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Glucose Dehydrogenase for the Regeneration of NADPH and NADH

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Microbial Enzymes and Biotransformations

Part of the book series: Methods in Biotechnology ((MIBT,volume 17))

Abstract

Glucose dehydrogenases (GDHs) occur in several organisms such as Bacillus megaterium and Bacillus subtilis. They accept both NAD+and NADP+as cofactor and can be used for the regeneration of NADH and NADPH. In order to demonstrate their applicability we coupled an NADP+-dependent, (R)-specific alcohol dehydrogenase (ADH) from Lactobacillus kefir with the glucose dehydrogenase from B. subtilis. The ADH reduces prochiral ketones stereoselectively to chiral alcohols. The reduction requires NADPH, which was regenerated by the glucose dehydrogenase. Glucose dehydrogenase from B. subtilis (EC 1.1.1.47) is a tetramer with a molecular weight of 126,000. The enzyme shows a pH optimum at 8.0 and a broad temperature optimum at 45-50°C. We investigated the conversion of acetophenone in a cell-free system with purified ADH and GDH. Furthermore, we constructed two plasmids containing the genes encoding ADH and GDH by inserting them one after the other. These two plasmids differ from each other in the order of the genes. Because of the low solubility of the compounds, we examined the reaction in a water/organic solvent two-phase system.

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References

  1. Pauly, H. E. and Pfleiderer, G. (1975) D-glucose dehydrogenase from Bacillus megaterium M 1286: purification, properties and structure. Hoppe Seylers Z Physiol. Chem. 356, 1613–1623.

    Article  PubMed  CAS  Google Scholar 

  2. Jany, K. D., Ulmer, W., Froschle, M., and Pfleiderer, G. (1984) Complete amino acid sequence of glucose dehydrogenase from Bacillus megaterium. FEBS Lett. 165, 6–10.

    Article  PubMed  CAS  Google Scholar 

  3. Heilmann, H. J., Magert, H. J., and Gassen, H. G. (1988) Identification and isolation of glucose dehydrogenase genes of Bacillus megaterium M 1286 and their expression in Escherichia coli. Eur. J. Biochem. 174, 485–490.

    CAS  Google Scholar 

  4. Mitamura, T., Urabe, I., and Okada, H. (1989) Enzymatic properties of isozymes and variants of glucose dehydrogenase from Bacillus megaterium. Eur. J. Biochem. 186, 389–393.

    Article  PubMed  CAS  Google Scholar 

  5. Fujita, Y., Ramaley, R., and Freese, E. (1977) Location and properties of glucose dehydrogenase in sporulating cells and spores of Bacillus subtilis. J. Bacteriol. 132, 282–293.

    PubMed  CAS  Google Scholar 

  6. Lampel, K. A., Uratani, B., Chaudhry, G. R., Ramaley, R. R, and Rudikoff, S. (1986) Characterization of the developmentally regulated Bacillus subtilis glucose dehydrogenase gene. J. Bacteriol. 166, 238–243.

    PubMed  CAS  Google Scholar 

  7. Hilt, W., Pfleiderer, G., and Fortnagel, P. (1991) Glucose dehydrogenase from Bacillus subtilis expressed in Escherichia coli. I: Purification, characterization and comparison with glucose dehydrogenase from Bacillus megaterium. Biochim. Biophys. Acta 1076, 298–304.

    Article  PubMed  CAS  Google Scholar 

  8. Adachi, O., Kazunobu, M., Shinagawa, E., and Ameyama, M. (1980) Crystallization and characterization of NADP-dependent D-glucose dehydrogenase from Gluconobacter suboxydans. Agric. Biol. Chem. 44, 301–308.

    CAS  Google Scholar 

  9. Bonete, M. J., Pire, C., Llorca, F. L, and Camacho, M. L. (1996) Glucose dehydrogenase from the halophilic archaeon Haloferax mediterranei: enzyme purification, characterisation and N-terminal sequence. FEBS Lett. 383, 227–229.

    Article  PubMed  CAS  Google Scholar 

  10. Smith, L. D., Budgen, N., Bungard, S. J., Danson, M. J., and Hough, D. W. (1989) Purification and characterization of glucose dehydrogenase from the thermoacidophilic archaebacterium Thermoplasma acidophilum. Biochem. J. 261, 973–977.

    PubMed  CAS  Google Scholar 

  11. Bright, J. R., Byrom, D., Danson, M. J., Hough, D. W., and Towner, P. (1993) Cloning, sequencing and expression of the gene encoding glucose dehydrogenase from the thermophilic archaeon Thermoplasma acidophilum. Eur. J. Biochem. 211, 549–554.

    Article  PubMed  CAS  Google Scholar 

  12. Giardina, P., de Biasi, M. G., de Rosa, M., Gambacorta, A., and Buonocore, V. (1986) Glucose dehydrogenase from the thermoacidophilic archaebacterium Sulfolobus solfataricus. Biochem. J. 239, 517–522.

