In situ extraction of intracellular l-asparaginase using thermoseparating aqueous two-phase systems

• Published in: Journal of Chromatography A 2007-04, Vol.1147 (1), p.127-134
• Main Authors: Zhu, Jian-Hang, Yan, Xi-Luan, Chen, Hong-Jun, Wang, Zhi-Hui
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Antineoplastic agents; Aqueous two-phase extraction system; Asparaginase - biosynthesis; Asparaginase - isolation & purification; Biological and medical sciences; Bioreactors; Biotechnology - methods; Cell disruption; Cell Extracts - isolation & purification; Cell Fractionation - methods; Enzymes and enzyme inhibitors; Escherichia coli - classification; Escherichia coli - growth & development; Fundamental and applied biological sciences. Psychology; General aspects; Hydrogen-Ion Concentration; Hydrolases; In situ extraction; Intracellular protein; Medical sciences; Pharmacology. Drug treatments; Polyethylene Glycols - chemistry; Process integration; Proteins - isolation & purification; Recombinant Proteins - isolation & purification; Temperature
• ISSN: 0021-9673
• DOI: 10.1016/j.chroma.2007.02.035

The feasibility and generic applicability of directly integrating conventional discrete operations of cell disruption by high pressure homogenizer and the product capture by aqueous two-phase extraction (ATPE) system have been demonstrated for the extraction of intracellular l-asparaginase from E. coli. In a side-by-side comparison with the conventional ATPE process, including cell disruption, centrifugal clarification and following ATPE, purification of l-asparaginase via this novel in situ ATPE process yielded a product of l-asparaginase with a higher specific activity of 94.8 U/(mg protein) and a higher yield of 73.3%, both of which in the conventional ATPE process were 78.6 U/(mg protein) and 52.1%, respectively. In the purification of l-asparaginase (p I = 4.9), product–debris interactions commonly diminish its recovery. It was demonstrated that immediate extraction of l-asparaginase in ATPE systems when it is released at pH 5.0 during cell disruption effectively increased its recovery in the top phase due to the reduced interaction between l-asparaginase and cell debris and the reduced degradation by contaminated protease. In addition, no clarification step and/or disruptate storage are required in this in situ ATPE, which reduced the number of unit operations and thus shortened the overall process time. This novel process has a good potential for the separation of other intracellular biological products.