Purification of recombinant phenylalanine dehydrogenase by partitioning in aqueous two-phase systems.
This study presents the partitioning and purification of recombinant Bacillus badius phenylalanine dehydrogenase (PheDH) in aqueous two-phase systems (ATPS) composed of polyethylene glycol 6000 (PEG-6000) and ammonium sulfate. A single-step operation of ATPS was developed for extraction and purification of recombinant PheDH from E. coli BL21 (DE3). The influence of system parameters including; PEG molecular weight and concentration, pH, (NH(4))(2)SO(4) concentration and NaCl salt addition on enzyme partitioning were investigated. The best optimal system for the partitioning and purification of PheDH was 8.5% (w/w) PEG-6000, 17.5% (w/w) (NH(4))(2)SO(4) and 13% (w/w) NaCl at pH 8.0. The partition coefficient, recovery, yield, purification factor and specific activity values were of 92.57, 141%, 95.85%, 474.3 and 10424.97 U/mg, respectively. Also the K(m) values for L-phenylalanine and NAD(+) in oxidative deamination were 0.020 and 0.13 mM, respectively. Our data suggested that this ATPS could be an economical and attractive technology for large-scale purification of recombinant PheDH.
Mohamadi
Hamid Shahbaz
HS
Young Researcher Club, Department of Biochemistry, Science & Research Campus, Islamic Azad University, Tehran, Iran.
Omidinia
Eskandar
E
D000594:Amino Acid Oxidoreductases / Q000302:isolation & purification*; D001407:Bacillus / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000302:isolation & purification; D014867:Water
D011994:Recombinant Proteins; D014867:Water; D000594:Amino Acid Oxidoreductases; C022342:phenylalanine oxidase