Molecular Cloning, E. coli Expression and Characterization of Thermostable Alanine Aminotransferase from Pyrococcus abyssi
Molecular Cloning, E. coli Expression and Characterization of Thermostable Alanine Aminotransferase from Pyrococcus abyssi
Muhammad Shahid Nadeem*, Jalaluddin Azam Khan and Firoz Anwar
ABSTRACT
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 25µM 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.
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