Applied Microbiology and Biotechnology

, Volume 102, Issue 7, pp 3159–3171 | Cite as

Aldopentoses as new substrates for the membrane-bound, pyrroloquinoline quinone-dependent glycerol (polyol) dehydrogenase of Gluconobacter sp.

  • Toshiharu Yakushi
  • Yuka Terada
  • Seishiro Ozaki
  • Naoya Kataoka
  • Yoshihiko Akakabe
  • Osao Adachi
  • Minenosuke Matsutani
  • Kazunobu Matsushita
Biotechnologically relevant enzymes and proteins


Membrane-bound, pyrroloquinoline quinone (PQQ)-dependent glycerol dehydrogenase (GLDH, or polyol dehydrogenase) of Gluconobacter sp. oxidizes various secondary alcohols to produce the corresponding ketones, such as oxidation of D-sorbitol to L-sorbose in vitamin C production. Substrate specificity of GLDH is considered limited to secondary alcohols in the D-erythro configuration at the next to the last carbon. Here, we suggest that L-ribose, D- and L-lyxoses, and L-tagatose are also substrates of GLDH, but these sugars do not meet the substrate specificity rule of GLDH. The oxygen consumption activity of wild-type Gluconobacter frateurii cell membranes depends on several kinds of sugars as compared with that of the membranes of a GLDH-negative variant. Biotransformation of those sugars with the membranes was examined to determine the reaction products. A time course measuring the pH in the reaction mixture and the increase or decrease in substrates and products on TLC suggested that oxidation products of L-lyxose and L-tagatose were ketones with unknown structures, but those of L-ribose and D-lyxose were acids. The oxidation product of L-ribose was purified and revealed to be L-ribonate by HRMS and NMR analysis. Biotransformation of L-ribose with the membranes and also with the whole cells produced L-ribonate in nearly stoichiometric amounts, indicating that the specific oxidation site in L-ribose is recognized by GLDH. Since purified GLDH produced L-ribonate without any intermediate-like compounds, we propose here a reaction model where the first carbon in the pyranose form of L-ribose is oxidized by GLDH to L-ribonolactone, which is further hydrolyzed spontaneously to produce L-ribonate.


Acetic acid bacteria Oxidative biotransformation Gluconobacter L-ribonic acid L-ribose 



We are grateful to Armin Ehrenreich (Technische Universität München, Germany) for kindly providing pKOS6b to us. We thank Yuka Narita, Takahiro Torikai, and Koichi Furuya (Yamaguchi University, Japan) for their technical assistances.


This study was funded by MEXT KAKENHI (grant numbers 17K07722 to TY; 2660068 to KM).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2018_8848_MOESM1_ESM.pdf (929 kb)
ESM 1 (PDF 929 kb)


