Expression and Functional Characterization of Pseudomonas aeruginosa Recombinant l.Asparaginase

Saeed, Hesham; Soudan, Hadeer; Sharkawy, Amany S. El; Farag, Aïda M.; Embaby, Amira M.; Ataya, Farid S.

doi:10.1007/s10930-018-9789-3

Cited by 19 publications

(7 citation statements)

References 51 publications

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“…The enzyme-substrate combinations were incubated for 10 min at 37°C before being stopped by adding 100 µl of 1.5 M TCA. The amount of ammonia emitted was estimated using Nessler's reagent [ 21 ] and an ammonium sulfate solution as a standard, after which the samples were centrifuged and used for ammonia estimation. An international unit (UI) of L-asparaginase is defined as the amount of enzyme necessary to release one micromole of ammonia per minute at saturating substrate concentration under the assay conditions [ 22 ].…”

Section: Methodsmentioning

confidence: 99%

Anticancer Activity of Extremely Effective Recombinant L-Asparaginase fromBurkholderia pseudomallei

Darwesh

Al-Awthan

Elfaki

et al. 2022

J. Microbiol. Biotechnol.

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L-asparaginase (E.C. 3.5.1.1) purified from bacterial cells is widely used in the food industry, as well as in the treatment of childhood acute lymphoblastic leukemia. In the present study, the Burkholderia pseudomallei L-asparaginase gene was cloned into the pGEX-2T DNA plasmid, expressed in E. coli BL21 (DE3) pLysS, and purified to homogeneity using Glutathione Sepharose chromatography with 7.26 purification fold and 16.01% recovery. The purified enzyme exhibited a molecular weight of ~33.6 kDa with SDS-PAGE and showed maximal activity at 50°C and pH 8.0. It retained 95.1, 89.6%, and 70.2% initial activity after 60 min at 30°C, 40°C, and 50°C, respectively. The enzyme reserved its activity at 30 o C and 37 o C up to 24 h. The enzyme had optimum pH of 8 and reserved 50% activity up to 24 h. The recombinant enzyme showed the highest substrate specificity towards L-asparaginase substrate, while no detectable specificity was observed for L-glutamine, urea, and acrylamide at 10 mM concentration. THP-1, a human leukemia cell line, displayed significant morphological alterations after being treated with recombinant L-asparaginase and the IC 50 of the purified enzyme was recorded as 0.8 IU. Furthermore, the purified recombinant Lasparaginase improved cytotoxicity in liver cancer HepG2 and breast cancer MCF-7 cell lines, with IC 50 values of 1.53 and 18 IU, respectively.

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Section: Methodsmentioning

confidence: 99%

Anticancer Activity of Extremely Effective Recombinant L-Asparaginase fromBurkholderia pseudomallei

Darwesh

Al-Awthan

Elfaki

et al. 2022

J. Microbiol. Biotechnol.

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“…L-asparaginase activity was frequently reported in plants, micro-organisms (bacteria, fungi, and actinomycetes), animals, and in the serum of certain rodents but was not isolated from a human source (Lalitha and Ramanjaneyulu, 2016). Many genera of bacteria, Bacillus circuans (Prakasham et al, 2010), Bacilus brevis (Narta et al, 2011), Pseudomonas flurescens (Sinha et al, 2015), Pseudomonas aeruginosa (Saeed et al, 2018), and Escherichia coli (Kante et al, 2019) are reported as Lasparaginase producers.…”

Section: Introductionmentioning

confidence: 99%

roduction of Chemotherapeutic Agent L-asparaginase from Gamma-Irradiated Pseudomonas aeruginosa WCHPA075019.

