A novel type alanine dehydrogenase from Helicobacter aurati: Molecular characterization and application.

A novel alanine dehydrogenase (ADH; EC.1.4.1.1) with high pyruvate reduced activity was isolated from Helicobacter aurati and expressed in Escherichia coli BL21 (DE3). The optimum pH of the reduction and oxidation reaction were 8.0 and 9.0, respectively, and the optimum temperature was 55 °C. With pyruvate and alanine as substrates, the specific activity of HAADH1 were 268 U·mg-1 and 26 U·mg-1, respectively. HAADH1 had a prominent substrate specificity for alanine (Km = 2.23 mM, kcat/Km = 8.1 s-1·mM-1). In the reduction reaction, HAADH1 showed the highest substrate affinity for pyruvate (Km = 0.56 mM, kcat/Km = 364 s-1·mM-1). Compared to pyruvate, oxaloacetic acid, 2-ketobutyric acid, 3-fluoropyruvate, α-ketoglutaric acids, glyoxylic acid showed a residual activity of 93.30%, 8.93%, 5.62%, 2.57%, 2.51%, respectively. Phylogenetic tree analysis showed that this is a new type of ADH which have a low sequence similarity to available ADH reported in references. 3-Fluoropyruvate was effectively reduced to 3-fluoro-L-alanine by whole-cell catalysis.