African Journal of
Biotechnology

  • Abbreviation: Afr. J. Biotechnol.
  • Language: English
  • ISSN: 1684-5315
  • DOI: 10.5897/AJB
  • Start Year: 2002
  • Published Articles: 12278

Full Length Research Paper

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

Trang Thi Hien Nguyen
  • Trang Thi Hien Nguyen
  • Institute of Biotechnology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Distr., Caugiay, 10000 Hanoi, Vietnam.
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Cuong Tien Nguyen
  • Cuong Tien Nguyen
  • Institute of Biotechnology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Distr., Caugiay, 10000 Hanoi, Vietnam.
  • Google Scholar
Thanh Sy Le Nguyen
  • Thanh Sy Le Nguyen
  • Institute of Biotechnology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Distr., Caugiay, 10000 Hanoi, Vietnam.
  • Google Scholar
Tuyen Thi Do
  • Tuyen Thi Do
  • Institute of Biotechnology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Distr., Caugiay, 10000 Hanoi, Vietnam.
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  •  Received: 21 April 2016
  •  Accepted: 19 July 2016
  •  Published: 03 August 2016

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  103 s-1, and 29.9  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.