Purification and characterization of alanine dehydrogenase from a marine bacterium, Vibrio proteolyticus

Abstract Alanine dehydrogenase of Vibrio proteolyticus DSM 30189 shows high activity toward β-hydroxypyruvate, and the enzyme is applicable to the production of l -serine. We have cloned the enzyme gene from the bacterium into Escherichia coli TG1 with a vector plasmid, pUC118. The enzyme was overproduced by the transformed cells and purified to homogeneity with a yield of 46%. The molecular mass of the enzyme was about 230 kDa and consisted of six identical subunits. The enzyme showed broad specificity toward α-keto acids in the reductive amination. The relative activities of the enzyme for pyruvate, β-fluoropyruvate, and β-hydroxypyruvate were 100%, 74%, and 54%, respectively. The enzyme retained more than 90% of the activity after incubation at 65 °C for 60 min in the presence of 2.0 M NaCl, but 98% of its original activity was lost in the absence of NaCl. RbCl, as well as NaCl, significantly stabilized the enzyme. On the other hand, LiCl and KCl were not as effective as stabilizers such as RbCl and NaCl.