Cloning, expression, purification and characterisation of Erwinia carotovora L-asparaginase in Escherichia coli

Pourhossein, Meraj; Korbekandi, Hassan

doi:10.4103/2277-9175.127995

Cited by 34 publications

(12 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Some divalent cations are essential cofactors for many catalytic reactions, and their presence may impact these reactions. Furthermore, some L-asparaginases from different sources are sensitive to urea 34 , 35 . Therefore, the effects of metal ions and urea on L-asparaginase were examined (Fig.…”

Section: Resultsmentioning

confidence: 99%

Simultaneous cell disruption and semi-quantitative activity assays for high-throughput screening of thermostable L-asparaginases

Zhang

et al. 2018

Sci Rep

View full text Add to dashboard Cite

L-asparaginase, which catalyses the hydrolysis of L-asparagine to L-aspartate, has attracted the attention of researchers due to its expanded applications in medicine and the food industry. In this study, a novel thermostable L-asparaginase from Pyrococcus yayanosii CH1 was cloned and over-expressed in Bacillus subtilis 168. To obtain thermostable L-asparaginase mutants with higher activity, a robust high-throughput screening process was developed specifically for thermophilic enzymes. In this process, cell disruption and enzyme activity assays are simultaneously performed in 96-deep well plates. By combining error-prone PCR and screening, six brilliant positive variants and four key amino acid residue mutations were identified. Combined mutation of the four residues showed relatively high specific activity (3108 U/mg) that was 2.1 times greater than that of the wild-type enzyme. Fermentation with the mutant strain in a 5-L fermenter yielded L-asparaginase activity of 2168 U/mL.

show abstract

Section: Resultsmentioning

confidence: 99%

Simultaneous cell disruption and semi-quantitative activity assays for high-throughput screening of thermostable L-asparaginases

Zhang

et al. 2018

Sci Rep

View full text Add to dashboard Cite

show abstract

“…atroseptica) (GenBank accession No. AAP92666.3) [ 55 , 56 ] and Rhodospirillum rubrum (GenBank accession No. QXG80441.1) [ 57 , 58 , 59 ] is about 27% and 33%, respectively.…”

Section: Resultsmentioning

confidence: 99%

A Novel L-Asparaginase from Hyperthermophilic Archaeon Thermococcus sibiricus: Heterologous Expression and Characterization for Biotechnology Application

Думина

Zhgun

Pokrovskaya

et al. 2021

IJMS

View full text Add to dashboard Cite

L-asparaginase (L-ASNase) is a vital enzyme with a broad range of applications in medicine and food industry. Drawbacks of current commercial L-ASNases stimulate the search for better-producing sources of the enzyme, and extremophiles are especially attractive in this view. In this study, a novel L-asparaginase originating from the hyperthermophilic archaeon Thermococcus sibiricus (TsA) was expressed in Escherichia coli, purified and characterized. The enzyme is optimally active at 90 °C and pH 9.0 with a specific activity of 2164 U/mg towards L-asparagine. Kinetic parameters KM and Vmax for the enzyme are 2.8 mM and 1200 µM/min, respectively. TsA is stable in urea solutions 0–6 M and displays no significant changes of the activity in the presence of metal ions Ni2+, Cu2+, Mg2+, Zn2+ and Ca2+ and EDTA added in concentrations 1 and 10 mmol/L except for Fe3+. The enzyme retains 86% of its initial activity after 20 min incubation at 90 °C, which should be enough to reduce acrylamide formation in foods processed at elevated temperatures. TsA displays strong cytotoxic activity toward cancer cell lines K562, A549 and Sk-Br-3, while normal human fibroblasts WI-38 are almost unsensitive to it. The enzyme seems to be a promising candidate for further investigation and biotechnology application.

show abstract

“…Kotzia and Labrou (2005) and Pourhossein and Korbekandi (2014) reported L-ASNase specific activity of 0.72 U mg −1 in the crude extract when working with the enzyme from Erwinia carotovora produced in E. coli [ 57 , 66 ]. A higher specific activity (1.08 U mg −1 ) was obtained in the present work.…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2020

View full text Add to dashboard Cite

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.

show abstract

Cloning, expression, purification and characterisation of Erwinia carotovora L-asparaginase in Escherichia coli

Cited by 34 publications

References 31 publications

Simultaneous cell disruption and semi-quantitative activity assays for high-throughput screening of thermostable L-asparaginases

Simultaneous cell disruption and semi-quantitative activity assays for high-throughput screening of thermostable L-asparaginases

A Novel L-Asparaginase from Hyperthermophilic Archaeon Thermococcus sibiricus: Heterologous Expression and Characterization for Biotechnology Application

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

Contact Info

Product

Resources

About