  1. Adachi O, Ano Y, Toyama H, Matsushita K (2006) Enzymatic preparation of metabolic intermediates, 3-dehydroquinate and 3-dehydroshikimate, in the shikimate pathway. Biosci Biotechnol Biochem 70:3081–3083 Epub 2006 Dec 7CrossRefPubMedGoogle Scholar
  2. Adachi O, Fujii Y, Ghaly MF, Toyama H, Shinagawa E, Matsushita K (2001) Membrane-bound quinoprotein D-arabitol dehydrogenase of Gluconobacter suboxydans IFO 3257: a versatile enzyme for the oxidative fermentation of various ketoses. Biosci Biotechnol Biochem 65:2755–2762CrossRefPubMedGoogle Scholar
  3. Adachi O, Matsushita K, Shinagawa E, Ameyama M (1980) Crystallization and characterization of NADP-dependent D-glucose dehydrogenase from Gluconobacter suboxydans. Agric Biol Chem 44:301–308. Google Scholar
  4. Adachi O, Toyama H, Matsushita K (1999) Crystalline NADP-dependent D-mannitol dehydrogenase from Gluconobacter suboxydans. Biosci Biotechnol Biochem 63:402–407. CrossRefPubMedGoogle Scholar
  5. Ameyama M, Shinagawa E, Matsushita K, Adachi O (1985) Solubilization, purification and properties of membrane-bound glycerol dehydrogenase from Gluconobacter industrius. Agric Biol Chem 49:1001–1010. Google Scholar
  6. Angyal SJ (1969) The composition and conformation of sugars in solution. Angew Chem Int Ed Engl 8:157–166. CrossRefGoogle Scholar
  7. Ano Y, Hours RA, Akakabe Y, Kataoka N, Yakushi T, Matsushita K, Adachi O (2017) Membrane-bound glycerol dehydrogenase catalyzes oxidation of D-pentonates to 4-keto-D-pentonates, D-fructose to 5-keto-D-fructose, and D-psicose to 5-keto-D-psicose. Biosci Biotechnol Biochem 81:411–418CrossRefPubMedGoogle Scholar
  8. Buchert J (1991) A xylose-oxidizing membrane-bound aldose dehydrogenase of Gluconobacter oxydans ATCC 621. J Biotechnol 18:103–113. CrossRefGoogle Scholar
  9. Dulley JR, Grieve PA (1975) A simple technique for eliminating interference by detergents in the Lowry method of protein determination. Anal Biochem 64:136–141CrossRefPubMedGoogle Scholar
  10. Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580CrossRefPubMedGoogle Scholar
  11. Hann RM, Tilden EB, Hudson CS (1938) The oxidation of sugar alcohols by Acetobacter suboxydans. J Am Chem Soc 60:1201–1203. CrossRefGoogle Scholar
  12. Kearney EB, Singer TP (1956) Studies on succinic dehydrogenase: I. Preparation and assay of the soluble dehydrogenase. J Biol Chem 219:963–975PubMedGoogle Scholar
  13. Kostner D, Peters B, Mientus M, Liebl W, Ehrenreich A (2013) Importance of codB for new codA-based markerless gene deletion in Gluconobacter strains. Appl Microbiol Biotechnol 97:8341–8349CrossRefPubMedGoogle Scholar
  14. Kovach ME, Elzer PH, Hill DS, Robertson GT, Farris MA, Roop RM 2nd, Peterson KM (1995) Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166:175–176CrossRefPubMedGoogle Scholar
  15. Kulhánek M (1989) Microbial dehydrogenations of monosaccharides. In: Neidleman SL (ed) Adv Appl Microbiol (vol 34). Academic Press, pp 141–182Google Scholar
  16. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefPubMedGoogle Scholar
  17. Liu X, Yuan Z, Adam Yuan Y, Lin J, Wei D (2011) Biochemical and structural analysis of Gox2181, a new member of the SDR superfamily from Gluconobacter oxydans. Biochem Biophys Res Commun 415:410–415. CrossRefPubMedGoogle Scholar
  18. Matsushita K, Fujii Y, Ano Y, Toyama H, Shinjoh M, Tomiyama N, Miyazaki T, Sugisawa T, Hoshino T, Adachi O (2003) 5-keto-D-gluconate production is catalyzed by a quinoprotein glycerol dehydrogenase, major polyol dehydrogenase, in Gluconobacter species. Appl Environ Microbiol 69:1959–1966CrossRefPubMedPubMedCentralGoogle Scholar
  19. Matsushita K, Toyama H, Adachi O (1994) Respiratory chains and bioenergetics of acetic acid bacteria. In: Rose AH, Tempest DW (eds) Adv Microb Physiol, vol vol 36. Academic Press, London, pp 247–301Google Scholar
