Production, purification and kinetic characterization of glutaminase free anti-leukemic L-asparaginase with low endotoxin level from novel soil isolate

Prakash, Pragya; Singh, Hare Ram; Jha, Santosh Kumar

doi:10.1080/10826068.2019.1692221

Cited by 27 publications

(11 citation statements)

References 45 publications

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“…SDS-PAGE results showed a single protein band between 25 and 35 kDa for GmAS-Nase, which is consistent with the predicted value. This result is similar to those for L-asparaginases from P. fluorescens (35 kDa) [40], B. altitudinis (35 kDa) [46], T. kodakarensis (32.6 kDa) [47], and P. furiosus (33.66 kDa) [45].…”

Section: Discussionsupporting

confidence: 82%

“…As expected, the specific activity of purified GmASNase was as high as 486.65 IU mg −1 , which is 31.30 times that of the crude enzyme (15.55 IU mg −1 ), and even higher than those of L-asparaginases from many other sources, such as P. fluorescens (26 IU mg −1 ) [40], S. brollosae (76.671 IU mg −1 ) [41], A. terreus (339.34 IU mg −1 ) [42], B. licheniformis (36.08 IU mg −1 ) [43], and B. amyloliquefaciens (4.75 IU mg −1 ) [31]. However, it is still lower than L-asparaginases from B. sonorensis (4438.62 IU mg −1 ) [44], P. furiosus (11,203.5 IU mg −1 ) [45], and B. altitudinis (800 IU mg −1 ) [46]. SDS-PAGE results showed a single protein band between 25 and 35 kDa for GmAS-Nase, which is consistent with the predicted value.…”

Section: Discussionmentioning

confidence: 97%

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Characterization of a Novel L-Asparaginase from Mycobacterium gordonae with Acrylamide Mitigation Potential

Chi

Chen

Jiao

et al. 2021

Foods

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L-asparaginase (E.C.3.5.1.1) is a well-known agent that prevents the formation of acrylamide both in the food industry and against childhood acute lymphoblastic leukemia in clinical settings. The disadvantages of L-asparaginase, which restrict its industrial application, include its narrow range of pH stability and low thermostability. In this study, a novel L-asparaginase from Mycobacterium gordonae (GmASNase) was cloned and expressed in Escherichia coli BL21 (DE3). GmASNase was found to be a tetramer with a monomeric size of 32 kDa, sharing only 32% structural identity with Helicobacter pylori L-asparaginases in the Protein Data Bank database. The purified GmASNase had the highest specific activity of 486.65 IU mg−1 at pH 9.0 and 50 °C. In addition, GmASNase possessed superior properties in terms of stability at a wide pH range of 5.0–11.0 and activity at temperatures below 40 °C. Moreover, GmASNase displayed high substrate specificity towards L-asparagine with Km, kcat, and kcat/Km values of 6.025 mM, 11,864.71 min−1 and 1969.25 mM−1min−1, respectively. To evaluate its ability to mitigate acrylamide, GmASNase was used to treat potato chips prior to frying, where the acrylamide content decreased by 65.09% compared with the untreated control. These results suggest that GmASNase is a potential candidate for applications in the food industry.

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Section: Discussionsupporting

confidence: 82%

Section: Discussionmentioning

confidence: 97%

Characterization of a Novel L-Asparaginase from Mycobacterium gordonae with Acrylamide Mitigation Potential

Chi

Chen

Jiao

et al. 2021

Foods

View full text Add to dashboard Cite

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“…Of interest are enzymes from the bacteria Leucosporidium scottii , Anoxybacillus flavithermus , extremophile yeast, Bacillus antitudinis , etc., living in high- and low-temperature and highly saline environments [ 80 , 149 , 150 , 151 ]. Recently L-ASNase from Melioribacter roseus , which belongs to the Ignavibacteriae Bacteroidetes/Chlorobi group, was purified and characterized.…”

