In situ extraction of intracellular L-asparaginase using thermoseparating aqueous two-phase systems.
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 (pI=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.
Zhu
Jian-Hang
JH
Key Laboratory of Poyang Lake Ecology and Bio-Resource Utilization of Ministry of Education, School of Environmental Science and Engineering, Nanchang University, Nanchang, Jiangxi 330031, China. envzjh@ncu.edu.cn
Yan
Xi-Luan
XL
Chen
Hong-Jun
HJ
Wang
Zhi-Hui
ZH
D001215:Asparaginase / Q000096:biosynthesis / Q000302:isolation & purification*; D019149:Bioreactors; D001709:Biotechnology / Q000379:methods*; D002457:Cell Extracts / Q000302:isolation & purification*; D002458:Cell Fractionation / Q000379:methods; D004926:Escherichia coli / Q000145:classification / Q000254:growth & development; D006863:Hydrogen-Ion Concentration; D011092:Polyethylene Glycols / Q000737:chemistry*; D011506:Proteins / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000302:isolation & purification*; D013696:Temperature
D002457:Cell Extracts; D011506:Proteins; D011994:Recombinant Proteins; D011092:Polyethylene Glycols; D001215:Asparaginase