  20. Meyer M, Schweiger P, Deppenmeier U (2013) Effects of membrane-bound glucose dehydrogenase overproduction on the respiratory chain of Gluconobacter oxydans. Appl Microbiol Biotechnol 97:3457–3466CrossRefPubMedGoogle Scholar
  21. Mientus M, Kostner D, Peters B, Liebl W, Ehrenreich A (2017) Characterization of membrane-bound dehydrogenases of Gluconobacter oxydans 621H using a new system for their functional expression. Appl Microbiol Biotechnol 101:3189–3200. CrossRefPubMedGoogle Scholar
  22. Mitchell P, Moyle J, Mitchell R (1979) Measurement of translocation of H+/O in mitochondria and submitochondrial vesicles. Methods Enzymol 55:627–640CrossRefPubMedGoogle Scholar
  23. Miyazaki T, Tomiyama N, Shinjoh M, Hoshino T (2002) Molecular cloning and functional expression of D-sorbitol dehydrogenase from Gluconobacter suboxydans IFO3255, which requires pyrroloquinoline quinone and hydrophobic protein SldB for activity development in E. coli. Biosci Biotechnol Biochem 66:262–270CrossRefPubMedGoogle Scholar
  24. Moonmangmee D, Adachi O, Ano Y, Shinagawa E, Toyama H, Theeragool G, Lotong N, Matsushita K (2000) Isolation and characterization of thermotolerant Gluconobacter strains catalyzing oxidative fermentation at higher temperatures. Biosci Biotechnol Biochem 64:2306–2315CrossRefPubMedGoogle Scholar
  25. Moonmangmee D, Fujii Y, Toyama H, Theeragool G, Lotong N, Matsushita K, Adachi O (2001) Purification and characterization of membrane-bound quinoprotein cyclic alcohol dehydrogenase from Gluconobacter frateurii CHM 9. Biosci Biotechnol Biochem 65:2763–2772CrossRefPubMedGoogle Scholar
  26. Nakano S, Ebisuya H (2016) Physiology of Acetobacter and Komagataeibacter spp.: acetic acid resistance mechanism in acetic acid fermentation. In: Matsushita K, Toyama H, Tonouchi N, Okamoto-Kainuma A (eds) Acetic acid bacteria: ecology and physiology. Springer Japan, Tokyo, pp 223–234Google Scholar
  27. Nishikimi M, Appaji Rao N, Yagi K (1972) The occurrence of superoxide anion in the reaction of reduced phenazine methosulfate and molecular oxygen. Biochem Biophys Res Commun 46:849–854. CrossRefPubMedGoogle Scholar
  28. Peters B, Mientus M, Kostner D, Junker A, Liebl W, Ehrenreich A (2013) Characterization of membrane-bound dehydrogenases from Gluconobacter oxydans 621H via whole-cell activity assays using multideletion strains. Appl Microbiol Biotechnol 97:6397–6412CrossRefPubMedGoogle Scholar
  29. Que L Jr, Gray GR (1974) Carbon-13 nuclear magnetic resonance spectra and the tautomeric equilibriums of ketohexoses in solution. Biochemistry 13:146–153. CrossRefPubMedGoogle Scholar
  30. Salusjärvi T, Povelainen M, Hvorslev N, Eneyskaya EV, Kulminskaya AA, Shabalin KA, Neustroev KN, Kalkkinen N, Miasnikov AN (2004) Cloning of a gluconate/polyol dehydrogenase gene from Gluconobacter suboxydans IFO 12528, characterisation of the enzyme and its use for the production of 5-ketogluconate in a recombinant Escherichia coli strain. Appl Microbiol Biotechnol 65:306–314CrossRefPubMedGoogle Scholar
  31. Sambrook J, Russel DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold SpringHarbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar
  32. Shinjoh M, Tomiyama N, Miyazaki T, Hoshino T (2002) Main polyol dehydrogenase of Gluconobacter suboxydans IFO 3255, membrane-bound D-sorbitol dehydrogenase, that needs product of upstream gene, sldB, for activity. Biosci Biotechnol Biochem 66:2314–2322CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Division of Agricultural Science, Graduate School of Science and Technology for InnovationYamaguchi UniversityYamaguchiJapan
  2. 2.Department of Biological Chemistry, Faculty of AgricultureYamaguchi UniversityYamaguchiJapan
  3. 3.Research Center for Thermotolerant Microbial ResourcesYamaguchi UniversityYamaguchiJapan

Personalised recommendations