l-leucine dehydrogenase fromBacillus cereus

Summary An improved method for the production ofl-leucine dehydrogenase is described employing a mutant with a constitutive enzyme and a fed-batch cultivation technique yielding high cell concentrations. Purification ofl-leucine dehydrogenase to homogeneity was carried out starting with 30 kgBacillus cereus cells by heat treatment at 63°C, followed by two liquid-liquid extraction steps and three conventional column chromatographies. Crystals have been obtained from the 95-fold purified enzyme. The molecular weight of the native enzyme was determined by sedimentation equilibrium and gel filtration studies to be 310 000 containing eight identical subunits with a molecular weight of 39 000. The sedimentation coefficient was estimated to 11.65 S. Branched-chain amino acids likel-leucine,l-valine orl-isoleucine are deaminated by the NAD-dependent enzyme. In the reverse reaction a variety of 2-ketoacids, especially 2-ketoisocaproate, 2-ketoisovalerate and 2-keto-3-methyl-valerate, were reductive aminated to the correspondingl-amino acids in the presence of 0.9 M ammonia. The amino acid composition for the subunit ofl-leucine dehydrogenase is presented.