Optimization, purification and characterization of recombinant L-asparaginase II in Escherichia coli

  • Trang T
  • Cuong T
  • Thanh S
  • et al.
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Abstract

We studied optimal L-asparaginase sequence from GenBank accession number X12746 encoding for L-asparaginase from Erwinia chrysanthemi NCPPB1125. The expression level of recombinant L-asparaginase was determined as 78% of the total proteins. The purified L-asparaginase had a molecular mass of 37 kDa with specific activity of 312.8 U/mg. Kinetic parameters, Km, Vmax, Kcat and Kcat/Km of purified enzyme were found to be 0.5 mM, 500 U/mg, 14.9 ï‚´ 103 s-1, and 29.9 ï‚´ 103 mM-1s-1, respectively. Temperature and pH optimum were observed at 45ºC and pH 7.5, respectively. The enzyme exhibited about 20 and 60% retention of activity following 100 min incubation at 55 or 40°C, respectively. The activity of enzyme was inhibited by EDTA, Hg2+, Cu2+, Ni2+, and enhanced by Mg2+. Detergents (Tween 20, Tween 80, Triton X-100, and Triton X-114) decreased enzyme activity. DTT and DMSO at appropriate concentrations enhanced enzyme activity. In vitro anti-cancer activity was performed using different tumor cell lines. Concentration of recombinant L-asparaginase at 50 µg/ml inhibited 45.32, 48.22, 53.68, 51.22% with HL-60, P388, P3X63Ag8, SP2/0-Ag14 cell lines. Recombinant L-asparaginase was expressed successfully in Escherichia coli with high expression level, had a high specific activity and antiproliferative effect on several tumor cell lines.   Key words: Characterization, Erwinia chrysanthemi, L-asparaginase, purification, tumor cell line.

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Trang, T. H. N., Cuong, T. N., Thanh, S. L. N., & Tuyen, T. D. (2016). Optimization, purification and characterization of recombinant L-asparaginase II in Escherichia coli. African Journal of Biotechnology, 15(31), 1681–1691. https://doi.org/10.5897/ajb2016.15425

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