Rapid one-step separation and purification of recombinant phenylalanine dehydrogenase in aqueous two-phase systems.
Phenylalanine dehydrogenase (PheDH; EC 1.4.1.20) is a NAD+-dependent enzyme that performs the reversible oxidative deamination of L-phenylalanine to phenylpyruvate. It plays an important role in detection and screening of phenylketonuria (PKU) diseases and production of chiral intermediates as well. The main goal of this study was to find a simple and rapid alternative method for purifying PheDH.



The purification of recombinant Bacillus sphaericus PheDH was investigated in polyethylene glycol (PEG) and ammonium sulfate aqueous two-phase systems (ATPS). The influences of system parameters including PEG molecular weight and concentration, pH and (NH4)2SO4 concentration on enzyme partitioning were also studied. The purity of enzyme was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis.



A single extraction process was developed for separation and purification of recombinant PheDH from E. coli BL21 (DE3). The optimized conditions for partitioning and purification of PheDH were 9% (w/w) PEG-6,000 and 16% (w/w) (NH4)2SO4 at pH 8.0. The partition coefficient, recovery, yield, purification factor and specific activity values were achieved 58.7, 135%, 94.42%, 491.93 and 9828.88 U/mg, respectively. Also, the Km values for L-phenylalanine and NAD+ in oxidative deamination were 0.21 and 0.13 mM, respectively.



The data presented in this paper demonstrated the potential of ATPS as a versatile and scaleable process for downstream processing of recombinant PheDH.
Shahbaz Mohammadi
Hamid
H
Young Researcher Club, Department of Biochemistry, Islamic Azad University, Tehran, Iran.
Omidinia
Eskandar
E
Dept. of Biochemistry, Pasteur Institute of Iran, Tehran 13164, Iran.
Taherkhani
Heshmatollah
H
Dept. of Parasitology and Mycology, School of Medicine, Gorgan University of Medical Sciences, Gorgan, Iran.
D000594:Amino Acid Oxidoreductases / Q000235:genetics / Q000302:isolation & purification*; D000645:Ammonium Sulfate; D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D011092:Polyethylene Glycols; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification*; D014867:Water
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D014867:Water; D011092:Polyethylene Glycols; D000594:Amino Acid Oxidoreductases; C022342:phenylalanine oxidase; D000645:Ammonium Sulfate