Thesis (M.Sc.)--Chulalongkorn University, 2015

Alanine dehydrogenase catalyzes the reversible reaction of pyruvate to alanine (PvRA and ALD) and non-reversible reaction of glyoxylate to glycine (GxRA). It plays a crucial role for sporulation in bacteria while it is required for pigment degradation in photosynthetic organisms. In this study, the putative gene encoding alanine dehydrogenase from halotolerant cyanobacterium Aphanothece halophytica (ApalaDH) was cloned and expressed in Escherichia coli. ApalaDH was successfully expressed into the expression vector pColdI and pColdTF but not in pTrcHis2C. Recombinant ApalaDH was purified homogeneity and then functionally characterized. Recombinant ApalaDH exhibited two catalytic activities of pyruvate to alanine and glycine to glyoxylate, which were different from the AlaDH from filamentous cyanobacterium Anabaena variabilis. The kinetic parameter Km of ApalaDH for pyruvate, alanine and glyoxylate were 0.22 ± 0.02, 0.72 ± 0.04 and 1.91 ± 0.43 mM, respectively. These Kms of ApalaDH suggested high affinity for all substrates. The expression level under salt stress was carried out by semi-quantitative RT-PCR. The result showed that ApalaDH expression increased approximately two folds under salt stress. Furthermore, in vivo analysis was performed. Under salt stress condition, the PvRA and GxRA activities were increased about 1.3- and 2.7-folds, respectively. These results implicated that ApalaDH would important under salt stress condition. To our knowledge, this is the first functional characterization of AlaDH in cyanobacteria.