Section: Alternative Approaches To the Development Of Antitumor L-asn...mentioning

confidence: 99%

Molecular Analysis of L-Asparaginases for Clarification of the Mechanism of Action and Optimization of Pharmacological Functions

et al. 2022

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L-asparaginases (EC 3.5.1.1) are a family of enzymes that catalyze the hydrolysis of L-asparagine to L-aspartic acid and ammonia. These proteins with different biochemical, physicochemical and pharmacological properties are found in many organisms, including bacteria, fungi, algae, plants and mammals. To date, asparaginases from E. coli and Dickeya dadantii (formerly known as Erwinia chrysanthemi) are widely used in hematology for the treatment of lymphoblastic leukemias. However, their medical use is limited by side effects associated with the ability of these enzymes to hydrolyze L-glutamine, as well as the development of immune reactions. To solve these issues, gene-editing methods to introduce amino-acid substitutions of the enzyme are implemented. In this review, we focused on molecular analysis of the mechanism of enzyme action and to optimize the antitumor activity.

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“…Enzyme isolated from different sources has shown variable biological activity e.g. L-asparaginase from Streptomyces rochei has 119.5 U/mg of proteins (El-Naggar and El-Shweihy, 2020), from Anoxybacillus flavithermus 165U/mg (Maqsood et al, 2020), and enzyme isolated from Bacillus altitudinis exhibited 800 U/mg (Prakash et al, 2020). L-asparaginase from different species has been intensively used for the treatment of leukaemia (Verma et al, 2007;Hourani et al, 2008;Lanvers-Kaminsky et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

Studies on the recombinant production and anticancer activity of thermostable L- asparaginase I from Pyrococcus abyssi

et al. 2022

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L-Asparaginase catalysing the breakdown of L-Asparagine to L-Aspartate and ammonia is an enzyme of therapeutic importance in the treatment of cancer, especially the lymphomas and leukaemia. The present study describes the recombinant production, properties and anticancer potential of enzyme from a hyperthermophilic archaeon Pyrococcus abyssi. There are two genes coding for asparaginase in the genome of this organism. A 918 bp gene encoding 305 amino acids was PCR amplified and cloned in BL21 (DE3) strain of E. coli using pET28a (+) plasmid. The production of recombinant enzyme was induced under 0.5mM IPTG, purified by selective heat denaturation and ion exchange chromatography. Purified enzyme was analyzed for kinetics, in silico structure and anticancer properties. The recombinant enzyme has shown a molecular weight of 33 kDa, specific activity of 1175 U/mg, KM value 2.05mM, optimum temperature and pH 80°C and 8 respectively. No detectable enzyme activity found when L-Glutamine was used as the substrate. In silico studies have shown that the enzyme exists as a homodimer having Arg11, Ala87, Thr110, His112, Gln142, Leu172, and Lys232 being the putative active site residues. The free energy change calculated by molecular docking studies of enzyme and substrate was found as ∆G – 4.5 kJ/mole indicating the affinity of enzyme with the substrate. IC50 values of 5U/mL to 7.5U/mL were determined for FB, caco2 cells and HepG2 cells. A calculated amount of enzyme (5U/mL) exhibited 78% to 55% growth inhibition of caco2 and HepG2 cells. In conclusion, the recombinant enzyme produced and characterized in the present study offers a good candidate for the treatment of cancer. The procedures adopted in the present study can be prolonged for in vivo studies.

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Production, purification and kinetic characterization of glutaminase free anti-leukemic L-asparaginase with low endotoxin level from novel soil isolate

Cited by 27 publications

References 45 publications

Characterization of a Novel L-Asparaginase from Mycobacterium gordonae with Acrylamide Mitigation Potential

Characterization of a Novel L-Asparaginase from Mycobacterium gordonae with Acrylamide Mitigation Potential

Molecular Analysis of L-Asparaginases for Clarification of the Mechanism of Action and Optimization of Pharmacological Functions

Studies on the recombinant production and anticancer activity of thermostable L- asparaginase I from Pyrococcus abyssi

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