Studies on efficient production of a novel l-asparaginase by a newly isolated Pseudomonas resinovorans IGS-131 and its heterologous expression in Escherichia coli

Mihooliya, Kanti N.; Nandal, J. K.; Kumari, Aruna; Nanda, Sidhanta; Verma, Himanshu; Sahoo, Debendra K.

doi:10.1007/s13205-020-2135-4

Cited by 22 publications

(18 citation statements)

References 23 publications

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“…The concentrations of 10 g L −1 and 0.45 mmol L −1 of lactose and IPTG, respectively, showed the highest L-ASNase activity values as demonstrated in Figure 1 . Several studies used IPTG as an inducer for L-ASNase production ( Table 2 ), with concentrations from 0.40 to 0.50 mmol L −1 being commonly used [ 33 , 34 , 35 , 36 ]. In previous studies, lactose was used at a concentration of 2 g L −1 reaching the specific activity 210 U mg −1 of the purified enzyme [ 37 ].…”

Section: Resultsmentioning

confidence: 99%

“…Few studies have reported the use of bioreactors for the production of L-ASNase by E. coli . Mihooliya and collaborators (2020) reported the achievement of OD 600nm 6 using a bioreactor with a capacity of 2 L and the cultivation was carried out in batch [ 34 ]. Roth and collaborators (2013) reached 30 g L −1 of dry weight using a bioreactor with a capacity of 2 L and the cultivation was carried out in fed-batch [ 58 ].…”

Section: Resultsmentioning

confidence: 99%

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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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Section: Resultsmentioning

confidence: 99%

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

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“…The organism Enterobacter aerogenes showed maximum l -asparaginase production in the presence of diammonium hydrogen phosphate and sodium citrate as nitrogen and carbon sources, respectively [ 34 ]. Pseudomonas resinovorans IGS-131 exhibited maximum production of l -asparaginase when Na 2 HPO 4 , KH 2 PO 4 , and NaCl were used as ion sources [ 35 ].…”

Section: Discussionmentioning

confidence: 99%

Response surface methodological optimization of l-asparaginase production from the medicinal plant endophyte Acinetobacter baumannii ZAS1

Abhini

Rajan

Zuhara

et al. 2022

Journal of Genetic Engineering and Biotechnology

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Background This study targets the enhanced production of l-asparaginase, an antitumor enzyme by Acinetobacter baumannii ZAS1. This organism is an endophyte isolated from the medicinal plant Annona muricata. Plackett–Burman design (PBD) and central composite design (CCD) were used for statistical optimization of media components. Results The organism exhibited 18.85 ± 0.2 U/mL enzyme activities in unoptimized media. Eight variables: l-asparagine, peptone, glucose, lactose, yeast extract, NaCl, MgSO4, and Na2HPO4 were screened by PBD. Among them, only four factors—l-asparagine, peptone, glucose, and Na2HPO4—were found to affect enzyme production significantly (p < 0.05). Furthermore, the best possible concentrations and interactive effects of the components that enhance this enzyme's output were chosen by using CCD on these selected variables. The results revealed that an optimized medium produces a higher concentration of enzymes than the unoptimized medium. After optimizing media components, the maximum l-asparaginase activity was 45.59 ± 0.36 U/mL, around the anticipated value of 45.04 ± 0.42 U/mL. After optimization of process parameters, it showed a 2.41-fold increase in the production of l-asparaginase by the endophyte Acinetobacter baumannii ZAS1. Conclusion The findings of this study indicated that an endophyte, Acinetobacter baumannii ZAS1 that produces l-asparaginase could be used to increase enzyme output. However, using the statistical methods Plackett-Burman design and central composite design of response surface methodology is a handy tool for optimizing media components for increased l-asparaginase synthesis.

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“…The copyright holder for this preprint this version posted January 2, 2023. ; https://doi.org/10.1101/2023.01.01.522448 doi: bioRxiv preprint 8.0) and operated at 25°C and 1.0 mL min -1 of flow rate. In our previous study, gel filtration chromatography with Superdex-75 SEC column (Hi-Load 16/600, Prep Grade, GE Healthcare, USA) and MALDI analysis were used to determine the molecular mass of PrASNase [23].…”

Section: Molecular Weight Determinationmentioning

confidence: 99%

Assessment of structural behaviour of new L-asparaginase and SAXS data-based evidence for catalytic activity in its monomeric form

Mihooliya

Nandal

Kalidas

et al. 2023

Preprint

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The present study reports the structural and functional characterization of a new glutaminase-free recombinant L-asparaginase (PrASNase) from Pseudomonas resinovorans IGS-131. PrASNase showed substrate specificity to L-asparagine, and its kinetic parameters, Km, Vmax, and kcat were 9.49x10-3 M, 25.13 IUmL-1min-1, and 3x103 s-1, respectively. The CD spectra showed that PrASNase consists of 30.9% alpha-helix and 69.1% other structures in its native form.FTIR was used for the functional characterization, and molecular docking predicted that the substrate interacts with serine, alanine, and glutamine in the binding pocket of PrASNase. Different from known asparaginases, structural characterization by small-angle X-ray scattering (SAXS) and analytical ultracentrifugation (AUC) unambiguously revealed PrASNase to exist as a monomer in solution at low temperatures and oligomerized to a higher state with temperature rise. Through SAXS studies and enzyme assay, PrASNase was found to be mostly monomer and catalytically active at 37°C. Furthermore, this glutaminase-free PrASNase showed killing effects against WIL2-S with IC50 of 7.4 ug.mL-1 and and TF-1.28 cells with IC50 of 5.6 ug.mL-1. This is probably the first report with significant findings of fully active L-asparaginase in monomeric form using SAXS and AUC and demonstrates the potential of PrASNase in inhibiting cancerous cells, making it a potential therapeutic candidate.

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Studies on efficient production of a novel l-asparaginase by a newly isolated Pseudomonas resinovorans IGS-131 and its heterologous expression in Escherichia coli

Cited by 22 publications

References 23 publications

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

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

Response surface methodological optimization of l-asparaginase production from the medicinal plant endophyte Acinetobacter baumannii ZAS1

Assessment of structural behaviour of new L-asparaginase and SAXS data-based evidence for catalytic activity in its monomeric form

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