Glycosylation of L-asparaginase from E. coli through yeast expression and site-directed mutagenesis

Lima, Guilherme Meira; Effer, Brian; Biasoto, Henrique Pellin; Feijoli, Veronica; Pessoa, Adalberto; Palmisano, Giuseppe; Monteiro, Gisele

doi:10.1016/j.bej.2020.107516

Cited by 18 publications

(8 citation statements)

References 41 publications

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“…Thus, there is a need to purify this enzyme and perform X-ray crystallography for a better understanding of the three-dimensional structure, as well as its kinetic and dynamic parameters. Lima et al (2020) cloned and purified a L-asparaginase from E. coli in P. pastoris, obtaining an activity of 2.98 U/mg of protein after 48 h [27]. Likewise, extracellular ANSase recombinant in P. pastoris was purified, expressing an activity of about 2.81 IU/mL in a work performed by Sajitha et al (2015) [28].…”

Section: Discussionmentioning

confidence: 99%

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l-Asparaginase Type II from Fusarium proliferatum: Heterologous Expression and In Silico Analysis

Cardoso,

Souza,

Rodrigues

et al. 2023

Pharmaceutics

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The search for new drug-producing microorganisms is one of the most promising situations in current world scientific scenarios. The use of molecular biology as well as the cloning of protein and compound genes is already well established as the gold standard method of increasing productivity. Aiming at this increase in productivity, this work aims at the cloning, purification and in silico analysis of l-asparaginase from Fusarium proliferatum in Komagataella phaffii (Pichia pastoris) protein expression systems. The l-asparaginase gene (NCBI OQ439985) has been cloned into Pichia pastoris strains. Enzyme production was analyzed via the quantification of aspartic B-hydroxamate, followed by purification on a DEAE FF ion exchange column. The in silico analysis was proposed based on the combined use of various technological tools. The enzymatic activity found intracellularly was 2.84 IU/g. A purification factor of 1.18 was observed. The in silico analysis revealed the position of five important amino acid residues for enzymatic activity, and likewise, it was possible to predict a monomeric structure with a C-score of 1.59. The production of the enzyme l-asparaginase from F. proliferatum in P. pastoris was demonstrated in this work, being of great importance for the analysis of new methodologies in search of the production of important drugs in therapy.

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Section: Discussionmentioning

confidence: 99%

“…Lima et al (2020) cloned and purified a L-asparaginase from E. coli in P. pastoris, obtaining an activity of 2.98 U/mg of protein after 48 h [27]. Likewise, extracellular ANSase recombinant in P. pastoris was purified, expressing an activity of about 2.81 IU/mL in a work performed by Sajitha et al (2015) [28].…”

Section: Discussionmentioning

confidence: 99%

l-Asparaginase Type II from Fusarium proliferatum: Heterologous Expression and In Silico Analysis

Cardoso,

Souza,

Rodrigues

et al. 2023

Pharmaceutics

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“…It is used widely to drive the heterologous protein expression due to its strict regulation and strong inducibility when methanol is used as the only carbon source (Yang & Zhang, 2018). Several studies have been conducted on L-ASNase production using P. pastoris as the host system (de Almeida Parizotto et al, 2021;Effer et al, 2019;Ferrara et al, 2006;Lima et al, 2020;Pillaca-Pullo et al, 2021;Rodrigues et al, 2019;Sajitha et al, 2015;Tien Cuong Nguyen, 2014). Although all the reviewed studies used pAOX1, de Almeida Parizotto et al ( 2021) used a methanol-oxygen control strategy in the bioreactor, resulting in twice the maximum volumetric activity compared to the pulse strategy.…”

Section: Engineering Of Promoters To Improve the Transcription Of Asn...mentioning

confidence: 99%

Current state of molecular and metabolic strategies for the improvement of L-asparaginase expression in heterologous systems

Lefin

Miranda

Beltrán

et al. 2022

Preprint

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L-asparaginase is one of the world’s most in-demand industrial enzymes, mainly due to its therapeutic properties and its use in the food industry. Over the years studies have been conducted to improve its specific activity and reduce side effects, driving new discoveries that promise safer, more efficient, and cheaper drugs for consumers. However, heterologous protein modifications or expression continue to be a problem during production. Therefore, addressing bottlenecks when attempting to express modified and/or heterologous proteins using the rational design of heterologous expression systems with modified hosts could be a promising strategy to improve L-asparaginase production. Problems such as inadequate protein folding, metabolic load on host cells, codon usage bias, repetitive sequences affecting translation, heavy molecular weight and/or multi-membrane domains formation; need great attention during the development of “bio-better” proteins. In this article, we address the current molecular and metabolic strategies that have been developed to improve the heterologous expression of L-asparaginase.

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“…In clinical practice, L-ASNase from two prokaryotic microorganisms are used, Escherichia coli and Dickeya dadantii (previously named as Erwinia chrysanthemi). E. coli L-ASNase formulations are used as line first treatment and E. chrysanthemi L-ASNase is used as second line treatment [8,10,11].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2020

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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 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−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.

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Glycosylation of L-asparaginase from E. coli through yeast expression and site-directed mutagenesis

Cited by 18 publications

References 41 publications

l-Asparaginase Type II from Fusarium proliferatum: Heterologous Expression and In Silico Analysis

l-Asparaginase Type II from Fusarium proliferatum: Heterologous Expression and In Silico Analysis

Current state of molecular and metabolic strategies for the improvement of L-asparaginase expression in heterologous systems

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

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