Investigation of chromatography and polymer/salt aqueous two-phase processes for downstream processing development of recombinant phenylalanine dehydrogenase.
This work presents a comprehensive study between the polymer/salt aqueous two-phase systems (ATPS) and chromatography process for downstream processing of recombinant Bacillus badius phenylalanine dehydrogenase (PheDH). First, the partitioning behavior of recombinant PheDH in polyethylene glycol (PEG)/K2HPO4 ATPS was examined. For comparative purpose, a classical chromatographic protocol was performed as well. Investigation of chromatography and ATPS procedures revealed that the ATPS comprising of 9% (w/w) PEG-6000, 16% (w/w) K2HPO4 and 16% (w/w) KCl with pH of 8.0, volume ratio (V ( R )) of 0.25, temperature of 25 degrees C and 40% (w/w) cell lysate ensured the most favorable approach for PheDH downstream process. A specific activity of 4,231.4 U/mg, a yield of 96.7% and a recovery of 162.0% were obtained. Furthermore, the shorter process time (4 vs. 48 h) and the lower total cost (4 vs. 20 euro) were additionally features that confirmed the suitability of proposed technique.
Omidinia
Eskandar
E
Department of Biochemistry, Pasteur Institute of Iran, 13164, Tehran, Iran.
Shahbaz Mohamadi
Hamid
H
Dinarvand
Rassoul
R
Taherkhani
Heshmat-Allah
HA
D000594:Amino Acid Oxidoreductases / Q000737:chemistry*; D001709:Biotechnology / Q000379:methods*; D002845:Chromatography / Q000379:methods*; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D011092:Polyethylene Glycols / Q000737:chemistry; D011108:Polymers / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry*; D012492:Salts / Q000737:chemistry; D014867:Water / Q000737:chemistry
D011108:Polymers; D011994:Recombinant Proteins; D012492:Salts; D014867:Water; D011092:Polyethylene Glycols; D000594:Amino Acid Oxidoreductases; C022342:phenylalanine oxidase