    PubMed  CAS  Google Scholar 

  13. Hummel, W. (1999) Large-scale applications of NAD(P)-dependent oxidoreductases: recent developments. Trends Biotechnol. 17, 487–492.

    Article  PubMed  CAS  Google Scholar 

  14. Hanson, R. L., Schwinden, M. D., Banerjee, A., Brzozowski, D. B., Chen, B. C., Patel, B. P., et al. (1999) Enzymatic synthesis of L-6-hydroxynorleucine. Bioorg. Med. Chem. 7, 2247–2252.

    Article  PubMed  CAS  Google Scholar 

  15. Lin, S.-S., Miyawaki, O., and Nakamura, K. (1999) Continuous production of Lcarnitine with NADH regeneration by a nanofiltration membrane reactor with coimmobilized L-carnitine dehydrogenase and glucose dehydrogenase. J. Biosci. Bioeng. 87, 361–364.

    Article  PubMed  CAS  Google Scholar 

  16. Kataoka, M., Yamamoto, K., Kawabata, H., Wada, M., Kita, K., Yanase, H., and Shimizu, S. (1999) Stereoselective reduction of ethyl 4-chloro-3-oxobutanoate by Escherichia coli transformant cells coexpressing the aldehyde reductase and glucose dehydrogenase genes. Appl. Microbiol. Biotechnol. 51, 486–490.

    Article  PubMed  CAS  Google Scholar 

  17. Eguchi, T., Kuge, Y., Inoue, K., Yoshikawa, N., Mochida, K., and Uwajima, T. (1992) NADPH regeneration by glucose dehydrogenase from Gluconobacter scleroides for l-leucovorin synthesis. Biosci. Biotechnol. Biochem. 56, 701–703.

    Article  PubMed  CAS  Google Scholar 

  18. Hummel, W. (1990) Enzyme-catalyzed synthesis of optically pure R(+)phenylethanol. Biotechnol. Lett. 12, 403–408.

    Article  CAS  Google Scholar 

  19. Wong, C.-H. and Whitesides, G. M. (1981) Enzyme-catalyzed organic synthesis: NAD(P)H cofactor regeneration by using glucose 6-phosphate and the glucose-6phosphate dehydrogenase from Leuconostoc mesenteroides. J. Am. Chem. Soc. 103, 4890–4899.

    Article  CAS  Google Scholar 

  20. Hummel, W., Boermann, F., and Kula, M.-R. (1989) Purification and characterization of an acetoin dehydrogenase from Lactobacillus kefir suitable for the production of (+)-acetoin. Biocatalysis 2, 293–308.

    Article  CAS  Google Scholar 

  21. Bradshaw, C. W., Hummel, W., and Wong, C.-H. (1992) Lactobacillus kefir alcohol dehydrogenase: a useful catalyst for synthesis. J. Org. Chem. 57, 1532–1536.

    Article  CAS  Google Scholar 

  22. Hummel, W. and Riebel, B. (1996) Chiral alcohols by enantioselective enzymatic oxidation. Enzyme Engineering 13, 713–716.

    Google Scholar 

  23. Kruse, W., Hummel, W., and Kragl, U. (1996) Alcohol-dehydrogenase-catalyzed production of chiral hydrophobic alcohols. A new approach leading to a nearly waste-free process. Recueil des Travaux Chimiques des Pays-Bas 115, 239–243.

    Article  CAS  Google Scholar 

  24. Wilms, B., Wiese, A., Syldatk, C., Mattes, R., and Altenbuchner, J. (2001) Development of an Escherichia coli whole cell biocatalyst for the production of Lamino acids. J. Biotechnol. 86, 19–30.

    Article  PubMed  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. Institute of Molecular Enzyme Technology, Heinrich-Heine-University Düsseldorf, Research Centre Jülich, Germany

    Andrea Weckbecker

  2. Institute of Molecular Enzyme Technology, Heinrich- Heine-University Düsseldorf, Research Centre Jülich, Germany

    Werner Hummel

Authors
  1. Andrea Weckbecker
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  2. Werner Hummel
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Editor information

Editors and Affiliations

  1. R & D Biology, Antibióticos S. A, León, Spain

    José Luis Barredo

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© 2005 Humana Press Inc.

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Weckbecker, A., Hummel, W. (2005). Glucose Dehydrogenase for the Regeneration of NADPH and NADH. In: Barredo, J.L. (eds) Microbial Enzymes and Biotransformations. Methods in Biotechnology, vol 17. Humana Press. https://doi.org/10.1385/1-59259-846-3:225

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  • DOI: https://doi.org/10.1385/1-59259-846-3:225

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-253-7

  • Online ISBN: 978-1-59259-846-5

  • eBook Packages: Springer Protocols

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