Synthesis of l-β-hydroxyvaline from α-keto-β-hydroxyisovalerate using leucine dehydrogenase from Bacillus species

α-Keto-β-bromoisovaleric acid or its ethyl ester was hydrolyzed with sodium hydroxide to α-keto-β-hydroxyisovalerate and converted in situ to l -β-hydroxyvaline by reaction with NADH and NH3 catalyzed by leucine dehydrogenase from Bacillus species. Methyl 2-chloro-3,3-dimethyloxiranecarboxylate and the corresponding isopropyl or 1,1-dimethylethyl esters were prepared by Darzens condensation. These glycidic esters, after hydrolysis by sodium bicarbonate and sodium hydroxide to α-keto-β-hydroxyisovalerate, were also converted to l -β-hydroxyvaline by leucine dehydrogenase. NAD was recycled to NADH with either formate dehydrogenase from Candida boidinii or glucose dehydrogenase from Bacillus megaterium. Polyethylene glycol-NADH was an effective reductant with formate dehydrogenase and dextran-NAD was effective with glucose dehydrogenase. Reductive amination activity for α-keto-β-hydroxyisovalerate was found in most Bacillus strains screened, including megaterium, subtilis, cereus, pumilus, licheniformis, thuringiensis, and brevis. Highest specific activity was found in B. sphaericus ATCC 4525. pH 8.5 was optimum for both glucose dehydrogenase and reductive amination of α-keto-β-hydroxyisovalerate by the B. sphaericus enzyme. The apparent Km for α-keto-β-hydroxyisovalerate was 11.5 m m compared to 1.06 m m for α-ketoisovalerate. The apparent Vmax with α-keto-β-hydroxyisovalerate was 41% of the value with α-ketoisovalerate, making the enzyme very suitable for the preparation of l -β-hydroxyvaline.