Development of Processes for Recombinant L-Asparaginase II Production by <i>Escherichia coli</i> Bl21 (De3): From Shaker to Bioreactors
Since 1961, L-asparaginase has been used to treat patients with acute lymphocytic leukemia. It rapidly depletes the plasma asparagine and deprives the blood cells of this circulating amino acid, essential for the metabolic cycles of cells. In the search for viable alternatives to produce L-asparagin...
| المؤلفون الرئيسيون: | , , , , , |
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| التنسيق: | مقال |
| اللغة: | English |
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MDPI AG
2020-12-01
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| سلاسل: | Pharmaceutics |
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| الوصول للمادة أونلاين: | https://www.mdpi.com/1999-4923/13/1/14 |
| _version_ | 1827699396254367744 |
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| author | Thaís Barros Larissa Brumano Marcela Freitas Adalberto Pessoa Nádia Parachin Pérola O. Magalhães |
| author_facet | Thaís Barros Larissa Brumano Marcela Freitas Adalberto Pessoa Nádia Parachin Pérola O. Magalhães |
| author_sort | Thaís Barros |
| collection | DOAJ |
| description | Since 1961, L-asparaginase has been used to treat patients with acute lymphocytic leukemia. It rapidly depletes the plasma asparagine and deprives the blood cells of this circulating amino acid, essential for the metabolic cycles of cells. In the search for viable alternatives to produce L-asparaginase, this work aimed to produce this enzyme from <i>Escherichia coli</i> in a shaker and in a 3 L bioreactor. Three culture media were tested: defined, semi-defined and complex medium. L-asparaginase activity was quantified using the β-hydroxamate aspartic acid method. The defined medium provided the highest L-asparaginase activity. In induction studies, two inducers, lactose and its analog IPTG, were compared. Lactose was chosen as an inducer for the experiments conducted in the bioreactor due to its natural source, lower cost and lower toxicity. Batch and fed-batch cultures were carried out to reach high cell density and then start the induction. Batch cultivation provided a final cell concentration of 11 g L<sup>−1</sup> and fed-batch cultivation produced 69.90 g L<sup>−1</sup> of cells, which produced a volumetric activity of 43,954.79 U L<sup>−1</sup> after lactose induction. L-asparaginase was produced in a shaker and scaled up to a bioreactor, increasing 23-fold the cell concentration and thus, the enzyme productivity. |
| first_indexed | 2024-03-10T13:49:14Z |
| format | Article |
| id | doaj.art-3bc8da91748646968a22a6c0a6c19f86 |
| institution | Directory Open Access Journal |
| issn | 1999-4923 |
| language | English |
| last_indexed | 2024-03-10T13:49:14Z |
| publishDate | 2020-12-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Pharmaceutics |
| spelling | doaj.art-3bc8da91748646968a22a6c0a6c19f862023-11-21T02:20:20ZengMDPI AGPharmaceutics1999-49232020-12-011311410.3390/pharmaceutics13010014Development of Processes for Recombinant L-Asparaginase II Production by <i>Escherichia coli</i> Bl21 (De3): From Shaker to BioreactorsThaís Barros0Larissa Brumano1Marcela Freitas2Adalberto Pessoa3Nádia Parachin4Pérola O. Magalhães5Department of Pharmacy, Health Sciences School, University of Brasília, Brasília 70910-900, BrazilDepartment of Biochemical and Pharmaceutical Technology, University of São Paulo, São Paulo 05508-000, BrazilDepartment of Pharmacy, Health Sciences School, University of Brasília, Brasília 70910-900, BrazilDepartment of Biochemical and Pharmaceutical Technology, University of São Paulo, São Paulo 05508-000, BrazilDepartment of Cell Biology, Institute of Biology, University of Brasília, Brasília 70910-900, BrazilDepartment of Pharmacy, Health Sciences School, University of Brasília, Brasília 70910-900, BrazilSince 1961, L-asparaginase has been used to treat patients with acute lymphocytic leukemia. It rapidly depletes the plasma asparagine and deprives the blood cells of this circulating amino acid, essential for the metabolic cycles of cells. In the search for viable alternatives to produce L-asparaginase, this work aimed to produce this enzyme from <i>Escherichia coli</i> in a shaker and in a 3 L bioreactor. Three culture media were tested: defined, semi-defined and complex medium. L-asparaginase activity was quantified using the β-hydroxamate aspartic acid method. The defined medium provided the highest L-asparaginase activity. In induction studies, two inducers, lactose and its analog IPTG, were compared. Lactose was chosen as an inducer for the experiments conducted in the bioreactor due to its natural source, lower cost and lower toxicity. Batch and fed-batch cultures were carried out to reach high cell density and then start the induction. Batch cultivation provided a final cell concentration of 11 g L<sup>−1</sup> and fed-batch cultivation produced 69.90 g L<sup>−1</sup> of cells, which produced a volumetric activity of 43,954.79 U L<sup>−1</sup> after lactose induction. L-asparaginase was produced in a shaker and scaled up to a bioreactor, increasing 23-fold the cell concentration and thus, the enzyme productivity.https://www.mdpi.com/1999-4923/13/1/14L-asparaginase<i>Escherichia coli</i>IPTGlactoseshakerbioreactor |
| spellingShingle | Thaís Barros Larissa Brumano Marcela Freitas Adalberto Pessoa Nádia Parachin Pérola O. Magalhães Development of Processes for Recombinant L-Asparaginase II Production by <i>Escherichia coli</i> Bl21 (De3): From Shaker to Bioreactors Pharmaceutics L-asparaginase <i>Escherichia coli</i> IPTG lactose shaker bioreactor |
| title | Development of Processes for Recombinant L-Asparaginase II Production by <i>Escherichia coli</i> Bl21 (De3): From Shaker to Bioreactors |
| title_full | Development of Processes for Recombinant L-Asparaginase II Production by <i>Escherichia coli</i> Bl21 (De3): From Shaker to Bioreactors |
| title_fullStr | Development of Processes for Recombinant L-Asparaginase II Production by <i>Escherichia coli</i> Bl21 (De3): From Shaker to Bioreactors |
| title_full_unstemmed | Development of Processes for Recombinant L-Asparaginase II Production by <i>Escherichia coli</i> Bl21 (De3): From Shaker to Bioreactors |
| title_short | Development of Processes for Recombinant L-Asparaginase II Production by <i>Escherichia coli</i> Bl21 (De3): From Shaker to Bioreactors |
| title_sort | development of processes for recombinant l asparaginase ii production by i escherichia coli i bl21 de3 from shaker to bioreactors |
| topic | L-asparaginase <i>Escherichia coli</i> IPTG lactose shaker bioreactor |
| url | https://www.mdpi.com/1999-4923/13/1/14 |
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