Dataset Information

Development of Processes for Recombinant L-Asparaginase II Production by Escherichia coli Bl21 (De3): From Shaker to Bioreactors.

ABSTRACT: 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 Escherichia coli 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 ?-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^-1 and fed-batch cultivation produced 69.90 g L^-1 of cells, which produced a volumetric activity of 43,954.79 U L^-1 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.

SUBMITTER: Barros T

PROVIDER: S-EPMC7823503 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Development of Processes for Recombinant L-Asparaginase II Production by <i>Escherichia coli</i> Bl21 (De3): From Shaker to Bioreactors.

Barros Thaís T Brumano Larissa L Freitas Marcela M Pessoa Adalberto A Parachin Nádia N Magalhães Pérola O PO

Pharmaceutics 20201224 1

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 ...[more]

PMID: 33374100

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Project description:The Escherichia coli B strain BL21(DE3) has had a profound impact on biotechnology through its use in the production of recombinant proteins. Little is understood, however, regarding the physiology of this important E. coli strain. We show here that BL21(DE3) totally lacks activity of the four [NiFe]-hydrogenases, the three molybdenum- and selenium-containing formate dehydrogenases and molybdenum-dependent nitrate reductase. Nevertheless, all of the structural genes necessary for the synthesis of the respective anaerobic metalloenzymes are present in the genome. However, the genes encoding the high-affinity molybdate transport system and the molybdenum-responsive transcriptional regulator ModE are absent from the genome. Moreover, BL21(DE3) has a nonsense mutation in the gene encoding the global oxygen-responsive transcriptional regulator FNR. The activities of the two hydrogen-oxidizing hydrogenases, therefore, could be restored to BL21(DE3) by supplementing the growth medium with high concentrations of Ni²⁺ (Ni²⁺-transport is FNR-dependent) or by introducing a wild-type copy of the fnr gene. Only combined addition of plasmid-encoded fnr and high concentrations of MoO₄²⁻ ions could restore hydrogen production to BL21(DE3); however, to only 25-30% of a K-12 wildtype. We could show that limited hydrogen production from the enzyme complex responsible for formate-dependent hydrogen evolution was due solely to reduced activity of the formate dehydrogenase (FDH-H), not the hydrogenase component. The activity of the FNR-dependent formate dehydrogenase, FDH-N, could not be restored, even when the fnr gene and MoO₄²⁻ were supplied; however, nitrate reductase activity could be recovered by combined addition of MoO₄²⁻ and the fnr gene. This suggested that a further component specific for biosynthesis or activity of formate dehydrogenases H and N was missing. Re-introduction of the gene encoding ModE could only partially restore the activities of both enzymes. Taken together these results demonstrate that BL21(DE3) has major defects in anaerobic metabolism, metal ion transport and metalloprotein biosynthesis.

Dataset Information

Development of Processes for Recombinant L-Asparaginase II Production by Escherichia coli Bl21 (De3): From Shaker to Bioreactors.

Publications

Development of Processes for Recombinant L-Asparaginase II Production by <i>Escherichia coli</i> Bl21 (De3): From Shaker to Bioreactors.

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