Skip to main content
Log in

Large scale production of d-lactate dehydrogenase for the stereospecific reduction of pyruvate and phenylpyruvate

  • Biotechnology
  • Published:
European journal of applied microbiology and biotechnology Aims and scope Submit manuscript

Summary

To initiate studies of the stereospecific reduction of pyruvate and phenylpyruvate to the corresponding d-2-hydroxyacids a limited screening was carried out for microorganisms possessing a high NADH-dependet d-lactate dehydrogenase activity. Lactobacillus confusus was found to produce the desired dehydrogenase, which showed also relatively high activity towards phenylpyruvate, so this strain was selected for large scale production of the enzyme.

A procedure for large scale purification of the enzyme starting with 24 kg wet cells is described including liquid-liquid extraction, ultrafiltration and chromatography on DEAE-cellulose, yielding a catalyst with specific activities of 216 U×mg−1 for pyruvate reduction and 15 U×mg−1 for phenyl-pyruvate reduction. A further tenfold purification can be achieved by affinity chromatography on Blue-Sepharose C-6B.

Parameters which are important for industrial application of the enzyme were determined: substrate specifity, pH and temperature optimum, temperature stability, stability at different pH-values, and the storage stability of the enzyme in crude extracts.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Similar content being viewed by others

References

  • Albertsson PÅ (1971) Partition of cell particles and macromolecules, 2nd ed. Wiley Interscience, New York

    Google Scholar 

  • Biochemica Information I (1973) 121–122, by Boehringer Mannheim GmbH

  • 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 72:248–254

    Google Scholar 

  • Bückmann AF, Kula M-R, Wichmann R, Wandrey C (1981) An efficient synthesis of high-molecular-weight NAD(H) derivatives suitable for continuous operation with coenzyme-dependent enzyme systems. J Appl Biochem 3:301–315

    Google Scholar 

  • Buchanan RE, Gibbons NE (1974) Bergey's manual of determinative bacteriology, 8th ed. Williams Wilkins, Baltimore

    Google Scholar 

  • De Man JC, Rogosa M, Sharpe ME (1960) A medium for the cultivation of lactobacilli. J Appl Bacteriol 23:130–135

    Google Scholar 

  • Elsden SR, Hilton MG, Wallter JM (1976) The End Products of the metabolism of aromatic amino acids by Clostridia. Arch Microbiol 107:283–288

    Google Scholar 

  • Hensel R, Mayr U, Stetter KO, Kandler O (1977) Comperative studies of lactic acid dehydrogenases in lactic acid bacteria. Arch Microbiol 112:81–93

    Google Scholar 

  • Jovin T, Chrambach A, Naughton MA (1964) An apparatus for preparative temperature-regulated polyacrylamide gel electrophoresis. Anal Biochem 9:351–369

    Google Scholar 

  • Kroner KH, Schütte H, Stach W, Kula M-R (1982) Scale-up of formate dehydrogenase by partition. J Chem Tech Biotechnol 32:130–137

    Google Scholar 

  • Kula M-R, Kroner KH, Hustedt H (1982) Purification of enzymes by liquid-liquid extraction. Adv Biochem Eng 24:73–118

    Google Scholar 

  • Lessie TG, Vander Wyk JC (1972) Multiple forms of Pseudomonas multivorans G6PDH and 6-phosphogluconate-DH. J Bacteriol 110:1107–1117

    Google Scholar 

  • Meister A (1959) Reduction of α, γ-diketo and α-keto acids catalyzed by muscle preparations and by crystalline lactic dehydrogenase. J Biol Chem 184:117–129

    Google Scholar 

  • Schütte H, Kroner KH, Hustedt H, Kula M-R (1983) Experiences with a 20 1 industrial bead mill for the disruption of microorganisms. Enzyme Microbiol Technol 5:143–148

    Google Scholar 

  • Schütte H, Kroner KH, Hustedt H, Kula M-R (1983) Disintegration of microorganisms in a 20 l industrial bead mill. B. Braun Melsungen AG., III. Rothenburger Symposium 1982 In: Lafferty RM (ed) Enzyme technology (in press)

  • Shapiro AL, Vinuela E, Maizel JV (1967) Molecular weight estimation of polypeptide chains by electrophoresis in SDS-Polyacrylamide gels. Biochem Biophys Res Commun 28:815–820

    Google Scholar 

  • Simon H, Bader J, Rambeck B, Krezdorn E, Tischer W (1979) Stereospezifische Hydrierung mit Mikroorganismen und immobilisierten Enzymsystemen. In: Dellweg HW (Hrsg) 4. Symposium Technische Mikrobiologie, Berlin 1979

  • Wichmann R, Wandrey C, Bückmann AF, Kula M-R (1981) Continuous enzymatic transformation in an enzyme membrane reactor with simultaneous NAD(H) regeneration. Biotechnol Bioeng 23:2789–2802

    Google Scholar 

  • Wichmann R, Wandrey C, Leuchtenberger W, Kula M-R, Bückmann AF (1982) Deutsche Offenlegungsschrift DOS 29 30 087.6, US-Patent 4.326.031 vom 20. 4. 1982, Verfahren zur Herstellung von optisch aktiven α-Hydroxycarbonsäuren

Download references

Author information

Authors and Affiliations

Authors

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hummel, W., Schütte, H. & Kula, MR. Large scale production of d-lactate dehydrogenase for the stereospecific reduction of pyruvate and phenylpyruvate. European J. Appl. Microbiol. Biotechnol. 18, 75–85 (1983). https://doi.org/10.1007/BF00500828

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00500828

Keywords

Navigation