, 14:33 | Cite as

Gene cloning and characterization of an aldehyde dehydrogenase from long-chain alkane-degrading Geobacillus thermoleovorans B23

  • Tomohisa Kato
  • Asuka Miyanaga
  • Shigenori Kanaya
  • Masaaki MorikawaEmail author
Original Paper


Geobacillus thermoleovorans B23 is capable of degrading long-chain alkanes at 70°C. Bt-aldh, an aldehyde dehydrogenase gene in B23, was located in the upstream region of p21 whose expression level was dramatically increased when alkane degradation was started (Kato et al. 2009, BMC Microbiol 9:60). Like p21, transcription level of Bt-aldh was also increased upon alkane degradation. Bt-Aldh (497 aa, MW = 53,886) was overproduced in Escherichia coli, purified, and characterized biochemically. Bt-Aldh acted as an octamer, required NAD+ as a coenzyme, and showed high activity against aliphatic long-chain aldehydes such as tetradecanal. The optimum condition for activity was 50–55°C and pH 10.0. The activity was elevated to two- to threefold in the presence of 2 mM Ba2+, Ca2+, or Sr2+, while Mg2+ and Zn2+ inhibited the enzyme activity. Bt-Aldh represents thermophilic aldehyde dehydrogenases responsible for degradation of long-chain alkanes.


Long-chain alkane degradation Aldehyde dehydrogenase Geobacillus thermoleovorans (Extreme) thermophilic microorganisms and their enzymology Biochemical characterisation Biodegradation of pollutants Biotechnology of thermophiles Enzymology Gene cloning and expression Isolation and characterization Thermophiles and thermophilic enzymes 



This work was supported by a grant from the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN), NEDO, and KAKENHI (19380189) to MM.


