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

Abstract

We studied optimal L-asparaginase sequence from GenBank accession number X12746 encoding for Lasparaginase from Erwinia chrysanthemi NCPPB1125. The expression level of recombinant Lasparaginase 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, K m , V max , K cat and K cat /K m of purified enzyme were found to be 0.5 mM, 500 U/mg, 14.9  10 3 s-1 , and 29.9  10 3 mM-1 s-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, Hg 2+ , Cu 2+ , Ni 2+ , and enhanced by Mg 2+. 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.

Current state of molecular and metabolic strategies for the improvement of L-asparaginase expression in heterologous systems

“…In this study, codons were optimized and inserted in a pET-21a(+) vector. After adjusting induction and purification conditions, the specific activity reached 312.8 U/mg, which increased 1.5-fold control activity ( Nguyen et al, 2016b ). Furthermore, the activity of three new uncharacterized extremophilic L-ASNases produced by psychrophilic fungus Sclerotinia borealis , thermoacidophilic crenarchaeon Acidilobus saccharovorans and thermophilic bacterium Melioribacter roseu.…”

Engineering and Expression Strategies for Optimization of L-Asparaginase Development and Production

“…The optimum temperature of the recombinant enzyme varies depending on the bacterial source and the expression host [138]. The optimal pH and temperature for L-ASNases from E. coli MTCC739 in E. coli BL21(DE3) are 37 • C. For example, the optimal temperature and pH for rASPG from the thermotolerant strain E. coli KH027 in E. coli DH5 were 43 • C and pH 6.…”

“…The optimal pH and temperature for L-ASNases from E. coli MTCC739 in E. coli BL21(DE3) are 37 • C. For example, the optimal temperature and pH for rASPG from the thermotolerant strain E. coli KH027 in E. coli DH5 were 43 • C and pH 6. For rASPG from E. coli W3110 in E. coli BL21(DE3), the conditions were 37 • C and pH 7.5 [138]. According to research by Borah et al (2012), L-ASNase can be produced from E. coli, with 55 • C being the optimal temperature for enzyme activity [139].…”

Importancia de la enzima "L-Asparaginasa" en la industria alimentaria y en salud pública

“…Igualmente, esta enzima también es una promesa estratégica en casos de leucemia mieloide crónica, ya que genera un aumento en la inhibición y apoptosis en las células K562 y KU812 (líneas celulares derivadas de leucemia mieloide) (Trang et al, 2016). De igual manera, hoy en día la L-Aase forma parte de varios protocolos oncológicos como: enfermedad Hodking, leucemia mielomonocítica aguda, leucemia linfocítica crónica, melanosarcoma, reticulosarcoma, leucemia linfoblástica crónica, leucemia mieloblástica, linfoma no hodgkin's…”