Single-Step Purification and Characterization of Recombinant Aspartase of Aeromonas media NFB-5
| Autor: | Mukesh Yadav, Ram Sarup Singh |
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| Rok vydání: | 2012 |
| Předmět: |
Fumaric acid
Entropy Molecular Sequence Data Bioengineering medicine.disease_cause Aspartate Ammonia-Lyase Applied Microbiology and Biotechnology Biochemistry Chromatography Affinity law.invention chemistry.chemical_compound Affinity chromatography law Enzyme Stability medicine Amino Acid Sequence Cloning Molecular Molecular Biology Magnesium ion Escherichia coli chemistry.chemical_classification Expression vector biology Temperature General Medicine Hydrogen-Ion Concentration biology.organism_classification Recombinant Proteins Enzyme Activation Molecular Weight Kinetics Enzyme chemistry Metals Recombinant DNA Aeromonas media Aeromonas Half-Life Biotechnology |
| Zdroj: | Applied Biochemistry and Biotechnology. 167:991-1001 |
| ISSN: | 1559-0291 0273-2289 |
| Popis: | Aspartase (L-aspartate ammonia-lyase; EC 4.3.1.1) catalyzes the reversible amination of fumaric acid to produce L-aspartic acid. Aspartase coding gene (aspA) of Aeromonas media NFB-5 was cloned, sequenced, and expressed with His tag using pET-21b(+) expression vector in Escherichia coli BL21. Higher expression was obtained with IPTG (1.5 mM) induction for 5 h at 37 °C in LB medium supplemented with 0.3% K2HPO4 and 0.3% KH2PO4. Recombinant His tagged aspartase was purified using Ni–NTA affinity chromatography and characterized for various biochemical and kinetic parameters. The purified aspartase showed optimal activity at pH 8.5 and 8.0 in the presence and absence of magnesium ions, respectively. The optimum temperature was determined to be 35 °C. The enzyme showed apparent K m and V max values for L-aspartate as 2.01 mM and 114 U/mg, respectively. The enzyme was stable in pH range of 6.5–9.5 and temperature up to 45 °C. Divalent metal ion requirement of enzyme was efficiently fulfilled by Mg2+, Mn2+, and Ca2+ ions. The cloned gene (aspA) product showed molecular weight of approximately 51 kDa by SDS-PAGE, which is in agreement with the molecular weight calculated from putative amino acid sequence. This is the first report on expression and characterization of recombinant aspartase from A. media. |
| Databáze: | OpenAIRE |
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