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Rapid one-step separation and purification of recombinant phenylalanine dehydrogenase in aqueous two-phase systems

Mohammadi, H.S. and Omidinia, E. and Taherkhani, H. (2008) Rapid one-step separation and purification of recombinant phenylalanine dehydrogenase in aqueous two-phase systems. Iranian Biomedical Journal, 12 (2). pp. 115-122. ISSN 1028852X (ISSN)

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Abstract

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 (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.

Item Type: Article
Additional Information: Unmapped bibliographic data: LA - English [Field not mapped to EPrints] J2 - Iran. Biomed. J. [Field not mapped to EPrints] C2 - 18506218 [Field not mapped to EPrints] AD - Dept. of Biochemistry, Science and Research Campus, Islamic Azad University, Tehran, Iran [Field not mapped to EPrints] AD - Dept. of Biochemistry, Pasteur Institute of Iran, Tehran 13164, Iran [Field not mapped to EPrints] AD - Dept. of Parasitology and Mycology, School of Medicine, Gorgan University of Medical Sciences, Gorgan, Iran [Field not mapped to EPrints] DB - Scopus [Field not mapped to EPrints]
Uncontrolled Keywords: Ammonium sulfate, Aqueous two-phase systems (ATPS), Phenylalanine dehydrogenase (PheDH), Polyethylene glycol 6,000 (PEG-6,000), Purification, amide, ammonium sulfate, macrogol, nicotinamide adenine dinucleotide, oxidoreductase, phenylalanine, recombinant enzyme, article, Bacillus sphaericus, concentration (parameters), deamination, enzyme activity, enzyme isolation, enzyme purification, Escherichia coli, molecular weight, nonhuman, partition coefficient, pH, polyacrylamide gel electrophoresis, Amino Acid Oxidoreductases, Ammonium Sulfate, Bacillus, Bacterial Proteins, Hydrogen-Ion Concentration, Polyethylene Glycols, Recombinant Proteins, Water, Bacillus sphaericus
Subjects: مقالات نمایه شده محققین دانشگاه در سایت ,Web of Science ,Scopus
Divisions: معاونت تحقیقات و فناوری
Depositing User: GOUMS
Date Deposited: 18 Apr 2015 05:34
Last Modified: 26 Feb 2017 07:53
URI: http://eprints.goums.ac.ir/id/eprint/2341

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