Molecular Cloning, E. coli Expression and Characterization of Thermostable Alanine Aminotransferase from Pyrococcus abyssi

Catalysing the reversible interconversion of L-alanine and alpha-ketoglutarate to pyruvate and L-glutamate in the presence of pyridoxal 5 phosphate, alanine aminotransferase (ALT) is an enzyme which operates at the cross roads of amino acid and carbohydrate metabolism. The enzyme has been reported from a wide range of organisms including animals, plants, fungi and microbes. The enzyme has a clinical applications in the diagnosis of many diseases. In the present study we have produced a recombinant of ALT from Pyrococcus abyssi in BL21 (DE3) strain of E. coli. The recombinant enzyme was purified by anion exchange chromatography, it displayed a 45kDa band on SDS-PAGE, with 58.1% final recovery, 15.3 fold purification and 139 U/mg specific activity. Maximum enzyme activity was found at pH 8 and above 90C, its KM and Vmax values were found 25 mu M L-alanine and 149 U/min/mg respectively. In silico studies have shown that the enzyme was found in a monomer structure. Molecular docking studies with potential molecules involved in the reaction catalysed have been conducted and binding energy values were calculated for each molecule including L-alanine, pyridoxal 5 phosphate, pyruvate, alpha ketoglutarate and L-glutamate. Present study provides the first report of ALT from Pyrococcus abyssi and suggests active site residues of enzyme from archaeal origin.