because of the lack of L-asparagine synthetase and thus cannot synthesize the necessary proteins that rely on L-asparagine 4. The survival and growth of tumor cells rely on the exogenous source of L-asparagine (consumed in a diet, absorbed and accessible in the circulating pool) 5. Thus, injection of the enzyme into cancer patients intravenously decreases L-asparagine blood levels and selectively damages tumor cells 6. Conversely, the normal cell...

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

“…The impact of this enzyme is evidenced by the high global demand, which reached $ 380 million in 2017 and is expected to increase up to $ 420 million by 2025 ( Alam et al, 2019 ). Towards attaining this vital importance, it is necessary to improve production conditions including cultural conditions and neutral supplements ( Ashok et al, 2019 , El-Gendy et al, 2017 , El-Naggar and El-Shweihy, 2020 ). Submerged fermentation (SMF) supported by many authors as the preferable fermentation method for fungal L-asparaginase synthesis from Trichoderma viride , Aspergillus aculeatus , Cladosporium sp., Rhizomucor miehei , Penicillium digitatum , Penicillium sp., Penicillium brevicompactum ( Cachumba et al, 2016 , Costa-Silva et al, 2019 ) at varied process parameters.…”

Bioprocess Optimization of Nutritional Parameters for Enhanced Anti-leukemic L-Asparaginase Production by Aspergillus candidus UCCM 00117: A Sequential Statistical Approach

“…L-asparaginase yield by Streptomyces rochei subsp. chromatogens NEAE-K reported by El-Naggar and El-Shweihy ( 2020 ) from a similar optimization study was 30%, although with lower fold of purification. The L-glutaminase activity of the enzyme also decreased from an initial activity of 82.131U in the sterile broth to a final activity of 0.087U, when L-asparaginase activity was 1891.64U with a specific activity of 476.48 U/mg.…”

“…The index was greater than 2 for all tested cell lines indicating low toxicity of the compound (Suffness and Pezzuto 1990 ). El-Naggar and El-Shweihy ( 2020 ) reported similar success (SI > 2) with Streptomyces rochei L-asparaginase against HepG-2 and MCF-7 cell lines. Anti-cancer substances with IC 50 less than 30 µg/mL have been reported to hold promise for cancer drug development (de Oliveira et al 2016 ).…”

“…L-asparaginase is frequently sourced from plants (Oza et al 2010 ; Mohamed et al 2015 ) and microorganisms (Sahu et al 2007 ; Hassan et al 2018 ). Foremost among microorganisms that produce L-asparaginases include species of Escherichia and Erwinia however; other bacteria including Bacillus velezensis (Mostafa et al 2019 ), Enterobacter cloacae (Husain et al 2016 ), Streptomyces rochei (El-Naggar and El-Shweihy 2020 ) and Geobacillus thermodenitrificans (Nadeem et al 2019 ) have also been reported. Species of the genera Aspergillus , Penicillium , Fusarium (Sarquis et al 2004 ) and Trichosporon (Ashok et al 2019 ) dominate literature as fungal L-asparaginase producers.…”

Microbial L-asparaginase for Application in Acrylamide Mitigation from Food: Current Research Status and Future Perspectives

“…El-Naggar and El-Shweihy (2020) evaluated L-asparaginase production from actinomycetes by using the agar plates with culture medium at a pH of 6.8–7, which consisted of L-asparagine and phenol red as indicators of pH value. L-asparaginase activity resulted in hydrolyzing L-asparagine into aspartic acid and ammonia that converted the phenol red from yellow to pink (alkaline condition) [ 35 ]. However, this method may produce false positives results [ 36 ].…”