:: Volume 12, Issue 2 (4-2008) ::
ibj 2008, 12(2): 115-122 Back to browse issues page
Rapid One-Step Separation and Purification of Recombinant Phenylalanine Dehydrogenase in Aqueous Two-Phase Systems
Hamid Shahbaz Mohammadi, Eskandar Omidinia *, Heshmatollah Taherkhani
Background: Phenylalanine dehydrogenase (PheDH EC 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 (NH4)2SO4 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) (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. Conclusion: The data presented in this paper demonstrated the potential of ATPS as a versatile and scaleable process for downstream processing of recombinant PheDH.
Keywords: Aqueous two-phase systems (ATPS), Ammonium sulfate, Phenylalanine dehydrogenase (PheDH), Purification
Full-Text [PDF 154 kb]      
Type of Study: Full Length | Subject: Related Fields
Send email to the article author

Add your comments about this article
Your username or Email:


XML     Print

Download citation:
BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks
Send citation to:

Shahbaz Mohammadi H, Omidinia E, Taherkhani H. Rapid One-Step Separation and Purification of Recombinant Phenylalanine Dehydrogenase in Aqueous Two-Phase Systems. ibj. 2008; 12 (2) :115-122
URL: http://ibj.pasteur.ac.ir/article-1-98-en.html

Volume 12, Issue 2 (4-2008) Back to browse issues page
Persian site map - English site map - Created in 0.18 seconds with 38 queries by YEKTAWEB 3879