Aqueous micellar two-phase system composed of hyamine-type hydrophobically modified ethylene oxide and application for cytochrome P450 BM-3 separation.
The hydrophobically modified ethylene oxide polymer, HM-EO, was modified with an alkyl halide to prepare a hyamine-type HM-EO, named N-Me-HM-EO, which could be used for forming N-Me-HM-EO/buffer aqueous micellar two-phase system. The critical micelle concentration of N-Me-HM-EO solution and the phase diagrams of N-Me-HM-EO/buffer systems were determined. By using this novel aqueous micellar two-phase system, the separation of cytochrome P450 BM-3 from cell extract was explored. The partitioning behavior of P450 BM-3 in N-Me-HM-EO/buffer systems was measured. The influences of some factors such as total proteins concentration, pH, temperature and salt concentration, on the partitioning coefficients of P450 BM-3 were investigated. Since the micellar aggregates in the N-Me-HM-EO enriched phase were positively charged, it was possible to conduct the proteins with different charges to top or bottom phases by adjusting pH and salt concentration in the system. A separation scheme consisting of two consecutive aqueous two-phase extraction steps was proposed: the first extraction with N-Me-HM-EO/buffer system at pH 8.0, and the second extraction in the same system at pH 6.0. The recovery of P450 BM-3 was 73.3% with the purification factor of 2.5. The results indicated that the aqueous micellar two-phase system composed of hyamine modified polysoap has a promising application for selective separation of biomolecules depending on the enhanced electrostatic interactions between micelles and proteins.
Deng
Gang
G
Department of Chemical and Biochemical Engineering, Zhejiang University, 310027 Hangzhou, China.
Lin
Dong-Qiang
DQ
Yao
Shan-Jing
SJ
Mei
Le-He
LH
D001426:Bacterial Proteins / Q000302:isolation & purification* / Q000378:metabolism; D001558:Benzethonium / Q000031:analogs & derivatives* / Q000737:chemistry; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification* / Q000378:metabolism; D005027:Ethylene Oxide / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008823:Micelles; D006899:Mixed Function Oxygenases / Q000302:isolation & purification* / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase
D001426:Bacterial Proteins; D008823:Micelles; D001558:Benzethonium; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases; D005027:Ethylene Oxide; C005106:methylbenzethonium chloride