چكيده لاتين :
Background: 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.
Methods: 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 (NH4hS04 concentration on enzyme
partitioning were also studied. The purity of enzyme was analyzed by sodium dodecyl sulfate polyacrylamide
gel electrophoresis. Results: 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) (NH4hS04 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 Ki; values for L-phenylalanine and NAD+ in oxidative deamination were 0.21
and 0.13 mM, respectively. Conclusion: The data presented in this paper demonstrated the potential of ATPS
as a versatile and scaleable process for downstream processing of recombinant PheDH. Iran. Biomed. J. 12 (2):
115-122,2008
