Abstract
Aspartate aminotransferase catalyzes the transfer of an amino group from l-aspartate to α-oxoglutarate. A gene encoding aspartate aminotransferase, ASTGt, from Geobacillus thermopakistaniensis was cloned and expressed in Escherichia coli. The purified recombinant ASTGt exhibited highest activity at 65 °C and pH 7.0. The activity was dependent on pyridoxal phosphate but not on any metal ions. Stoichiometry of purified ASTGt demonstrated that 0.1 pyridoxal phosphate was attached per subunit of the enzyme. Determination of molecular weight by gel filtration chromatography indicated that ASTGt existed in a dimeric form in solution. Thermostability experiments showed no significant change in activity even after 16 h incubation at 65 °C. ASTGt exhibited apparent Vmax and Km values of 120 μmol min−1 mg−1 and 1.5 mM, respectively, against l-aspartate. Substrate specificity experiments indicated the highest relative activity against aspartate (100%) followed by tyrosine (27%) and proline (16%). To the best of our knowledge, this is the first report on cloning and characterization of an AST from genus Geobacillus.
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