2021

JJBS

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Because of the dangers and painful effects of chemotherapeutic drugs, the need for therapeutic agents with less adverse effects will increase several times in the coming years. L-asparaginase enzyme is an effective antitumor agent, especially acute lymphoblastic leukemia, with no side effects compared to other chemotherapeutic agents. Microorganisms are emerging as a safer source of L-asparaginase. Therefore, the findings of new L-asparaginase-producing bacterial strains with high yield for therapeutic applications become necessary. From twenty bacterial isolates tested for their L-asparaginase activity, 16S rRNA sequencing for the most potent isolate showed that the selected isolate had 100% identity to pseudomonas aeruginosa strain (accession number: WCHPA075019). In the presence of L-asparagine (1%) and glucose (1%) as nitrogen and carbon sources at a low dose of gamma radiation (0.75 kGy), the maximum productivity of Lasparaginase was reached after 2 days at 35 ° C, pH 7.6 under shaking at 200 rpm. Purification of L-asparaginase with 70% ammonium sulphate, followed by Sephadex G100 increases enzyme purity by 1.5-fold after gel filtration. Pure Lasparaginase had a molecular weight of 123 kDa by SDS-PAGE. The maximum activity of the enzyme against L-asparagine was detected at 35°C and pH 9.0 after 30 min and 200 mM substrate. L-asparaginase activated in the presence of metal ions such as K+, and Na+, not affected when exposed to EDTA and strongly inhibited in the presence of Ba2+, and Cd2+. The anticancer activity of the purified enzyme was tested in vitro against three types of cell line carcinoma. The growth inhibition of L-asparaginase for HEPG2 carcinoma cell line (IC50 value of 3.5 µg/ml) was greater than the inhibition of HCT and MCF-7 carcinoma cell lines with IC50 value of 3.8 and 12.5 µg/ml, respectively relative to the growth of the untreated control cells.

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“…Nessler's reagent is widely used to quantify ASNase activity, yet little attention has been paid to the effects of salts present in the analyte that can yield erroneous estimates of enzyme activity 22–28 . Here, we determined that the salts of Co 2+ , Fe 2+ , Mg 2+ , Mn 2+ and Ni 2+ interfered with NH 3 quantification using Nessler's reagent, while the salts of Ca 2+ , Cu 2+ , Mo 2+ , Na + and Zn 2+ did not.…”

Section: Resultsmentioning

confidence: 89%

An improved method for simple and accurate colorimetric determination of l‐asparaginase enzyme activity using Nessler's reagent

Simas

Kleingesinds

Pessoa

et al. 2021

J of Chemical Tech & Biotech

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BACKGROUND Colorimetric determination of l‐asparaginase enzyme activity most commonly uses Nessler's reagent, but often yields unreliable results due to precipitation of tetraiodomercurate salts. Interference caused by some commonly used inorganic and organic compounds of biochemical buffers, reagents and microbiology fermentation media was determined. The stabilising effects of tartrate and polyvinyl alcohol upon the impact of these salts was next evaluated using the following two‐step assay in 96‐well microtitre plates: 160 μL of 50 mmol L−1 Tris–HCl pH 8.6, 32 μL of 100 mmol L−1 l‐asparagine, 16 μL analyte and 128 μL water were added to each well. After incubation at 37 °C for 1 h, 16 μL of 1.5 mol L−1 trichloroacetic acid was added to stop the reaction. A 35 μL volume was next transferred to a fresh well containing 35 μL Nessler's reagent, 35 μL stabiliser solution (4 mmol L−1 disodium tartrate and 10 mg L−1 polyvinyl alcohol) and 245 μL water. After incubation at room temperature for 10 min, absorbance was recorded at 436 nm and the enzyme activity calculated by extrapolation of a (NH4)2SO4 calibrated standard curve. RESULTS Addition of tartrate and polyvinyl alcohol proved to be effective in reducing interference caused by precipitation of salts, allowing determination of l‐asparaginase activity in a range from 0.050 to 0.796 IU mL−1, ideal for monitoring enzyme production in fermentations. CONCLUSION A method is offered for rapid and reliable determination of l‐asparaginase activity, particularly in complex samples such as microbial fermentation broths where existing methodologies do not account for interferences that frequently yield overvalues. © 2020 Society of Chemical Industry

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Expression and Functional Characterization of Pseudomonas aeruginosa Recombinant l.Asparaginase

Cited by 19 publications

References 51 publications

Anticancer Activity of Extremely Effective Recombinant L-Asparaginase fromBurkholderia pseudomallei

Anticancer Activity of Extremely Effective Recombinant L-Asparaginase fromBurkholderia pseudomallei

roduction of Chemotherapeutic Agent L-asparaginase from Gamma-Irradiated Pseudomonas aeruginosa WCHPA075019.

An improved method for simple and accurate colorimetric determination of l‐asparaginase enzyme activity using Nessler's reagent

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