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Characterization of thermotolerant Acetobacter pasteurianus strains and their quinoprotein alcohol dehydrogenases

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Abstract

We isolated several thermotolerant Acetobacter species of which MSU10 strain, identified as Acetobacter pasteurianus, could grow well on agar plates at 41°C, tolerate to 1.5% acetic acid or 4% ethanol at 39°C, similarly seen with A. pasteurianus SKU1108 previously isolated. The MSU10 strain showed higher acetic acid productivity in a medium containing 6% ethanol at 37°C than SKU1108 while SKU1108 strain could accumulate more acetic acid in a medium supplemented with 4–5% ethanol at the same temperature. The fermentation ability at 37°C of these thermotolerant strains was superior to that of mesophilic A. pasteurianus IFO3191 strain having weak growth and very delayed acetic acid production at 37°C even at 4% ethanol. Alcohol dehydrogenases (ADHs) were purified from MSU10, SKU1108, and IFO3191 strains, and their properties were compared related to the thermotolerance. ADH of the thermotolerant strains had a little higher optimal temperature and heat stability than that of mesophilic IFO3191. More critically, ADHs from MSU10 and SKU1108 strains exhibited a higher resistance to ethanol and acetic acid than IFO3191 enzyme at elevated temperature. Furthermore, in this study, the ADH genes were cloned, and the amino acid sequences of ADH subunit I, subunit II, and subunit III were compared. The difference in the amino acid residues could be seen, seemingly related to the thermotolerance, between MSU10 or SKU1108 ADH and IFO 3191 ADH.

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References

  • Adachi O, Tayama K, Shinagawa E, Matsushita K, Ameyama M (1978a) Purification and characterization of particulate alcohol dehydrogenase from Gluconobacter suboxydans. Agric Biol Chem 42:2045–2056

    CAS  Google Scholar 

  • Adachi O, Miyagawa E, Shinagawa E, Matsushita K, Ameyama M (1978b) Purification and properties of particulate alcohol dehydrogenase from Acetobacter aceti. Agric Biol Chem 42:2331–2340

    CAS  Google Scholar 

  • Adachi O, Moonmangmee D, Toyama H, Yamada M, Shinagawa E, Matsushita K (2003) New developments in oxidative fermentation. Appl Microbiol Biotechnol 60:643–653

    CAS  Google Scholar 

  • Ameyama M (1982) Enzymatic microdetermination of d-glucose, d-fructose, d-gluconate, 2-keto-d-gluconate, aldehyde, and alcohol with membrane bound dehydrogenases. In: Wood WA (ed) Methods in enzymology, vol 89. Academic, New York, pp 20–29

    Google Scholar 

  • Chinnawirotpisan P, Theeragool G, Limtong S, Toyama H, Adachi O, Matsushita K (2003) Quinoprotein alcohol dehydrogenase is involved in catabolic acetate production, while NAD-dependent alcohol dehydrogenase in ethanol assimilation in Acetobacter pasteurianus SKU1108. J Biosci Bioeng 96:564–571

    Article  CAS  Google Scholar 

  • Cleenwerck I, Vandemeulebroecke K, Janssens D, Swings J (2002) Re-examination of The genus Acetobacter, with descriptions of Acetobacter cerevisiae sp. nov. and Acetobacter malorum sp. nov. Int J Syst Evol Microbiol 52:1551–1558

    Article  CAS  Google Scholar 

  • Cleenwerck I, Camu N, Engelbeen K, De Winter T, Vandemeulebroecke K, De Vos P, De Vuyst L (2007) Acetobacter ghanensis sp. nov., a novel acetic acid bacterium isolated from traditional heap fermentations of Ghanaian cocoa beans. Int J Syst Evol Microbiol 57:1647–1652

    Article  CAS  Google Scholar 

  • De Ley J, Gillis M, Swings J (1984) In: Krieg NR, Holt JG (ed) Bergey's manual of systematic bacteriology, vol. 1. Williams & Wilkins, Baltimore pp 267–278

  • Dully JR, Grieve PA (1975) A simple technique for eliminating interference by detergents in the Lowry method of protein determination. Anal Biochem 64:136–141

    Article  Google Scholar 

  • Frébortová J, Matsushita K, Yakushi T, Toyama H, Adachi O (1997) Quinoprotein alcohol dehydrogenase of acetic acid bacteria: kinetic study on the enzyme purified from Acetobacter methanolicus. Biosci Biotechnol Biochem 61:459–46

    Article  Google Scholar 

  • Greenberg DE, Porcella SF, Stock F, Wong A, Conville PS, Murray PR, Holland SM, Zelazny AM (2006) Granulibacter bethesdensis gen. nov., sp. nov., a distinctive pathogenic acetic acid bacterium in the family Acetobacteraceae. Int J Syst Evol Microbiol 56:2609–2616