  1. Abriola DP, Fields R, Stein S, MacKerell AD, Pietruszuko R (1987) Active site of human liver aldehyde dehydrogenase. Biochemistry 26:5679–5684CrossRefPubMedGoogle Scholar
  2. Andersson CS, Högbom M (2009) A Mycobacterium tuberculosis ligand-binding Mn/Fe protein reveals a new cofactor in a remodeled R2-protein scaffold. Proc Natl Acad Sci USA 106:5633–5638CrossRefPubMedGoogle Scholar
  3. Black S (1951) Yeast aldehyde dehydrogenase. Arch Biochem Biophys 34:86–97CrossRefGoogle Scholar
  4. Bordelon T, Montequdo SK, Pakhomova S, Oldham ML, Newcomer ME (2004) A disorder to order transition accompanies catalysis in retinalaldehyde dehydrogenase typeII. J Biol Chem 279:43085–43091CrossRefPubMedGoogle Scholar
  5. Eaton RW (1997) p-Cymene catabolic pathway in Pseudomonas putida F1: cloning and characterization of DNA encoding conversion of p-cymene to p-cumate. J Bacteriol 179:3171–3180PubMedGoogle Scholar
  6. Feng L, Wang W, Cheng J, Ren Y, Zhao G, Gao C, Tang Y, Liu X, Han W, Peng X, Liu R, Wang L (2007) Genome and proteome of long-chain alkane degrading Geobacillus thermodenitrificans NG80–2 isolated from a deep-subsurface oil reservoir. Proc Natl Acad Sci USA 104:5602–5607CrossRefPubMedGoogle Scholar
  7. Fox BG, Shanklin J, Ai J, Loehr TM, Sanders-Loehr J (1994) Resonance Raman evidence for an Fe–O–Fe center in stearoyl-ACP desaturase. Primary sequence identity with other diiron-oxo proteins. Biochemistry 33:12776–12786CrossRefPubMedGoogle Scholar
  8. Geissdörfer W, Kok RG, Ratajczak A, Hellingwerf KJ, Hillen W (1999) The genes rubA and rubB for alkane degradation in Acinetobacter sp. strain ADP1 are in an operon with estB, encoding an esterase, and oxyR. J Bacteriol 181:4292–4298PubMedGoogle Scholar
  9. Harris TK, Davidson VL (1994) Replacement of enzyme-bound calcium with strontium alters the kinetic properties of methanol dehydrogenase. Biochem J 300:175–182PubMedGoogle Scholar
  10. Ishige T, Tani A, Sakai Y, Kato N (2000) Long-chain aldehyde dehydrogenase that participates in n-alkane utilization and wax ester synthesis in Acinetobacter sp. strain M-1. Appl Environ Microbiol 66:3481–3486CrossRefPubMedGoogle Scholar
  11. Kanamori T, Rashid N, Morikawa M, Atomi H, Imanaka T (2002) Oleomonas sagaranensis gen. nov., sp nov., represents a novel genus in the alpha-Proteobacteria. FEMS Microbiol Lett 217:255–261PubMedGoogle Scholar
  12. Kato T, Haruki M, Imanaka T, Morikawa M, Kanaya S (2001) Isolation and characterization of long-chain-alkane degrading Bacillus thermoleovorans from deep subterranean petroleum reservoirs. J Biosci Bioeng 91:64–70CrossRefPubMedGoogle Scholar
  13. Kato T, Miyanaga A, Morikawa M, Kanaya S (2009) Alkane inducible proteins in Geobacillus thermoleovorans B23. BMC Microbiol 9:60CrossRefPubMedGoogle Scholar
  14. Kunst F, Ogasawara N, Moszer I, Albertini AM, Alloni G et al (1997) The complete genome sequence of the gram-positive bacterium Geobacillus subtilis. Nature 390:249–256CrossRefPubMedGoogle Scholar
  15. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefPubMedGoogle Scholar
  16. Lamb AL, Newcomer ME (1999) The structure of retinal dehydrogenase type II at 2.7 A resolution: Implications for retinal specificity. Biochemistry 38:6003–6011CrossRefPubMedGoogle Scholar
  17. Li L, Liu X, Yang W, Xu F, Wang W, Feng L, Bartlam M, Wang L, Rao Z (2008) Crystal structure of long-chain alkane monooxygenase (LadA) in complex with coenzyme FMN: unveiling the long-chain alkane hydroxylase. J Mol Biol 376:453–465CrossRefPubMedGoogle Scholar
  18. Maeng JH, Sakai Y, Ishige T, Tani Y, Kato N (1996) Diversity of dioxygenases that catalyze the first step of oxidation of long-chain n-alkanes in Acinetobacter sp. strain M-1. FEMS Microbiol Lett 141:177–182CrossRefGoogle Scholar
  19. Okibe N, Amada K, Hirano S, Haruki M, Imanaka T, Morikawa M, Kanaya S (1999) Gene cloning and characterization of aldehyde dehydrogenase from a petroleum-degrading bacterium, strain HD-1. J Biosci Bioeng 88:7–11CrossRefPubMedGoogle Scholar
  20. Poelarends GJ, Zandstra M, Bosma T, Kulakov LA, Larkin MJ, Marchesi JR, Weightman AJ, Janssen DB (2000) Haloalkane-utilizing Rhodococcus strains isolated from geographically distinct locations possess a highly conserved gene cluster encoding haloalkane catabolism. J Bacteriol 182:2725–2731CrossRefPubMedGoogle Scholar
  21. Priefert H, Krüger N, Jendrossek D, Schmidt B, Steinbüchel A (1992) Identification and molecular characterization of the gene coding for acetaldehyde dehydrogenase II (acoD) of Alcaligenes eutrophus. J Bacteriol 174:899–907PubMedGoogle Scholar
  22. Privitera O, Sisto F, Giuffrida V, Puntorieri M, Cascone C, Di Silvestro I, Rappazzo G, Stefani S (1999) Reverse transcription polymerase chain reaction method for the detection of glycopeptide resistance in enterococci. J Microbiol Methods 35:95–100CrossRefPubMedGoogle Scholar
  23. Reddy KJ (1995) Isolation of RNA from gram-positive bacteria. In: Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (eds) Current protocols in molecular biology, vol 1. 4.4.3. Wiley, TorontoGoogle Scholar
  24. Rueter P, Rabus R, Wilkes H, Aeckersberg F, Rainey FA, Jannasch HW, Widdel F (1994) Anaerobic oxidation of hydrocarbons in crude oil by new types of sulphate-reducing bacteria. Nature 372:455–458CrossRefPubMedGoogle Scholar
  25. Sambrook J, Fritsch EF, Maniatis T (eds) (1989) Molecular cloning. a laboratory manual 2nd edition. Cold Spring Harbor Laboratory, Cold Spring HarborGoogle Scholar
  26. Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW (1990) Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol 185:60–89CrossRefPubMedGoogle Scholar
  27. Takami H, Nishi S, Lu J, Shimamura S, Takaki Y (2004a) Genomic characterization of thermophilic Geobacillus species isolated from the deepest sea mud of Mariana Trench. Extremophiles 8:351–356CrossRefPubMedGoogle Scholar
  28. Takami H, Takaki Y, Chee GJ, Nishi S, Shimamura S, Suzuki H, Matsui S, Uchiyama I (2004b) Thermoadaptation trait revealed by the genome sequence of thermophilic Geobacillus kaustophilus. Nucleic Acids Res 32:6292–6303CrossRefPubMedGoogle Scholar
  29. Takei D, Washio K, Morikawa M (2008) Identification of alkane hydroxylase genes in Rhodococcus sp. strain TMP2 that degrades a branched alkane. Biotechnol Lett 30:1447–1452CrossRefPubMedGoogle Scholar
  30. van Beilen JB, Wubbolts MG, Witholt B (1994) Genetics of alkane oxidation by Pseudomonas oleovorans. Biodegradation 5:161–174CrossRefPubMedGoogle Scholar
  31. von Bahr-Lindström H, Jeck R, Woenckhaus C, Sohn S, Hempel J, Jörnvall H (1985) Characterization of the coenzyme binding site of liver aldehyde dehydrogenase: differential reactivity of coenzyme analogues. Biochemistry 24:5847–5851CrossRefGoogle Scholar
  32. Whyte LG, Smits TH, Labbé D, Witholt B, Greer CW, van Beilen JB (2002) Gene cloning and characterization of multiple alkane hydroxylase systems in Rhodococcus strains Q15 and NRRL B-16531. Appl Environ Microbiol 68:5933–5942CrossRefPubMedGoogle Scholar
  33. Zarilla KA, Perry JJ (1987) Geobacillus thermoleovorans, sp. nov., a species of obligately thermophilic hydrocarbon utilizing endospore-forming bacteria. Syst Appl Microbiol 9:258–264Google Scholar
  34. Zhao D, McCaffery P, Ivins KJ, Neve RL, Hogan P, Chin WW, Dräger UC (1996) Molecular identification of a major retinoic-acid-synthesizing enzyme, a retinaldehyde-specific dehydrogenase. Eur J Biochem 240:15–22CrossRefPubMedGoogle Scholar

Copyright information

© Springer 2009

Authors and Affiliations

  • Tomohisa Kato
    • 1
  • Asuka Miyanaga
    • 1
  • Shigenori Kanaya
    • 1
  • Masaaki Morikawa
    • 1
    • 2
    Email author
  1. 1.Department of Material and Life Science, Graduate School of EngineeringOsaka UniversityOsakaJapan
  2. 2.Division of Biosphere Science, Graduate School of Environmental ScienceHokkaido UniversitySapporoJapan

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