Applied Microbiology and Biotechnology

, Volume 21, Issue 1–2, pp 7–15 | Cite as

d-2-hydroxyisocaproate dehydrogenase from Lactobacillus casei

A new enzyme suitable for stereospecific reduction of 2-ketocarboxylic acids
  • Werner Hummel
  • Horst Schütte
  • Maria-Regina Kula


The new enzyme d-2-hydroxyisocaproate dehydrogenase (NAD+-dependent) was detected in strains of the genus Lactobacillus and related genera. Straight and branched chain aliphatic as well as aromatic 2-ketocarboxylic acids are stereospecifically reduced to the corresponding d-2-hydroxycarboxylic acids according to the following equation:R-CO-COOH + NADH + H+ ⇌ R-CHOH-COOH + NAD+

The enzyme is called d-hydroxyisocaproate dehydrogenase by us because 2-ketoisocaproate is the substrate with the lowest KM-value. NAD(H) as a cofactor cannot be replaced by NADP(H). Because of its broad substrate specificity we chose the strain Lactobacillus casei ssp. pseudoplantarum (DSM 20 008) for enzyme production and characterization. d-2-hydroxyisocaproate dehydrogenase could be purified 180-fold starting with 500 g of wet cells.

The purification procedure involved liquid-liquid extraction with aqueous two-phase systems and ion-exchange chromatography. At this stage the enzyme has a specific activity of 25 U/mg and can be used for technical applications. Further purification up to a homogeneous protein with a specific activity of 110 U/mg can be achieved by chromatography on Amberlite CG 50 at pH 3.5. Properties important for technical application of the d-HicDH were investigated, especially the substrate specificity and the optimum pH- and temperature ranges for activity and stability of the catalist.


Enzyme NADH Lactobacillus Substrate Specificity Enzyme Production 
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  1. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254Google Scholar
  2. Bückmann AF, Kula M-R, Wichmann R, Wandrey C (1981) An efficient synthesis of high-molecular-weight NAD(H). J Appl Biochem 3:301–315Google Scholar
  3. Hensel R, Mayr U, Stetter KO, Kandler O (1977) Comperative studies of lactic acid dehydrogenases in lactic acid bacteria. Arch Microbiol 112:81–93Google Scholar
  4. Horio T, Yamashita J (1983) Hydrophobe purifies commercial enzymes in single step. Newswatch, Jan 17, p 8Google Scholar
  5. Hummel W, Schütte H, Kula M-R (1983) Large-scale production of d-lactate dehydrogenase for the stereospecific reduction of pyruvate and phenylpyruvate. Eur J Appl Microbiol Biotechnol 18:75–85Google Scholar
  6. Jovin T, Chrambach A, Maughton MA (1964) An apparatus for preparative temperature-regulated polyacrylamide gel electrophoresis. Anal Biochem 9:351–369Google Scholar
  7. Kroner KH, Schütte H, Stach W, Kula M-R (1982) Scale-up of formate dehydrogenase by partition. J Chem Technol Biotechnol 32:130–137Google Scholar
  8. Kula M-R, Kroner KH, Hustedt H (1982) Purification of enzymes by liquid-liquid extraction. Adv Biochem Eng 24:73–118Google Scholar
  9. deMan JC, Rogosa M, Sharpe ME (1960) A medium for the cultivation of lactobacilli. J Appl Bacteriol 23:130–135Google Scholar
  10. Meister A (1950) Reduction of α,γ-diketo and α-keto acids catalyzed by muscle preparations and by cristalline lactic dehydrogenase. J Biol Chem 184:117–129Google Scholar
  11. Schütte H, Hummel W, Kula M-R (1984) l-2-hydroxyisocaproate dehydrogenase — A new enzyme from Lactobacillus confusus for the stereospecific reduction of 2-ketocarboxylic acids. Appl Microbiol Biotechnol 19:167–176Google Scholar
  12. Shapiro AL, Vinuela E, Maizel JV (1967) Molecular weight estimation of polypeptide chains by electrophoresis in SDS-Polyacrylamide gels. Biochem Biophys Res Commun 28:815–820Google Scholar
  13. Wichmann R, Wandrey C, Leuchtenberger W, Kula M-R, Bückmann AF (1982) Deutsche Offenlegungsschrift DOS 2930087.6, US-Patent 4.326.031 vom 20. 4. 1982, Verfahren zur Herstellung von optisch aktiven α-HydroxycarbonsäurenGoogle Scholar
  14. Wichmann R, Hummel W, Schütte H, Bückmann AF, Wandrey C, Kula M-R (1983) Enzymatic production of optically active 2-hydroxy acids. In: Laskin A (ed) Enzyme engineering VII. Ann NY Acad Sci (in press)Google Scholar

Copyright information

© Springer-Verlag 1985

Authors and Affiliations

  • Werner Hummel
    • 1
  • Horst Schütte
    • 1
  • Maria-Regina Kula
    • 1
  1. 1.Gesellschaft für Biotechnologische Forschung mbHBraunschweigFederal Republic of Germany

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