Applied Microbiology and Biotechnology

, Volume 62, Issue 4, pp 356–361 | Cite as

2-(2′-Hydroxyphenyl)benzene sulfinate desulfinase from the thermophilic desulfurizing bacterium Paenibacillus sp. strain A11-2: purification and characterization

  • J. KonishiEmail author
  • K. Maruhashi
Original Paper


2-(2′-Hydroxyphenyl)benzene sulfinate (HPBSi) desulfinase (TdsB), which catalyzes the final step of desulfurization of dibenzothiophene (DBT), was purified from a thermophilic DBT- and benzothiophene (BT)-desulfurizing bacterium: Paenibacillus sp. strain A11-2. The molecular mass of the purified enzyme was 31 kDa and 39 kDa by gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis, respectively, suggesting a monomeric structure. The optimal temperature and pH for the reaction involving TdsB was 55°C and the enzyme was more resistant to heat treatment than DszB, a counterpart purified from Rhodococcus erythropolis. The optimum pH for TdsB activity was pH 8. TdsB converted HPBSi to 2-hydroxybiphenyl (2-HBP) and sulfite stoichiometrically. The Km and kcat values for HPBSi were 0.33 mM and 0.32 s−1, respectively. TdsB was inactivated by SH reagents such as p-chloromercuribenzoic acid and 5,5′-dithio-bis-2-nitrobenzoic acid, but was not inhibited by chelating reagents such as EDTA and o-phenanthroline. TdsB was also inhibited by o-hydroxystyrene, the final desulfurized product of BT. However, 2-HBP and its derivatives showed only a weak inhibitory effect. TdsB desulfurized 2-(2′-hydroxyphenyl)ethen-1-sulfinate to yield o-hydroxystyrene, but DszB could not. A site-directed mutagenesis study revealed the cysteine residue at position 17 to be essential to the catalytic activity of TdsB.


Desulfurization Organosulfur Compound Rhodococcus Erythropolis Hydroxy Compound Biodesulfurization 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



We thank all the staff of the Bio-Refining Process Laboratory for discussions. This work was supported by the Ministry of Economy, Trade and Industry, Japan.


  1. Denome SA, Olson ES, Young KD (1993) Identification and cloning of genes involved in specific desulfurization of dibenzothiophene by Rhodococcus sp. strain IGTS8. Appl Environ Microbiol 59:2837–2843Google Scholar
  2. Gallagher JR, Olson ES, Stanley DC (1993) Microbial desulfurization of dibenzothiophene: a sulfur-specific pathway. FEMS Microbiol Lett 107:31–36PubMedGoogle Scholar
  3. Gray KA, Pogrebinsky OS, Mrachko GT, Xi L, Monticello DJ, Squires CH (1996) Molecular mechanisms of biocatalytic desulfurization of fossil fuels. Nat Biotechnol 14:1705–1709Google Scholar
  4. Hanson G, Kemp DS (1981) Convenient routes to 4,4″ functionalised o-terphenyls and 2,2′functionalised biphenyls. Org Chem 46:5441–5443Google Scholar
  5. Ishii Y, Konishi J, Okada H, Hirasawa K, Onaka T, Suzuki M (2000) Operon structure and functional analysis of the genes encoding thermophilic desulfurizing enzymes of Paenibacillus sp. A11–2. Biophys Biochem Res Commun 270:81–88Google Scholar
  6. Izumi Y, Ohshiro T, Ogino H, Hine Y, Shimao M (1994) Selective desulfurization of dibenzothiophene by Rhodococcus erythropolis D-1. Appl Environ Microbiol 60:223–226Google Scholar
  7. Kilbane JJ (1989) Desulfurization of coals: the microbial solution. Trends Biotechnol 7:97–101Google Scholar
  8. Konishi J, Ishii Y, Onaka T, Okumura K, Suzuki M (1997) Thermophilic carbon-sulfur-bond-targeted biodesulfurization. Appl Environ Microbiol 63:3164–3169Google Scholar
  9. Konishi J, Ishii Y, Onaka T, Ohta Y, Suzuki M, Maruhashi K (2000) Purification and characterization of dibenzothiophene sulfone monooxygenase and FMN-dependent NADH oxidoreductase from the thermophilic bacterium Paenibacillus sp. strain A11–2. J Biosci Bioeng 90:607–613Google Scholar
  10. Konishi J, Ishii Y, Onaka T, Maruhashi K (2002) Purification and characterization of the monooxygenase catalyzing sulfur-atom specific oxidation of dibenzothiophene and benzothiophene from the thermophilic bacterium Paenibacillus sp. strain A11-2. Appl Microbiol Biotechnol 60:128–133CrossRefPubMedGoogle Scholar
  11. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685PubMedGoogle Scholar
  12. McFarland BL (1999) Biodesulfurization. Curr Opin Microbiol 2:257–264PubMedGoogle Scholar
  13. Nakayama N, Matsubara T, Ohshiro T, Moroto Y, Kawata Y, Koizumi K, Hirakawa Y, Suzuki M, Maruhashi K, Izumi Y, Kurane R (2002) A novel enzyme, 2′-hydroxybiphenyl-2- sulfinate desulfinase (DszB), from a dibenzothiophene-desulfurizing bacterium Rhodococcus erythropolis KA2–5-1: gene overexpression and enzyme characterization. Biochim Biophys Acta 1598:122–130CrossRefPubMedGoogle Scholar
  14. Ohshiro T, Izumi Y (1999) Microbial desulfurization of organic sulfur compounds in petroleum. Biosci Biotechnol Biochem 63:1–9PubMedGoogle Scholar
  15. Ohshiro T, Suzuki K, Izumi Y (1996) Regulation of dibenzothiophene degrading enzyme activity of R. erythropolis D-1. J Ferment Bioeng 81:121–124CrossRefGoogle Scholar
  16. Omori T, Monna L, Saiki Y, Kodama T (1992) Desulfurization of dibenzothiophene by Corynebacterium sp. strain SY1. Appl Environ Microbiol 58:911–915PubMedGoogle Scholar
  17. Piddington CS, Kovachvich BR, Rambosek J (1995) Sequence and molecular characterization of a DNA region encoding the dibenzothiophene desulfurization operon of Rhodococcus sp. strain IGTS8. Appl Environ Microbiol 64:2327–2331Google Scholar
  18. Wang P, Kraviec S (1994) Desulfurization of dibenzothiophene to 2-hydroxybiphenyl by some newly isolated bacterial strains. Arch Microbiol 161:266–271CrossRefGoogle Scholar
  19. Webb R (1963) Enzyme and metabolic inhibitors, vol 1. Academic Press, New York, p 78Google Scholar

Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  1. 1.Bio-Refining Process LaboratoryJapan Cooperation CenterShimizuJapan

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