Abstract
The present work elucidates the production of recombinant human asparaginase (rhASP) under optimized fermentation and downstream processes in Escherichia coli. The maximum biomass yield of 6.7 g/L was achieved with fed-batch fermentation. The highest rhASP inclusion bodies recovery yield (91%) was achieved with the optimized lysis conditions. The 8.0 M urea at pH 8.5 has shown efficient solubilization (94%) of rhASP inclusion bodies. The refolding efficiency of rhASP increased at pH 8.5 (84%) and temperature 25°C (86%). The diluted rhASP solution was concentrated and partially purified (92%) using cross flow filtration. A single step ion exchange chromatography is successfully achieved the maximum purity of ≥ 97%. The molecular mass of purified rhASP is confirmed as 34.1 kDa by mass spectrometry. The secondary structure of rhASP is characterized by FT-IR spectroscopy based on the structural elements. Finally, cell proliferative assay of purified rhASP is signifies the similar biological activity over the standard.
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