Molecular Cloning, E. coli Expression and Characterization of Thermostable Alanine Aminotransferase from Pyrococcus abyssi
Muhammad Shahid Nadeem*, Jalaluddin Azam Khan and Firoz Anwar
Department of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah 21589, Saudi Arabia
* Corresponding author: mhalim@kau.edu.sa
Fig. 1.
Sequence of P. abyssi gene coding for ALT with restriction sites for NdeI, NcoI inside the open reading frame (ORF) and restriction site of BamHI.
Fig. 2.
SDS-PAGE of purified enzymes, M, protein marker; P, purified enzyme; E1, E2, E3, E4, and E5- extract of experimental cells with gene expression; N, negative control.
Fig. 3.
Kinetic properties of purified recombinant ALT indicating maximum activity at pH 8, above 90◦C and in the presence of Mg2+ ions. KM and Vmax values were calculated as 25µM of L-alanine and 149.25 U/mg, respectively.
Fig. 4.
3D model of ALT-PA represented as cartoon (Green) (Visualized by PyMol).
Fig. 5.
Docked complexes showing alanine aminotransferase in green pyridoxal-5-phosphate (A), glutamate (C), α ketoglutarate (E), alanine (G) and pyruvate (I) as red sticks. Zoomed view of ALT-pyridoxal-5-phosphate docked complex (B), ALT-glutamate (D), ALT- α-ketoglutarate (F) ALT-alanine (H) ALT-pyruvate docked complex (J) with interactive residues under 5Å (Visualization by PyMol).