Enzymatic synthesis of optically pure (R)-(-)-mandelic acid and other 2-hydroxycarboxylic acids : Screening for the enzyme, and its purification, characterization and use.

An enzymethat reduces benzoylformate with NADH to form (i?)-mandelate was extracted from cells of Streptococcusfaecalis IFO 12964 and purified to more than 95% purity as evidenced by gel electrophoresis.Physicochemical and enzymic properties were studied.From the substrate specificity, weconcluded that the enzymewas a kind of (i?)-2-hydroxyisocaproate dehydrogenase.Optically pure (R)-( -)-mandelic acid was prepared with the enzyme, NADH,and alcohol, formate or glucose dehydrogenase in 84 ~93% yield.Five (i?)-2-hydroxyalkanoic acids (C4 ~C6) or their Ba salts, (R)-( +)-3-phenyllactic acid and (S)-(-)-3-chlorolactic acid were also prepared with the enzyme.In this paper we report that an appropriate enzymewas found in Streptococcus faecalis, purified, and characterized.Optically pure (R)-(-)-mandelic acid and other 2-hydroxycarboxylic acids were prepared with the enzyme. MATERIALS AND METHODS Chemicals.The following chemicals were purchased from the makers listed below: benzoylformic acid, (+)and ( -)-mandelic acid, from Aldrich Chemical Co.(Milwaukee); Na phenylpyruvate, Na 2-ketoisocaproate, Na 2ketoisovalerate, Na 2-keto-3-methylvalerate, bromopyruvic acid, L-3-phenyllactic acid, and L-2-hydroxyisocaproic acid, from Sigma Chemical Co.(St.Louis); Na 2-ketocaproate and Na 2-ketovalerate, from Nakarai Chemicals, Ltd. (Kyoto); (R)-(+)-a-methoxy-a-trifluoromethylphenylacetic acid and TLCglass plates coated with silica gel F254 (0.25mm thick), from Merck Japan Ltd. (Tokyo); ( ± )-mandelic acid and 2-ketobutyric acid,