    Article  CAS  Google Scholar 

  • Gómez-Manzo S, Contreras-Zentella M, González-Valdez A, Sosa-Torres M, Arreguín-Espinoza R, Escamilla-Marván E (2008) The PQQ-alcohol dehydrogenase of Gluconacetobacter diazotrophicus. Int J Food Microbiol 125:71–78

    Article  CAS  Google Scholar 

  • Inoue T, Sunagawa M, Mori A, Imai C, Fukuda M, Takagi M, Yano K (1989) Cloning and sequencing of the gene encoding the 72-kilodalton dehydrogenase subunit of alcohol dehydrogenase from Acetobacter aceti. J Bacteriol 171:3115–3122

    CAS  Google Scholar 

  • Jojima Y, Mihara Y, Suzuki S, Yokozeki K, Yamanaka S, Fudou R (2004) Saccharibacter floricola gen. nov., sp. nov., anovel osmophilic acetic acid bacterium isolated from pollen. Int J Syst Evol Microbiol 54:2263–2267

    Article  CAS  Google Scholar 

  • Kondo K, Horinuochi S (1997) Characterization of the genes encoding the three component membrane bound alcohol dehydrogenase from Gluconobacter suboxydans and their expression in Acetobacter pasteurianus. Appl Environ Microbiol 63:1131–1138

    CAS  Google Scholar 

  • Kondo K, Beppu T, Horinouchi S (1995) Cloning, sequencing, and characterization of the gene encoding the smallest subunit of the three-component membrane-bound alcohol dehydrogenase from Acetobacter pasteurianus. J Bacteriol 177:5048–5055

    CAS  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  CAS  Google Scholar 

  • Lisdiyanti P, Kawasaki H, Seki T, Yamada Y, Uchimura T, Komagata K (2000) Systematic study of the genus Acetobacter with descriptions of Acetobacter indonesiensis sp. nov., Acetobacter tropicalis sp. nov., Acetobacter orleanensis (Henneberg 1906) comb. nov., Acetobacter lovaniensis (Frateur 1950) comb. nov., and Acetobacter estunensis (Carr 1958) comb. nov. J Gen Appl Microbiol 46:147–165

    Article  CAS  Google Scholar 

  • Loganathan P, Nair S (2004) Swaminatania salitorans gen. nov., sp. nov., a salt-tolerant, nitrogen-fixing and phosphate-solubilizing bacterium from wild rice (Porterisia coarctata Tateoka). Int J Syst Evol Microbiol 54:1185–1190

    Article  CAS  Google Scholar 

  • Matsushita K, Takaki E, Shinogawa M, Ameyama M, Adachi O (1992) Ethanol oxidase respiratory chain of acetic acid bacteria. Reactivity with ubiquinone of pyrroloquinoline quinone-dependent alcohol dehydrogenase purified from Acetobacter aceti and Gluconobacter suboxydans. Biosci Biotechnol Biochem 56:304–310

    Article  CAS  Google Scholar 

  • Matsushita K, Toyama H, Adachi O (1994) Respiratory chain and bioenergetics of acetic acid bacteria. In: Rose AH, Tempest DW (eds) Advances in microbial physiology, vol 36. Academic, London, pp 247–301

    Google Scholar 

  • Matsushita K, Yakushi T, Toyama H, Shinagawa E, Adachi O (1996) Function of multiple heme c moieties in intramolecular electron transport and ubiquinone reduction in the quinohemoprotein alcohol dehydrogenase cytochrome c complex of Gluconobacter suboxydans. J Biol Chem 271:4850–4857

    Article  CAS  Google Scholar 

  • Matsushita K, Kobayashi Y, Mizuguchi M, Toyama H, Adachi O, Sakamoto K, Miyoshi H (2008) A tightly bound quinone functions in ubiquinone reaction site of quinoprotein alcohol dehydrogenase of an acetic acid bacterium, Gluconobacter suboxydans. Biosci Biotechnol Biochem 72:2723–2731

    Article  CAS  Google Scholar 

  • Ndoye B, Lebecque S, Dubois-Dauphin R, Tounkara L, Guiro A-T, Kere C, Diawara B, Thonart P (2006) Thermoresistant properties of acetic acids bacteria isolated from tropical products of sub-Saharan Africa and destined to industrial vinegar. Enzyme Microbial Technol 39:916–923

    Article  CAS  Google Scholar 

  • Ohmori S, Masai H, Arima K, Beppu T (1980) Isolation and identification of acetic acid bacteria for submerged acetic acid fermentation at high temperature. Agric Biol Chem 44:2901–2906

    CAS  Google Scholar 

  • Okumura H, Uozumi T, Beppu T (1985) Construction of plasmid vector and genetic transformation system for Acetobacter aceti. Agric Biol Chem 49:1011–1017

    CAS  Google Scholar 

  • Saeki A, Theeragool G, Matsushita K, Toyama H, Adachi O (1997) Development of thermotolerant acetic acid bacteria useful for vinegar fermentation at higher temperatures. Biosci Biotechnol Biochem 61:138–145

