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Studies on the Recombinant Production in E. coli and Characterization of Pharmaceutically Important Thermostable L-Asparaginase from Geobacillus thermodenitrificans

Studies on the Recombinant Production in E. coli and Characterization of Pharmaceutically Important Thermostable L-Asparaginase from Geobacillus thermodenitrificans

Muhammad Shahid Nadeem*, Maryam A. Al-Ghamdi and Jalaluddin Azam Khan

Department of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah 21589, Saudi Arabia

*      Corresponding author: mhalim@kau.edu.sa

 

 

ABSTRACT

L-asparaginase is ubiquitously found in microbes, animals and plants. The asparaginase of bacterial origin has been extensively applied in the treatment of childhood lymphomas and leukaemia. However, the glutaminase activity of enzyme can cause serious side-effects triggering a search for highly specific and more stable enzyme. In the present study the gene consisting of 972bp coding for 323 amino acids was PCR amplified from Geobacillus thermodenitrificans DSM-465 was PCR amplified and recombinant plasmid pET22b-Asn was prepared. L-asparaginase was produced in BL21 (DE3) RIL codon plus strain of E. coli as heterologous protein under 0.3mM IPTG. The recombinant enzyme was purified by DEAE-Sephadex colum based anion-exchange chromatography. On SDS-PAGE, the enzyme exhibited a molecular weight of about 36 KDa. Its specific activity was 1650 U per mg of protein with about 5% glutaminase activity and no activity against D-asparagine. Optimal enzyme activity was found at 75°C and pH 9. The KM value of 5.9mM was found for L-asparagine. In silico studies have shown that the enzyme exists as a homotetramer. Molecular docking studies have revealed that Gly199, Lys300, Arg198, Arg197, Tyr201, Tyr323 are putative active site residues interacting the substrate. Anti-proliferative effect of purified enzyme was determined quantitatively using non-cancerous human fibroblast cells (FB) and hepatocellular carcinoma cells (HepG2). A dose of 5U/ml of enzyme has shown an anti-proliferative effect up to 65 percent on HepG2 cells.
 

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