Extracellular Production of Recombinant L-Asparaginase II in Escherichia coli: Medium Optimization Using Response Surface Methodology

摘要

In this study a strategy based on using signal sequence and statistically optimized medium was applied for efficient extracellular production of the recombinant form of the therapeutic enzyme l-asparaginase. l-Asparaginase from E. coli is an important enzyme used in the cancer treatment, which has been produced and studied previously by other researchers. Nevertheless, to date no study has investigated the extracellular production of recombinant l-asparaginase in the culture medium optimized using response surface methodology. For this purpose, in this study at first, a complete gene of l-asparaginase II with its own signal sequence from a locally isolated E. coli was cloned and expressed in E. coli BL21 (DE3). Subsequently, the production of the active form of recombinant l-asparaginase was evaluated by asparaginase activity assay in the culture media 4 h after induction with IPTG in 250 mL shake flasks to evaluate the effect of eleven nutrient factors on the extracellular l-asparaginase production. Statistical design of experiments was employed in the screening stage and the most effective nutrients were selected for further optimization using central composite face design. Analysis of the results revealed a mature protein with correct N-terminal amino acids of l-asparaginase II in the culture medium. The highest enzyme activity of 17,386 U/L was resulted in the optimized medium consisting of 7.75 g/L tryptone, 9 g/L yeast extract, 5.25 g/L peptone and 0.6 g/L calcium chloride at shake flask level.