    Article  CAS  Google Scholar 

  • Sievers M, Swings J (2005) In: Brenner DJ, Krieg NR, Staley JT, Garrity GM (eds) Bergey's manual of systematic bacteriology, 2nd edn, Vol. 2. Part C. Springer, pp. 41–54

  • Sokollek SJ, Hertel C, Hammes WP (1998) Description of Acetobacter oboediens sp. nov. and Acetobacter pomorum sp. nov., two new species isolated from industrial vinegar fermentations. Int J Syst Bacteriol 48:935–940

    Article  CAS  Google Scholar 

  • Tayama K, Fukaya M, Okumura H, Kawamura Y, Beppu T (1989) Purification and characterization of membrane-bound alcohol dehydrogenase from Acetobacter polyoxogenase sp. nov. Appl Microbiol Biotechnol 32:181–185

    Article  CAS  Google Scholar 

  • Takemura H, Kondo K, Horinouchi S, Beppu T (1993) Induction by ethanol of alcohol dehydrogenase activity in Acetobacter pasteurianus. J Bacteriol 175:6857–6866

    CAS  Google Scholar 

  • Thompson DJ, Higgins DG, Gibson TG (1994) Clustal W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acid Res 22:4673–4680

    Article  CAS  Google Scholar 

  • Thomas PE, Ryan D, Levin W (1976) An improved staining procedure for the detection of the peroxidase activity of cytochrome P-450 on sodium dodecyl sulfate polyacrylamide gels. Anal Biochem 75:168–176

    Article  CAS  Google Scholar 

  • Toyama H, Mathews FS, Adachi O, Matsushita K (2004) Quinoprotein alcohol dehydrogenases: structure, function, and physiology. Arch Biochem Biophys 428:10–21

    Article  CAS  Google Scholar 

  • Trcek J, Toyama T, Czuba J, Misiewicz A, Matsushita K (2006) Correlation between acetic acid resistance and characteristics of PQQ-dependent ADH in acetic acid bacteria. Appl Microbiol Biotechnol 70:366–373

    Article  CAS  Google Scholar 

  • Yukphan P, Malimas T, Potacharoen W, Tanasupawat S, Tanticharoen M, Yamada Y (2005) Neoasaia chiangmaiensis gen. nov., sp. novel osmotolerant acetic acid bacterium in the α-Proteobacteria. J Gen Appl Microbiol 51:301–311

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences. Part of this work was carried out through collaboration in a Core University Program between Yamaguchi University and Kasetsart University supported by The Japan Society for the Promotion of Science (JSPS) and the National Research Council of Thailand (NRCT). W. K. would like to thank Mahasarakham University for the financial support to pursue her Ph.D. program. We are grateful to Dr. Mamoru Yamada for his helpful discussion during the course of this work.

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Authors and Affiliations

  1. Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi, 753-8515, Japan

    Watchara Kanchanarach, Toshiharu Yakushi, Osao Adachi & Kazunobu Matsushita

  2. Department of Microbiology, Faculty of Science, Kasetsart University, Bangkok, 10900, Thailand

    Gunjana Theeragool

  3. Department of Bioscience and Biotechnology, Faculty of Agriculture, University of the Ryukyus, Okinawa, 903-0213, Japan

    Hirohide Toyama

Authors
  1. Watchara Kanchanarach
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  2. Gunjana Theeragool
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  3. Toshiharu Yakushi
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  4. Hirohide Toyama
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  5. Osao Adachi
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  6. Kazunobu Matsushita
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Correspondence to Kazunobu Matsushita.

Electronic supplementary materials

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Table S1

Growth characteristics of acetic acid bacteria (22 isolated strains; DOC 63.5 kb)

Table S2

Utilization of carbon sources (DOC 35 kb)

Table S3

Comparison of 16S rRNA sequences of Acetobacter sp. MSU10 and MSU22 with those of other acetic acid bacteria (DOC 40 kb)

Table S4

Purification summary of ADHs from A. pasteurianus strains (DOC 38 kb)

Table S5

Substrate specificity of ADHs from A. pasteurianus strains (DOC 39.5 kb)

Fig. S1

An alignment of amino acid sequence of ADH subunit I (a), subunit II (b), and subunit III (c) from A. pasteurianus MSU10, SKU1108, and IFO3191. The black bar and white letters indicate different amino acid residues. W-motifs (W1–W8) are indicated as black boxes. Amino acid residues involved in PQQ binding are bold letters, and those involved in heme c binding are gray areas (Toyama et al. 2004; DOC 48 kb)

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Kanchanarach, W., Theeragool, G., Yakushi, T. et al. Characterization of thermotolerant Acetobacter pasteurianus strains and their quinoprotein alcohol dehydrogenases. Appl Microbiol Biotechnol 85, 741–751 (2010). https://doi.org/10.1007/s00253-009-2203-5

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