Abstract
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.
Highlights
L- asparaginase (EC 3.5.1.1) is the enzyme that catalysis the hydrolytic deamination of L-asparagine to produce L-aspartate and ammonia (Pritsa and Kyriakidis, 2001; Borek et al, 2004)
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)
The anticancer activity of L-asparaginase is based on the hydrolytic deamination of L-asparagine, the normal cells can produce L-asparagine
Summary
L- asparaginase (EC 3.5.1.1) is the enzyme that catalysis the hydrolytic deamination of L-asparagine to produce L-aspartate and ammonia (Pritsa and Kyriakidis, 2001; Borek et al, 2004). The anticancer activity of L-asparaginase is based on the hydrolytic deamination of L-asparagine, the normal cells can produce L-asparagine. It becomes an essential amino acid for cancer cells due to absence of asparagine synthase activity in these cancer cells (Kotzia and Labrou, 2007; Dhankhar et al, 2020). In addition to its anticancer activity the enzyme has an ability to prohibit or reduce the production of toxic compounds such as acrylamide in the baked or fried foods. Recombinant DNA technology has made it possible to produce bulk quantities of enzymes and proteins of pharmaceutical importance in E. coli and Bacillus species. The present study was aimed to characterize the recombinant L-asparaginase I from Pyrococcus abyssi and its effectiveness as anticancer molecule
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