Applied Microbiology and Biotechnology

, Volume 61, Issue 5–6, pp 517–522 | Cite as

Cloning and sequence analysis of the ces10 gene encoding a Sphingomonas paucimobilis esterase

  • P. A. Videira
  • A. M. Fialho
  • A. R. Marques
  • P. M. Coutinho
  • I. Sá-CorreiaEmail author
Original Paper


The ces10 gene of the gellan gum-producing strain Sphingomonas paucimobilis ATCC 31461 was cloned and sequenced. Multi-sequence alignment of the deduced protein indicated that Ces10 belongs to the serine hydrolase family with a potential catalytic triad comprising Ser153 (within the G-X-S-X-G consensus sequence), His75 and Asp125. The mixed block results obtained following pattern search and the low identities detected in a BLAST analysis indicate that Ces10 is significantly different from other characterised bacterial esterases/lipases. Nevertheless, the Ces10 amino acid sequence showed 45% similarity with Rhodococcus sp. heroin esterase and 48% with Bacillus subtilis p-nitrobenzyl esterase. Ces10, with a predicted molecular mass of 30,641 Da, was overproduced in Escherichia coli and purified to homogeneity in a histidine-tagged form. Enzyme assays using p-nitrophenyl-esters (p-NP-esters) with different acyl chain-lengths as the substrate confirmed the anticipated esterase activity. Ces10 exhibited a marked preference for short-chain fatty acids, yielding the highest activity with p-NP-propionate (optimal pH 7.4, optimal temperature 37 °C).


Lipolytic Enzyme Triacetine Ces10 Gene Sphingomonas Paucimobilis Ces10 Protein 
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.



This work was supported by the Fundação para a Ciência e Tecnologia, Portugal (project POCTI/35733/1999) and by post-doctoral and PhD grants to P.A.V. (SFRH/BPD/5710/2001) and A.R.M. (PRAXIS XXI/BD/18146/98)].


  1. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402PubMedGoogle Scholar
  2. Arpigny JL, Jaeger KE (1999) Bacterial lipolytic enzymes: classification and properties. Biochem J 343:177–183PubMedGoogle Scholar
  3. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  4. Coutinho PM, Henrissat B (1999) Carbohydrate-active enzymes: an integrated database approach. In: Gilbert HJ, Davies G, Henrissat B, Svensson B (eds) Recent advances in carbohydrate bioengineering. The Royal Society of Chemistry, Cambridge, pp 3–12Google Scholar
  5. Cygler M, Schrag JD (1997) Structure as a basis for understanding the interfacial properties of lipases. Methods Enzymol 284:3–27PubMedGoogle Scholar
  6. Frederickson JK, Balkwill DL, Drake GR, Romine MF, Ringelberg DB, White DC (1995) Aromatic-degrading Sphingomonas isolates from the deep subsurface. Appl Environ Microbiol 61:1917–1922PubMedGoogle Scholar
  7. Kang KS, Veeder GT, Mirrasoul PJ, Kaneko T, Cottrell W (1982) Agar like polysaccharide produced by a Pseudomonas species: production and basic properties. Appl Environ Microbiol 43:1086–1091Google Scholar
  8. Klm H, Nishiyama M, Kunito T, Senoo K, Kawahara K, Murakami K, Oyaizu H (1998) High population of Sphingomonas species on plant surface. J Appl Microbiol 85:731–736Google Scholar
  9. Marques AR, Coutinho PM, Videira P, Fialho AM, Sá-Correia I (2002) Sphingomonas paucimobilis beta-glucosidase Bgl1: a member of a new bacterial subfamily in family 1 of glycoside hydrolases. Biochem J DOI 10.1042/BJ20021249Google Scholar
  10. Ollis DL, Cheah E, Cygler M, Dijkstra B, Frolow F, Franken SM, Harel M, Remington SJ, Silman I, Schrag JD, Sussman JL, Verschueren KHG, Goldman A (1992) The alpha/beta hydrolase fold. Protein Eng 5:197–211PubMedGoogle Scholar
  11. Pleiss J, Fischer M, Peiker M, Thiele C, Schmid RD (2000) Lipase engineering database: understanding and exploiting sequence–structure–function relationships. J Mol Catal B Enzym 10:491–508CrossRefGoogle Scholar
  12. Rathbone DA, Holt PJ, Lowe CR, Bruce NC (1997) Molecular analysis of the Rhodococcus sp. strain H1 her gene and characterization of its product, a heroin esterase, expressed in Escherichia coli. Appl Environ Microbiol 63:2062–2066PubMedGoogle Scholar
  13. Sá-Correia I, Fialho AM, Videira P, Moreira LM, Marques AR, Albano H (2002) Gellan gum biosynthesis in Sphingomonas paucimobilis ATCC 31461: genes, enzymes and exopolysaccharide production engineering. J Ind Microbiol Biotechnol 29:170–176PubMedGoogle Scholar
  14. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.Google Scholar
  15. Schirmer F, Ehrt S, Hillen W (1997) Expression, inducer spectrum, domain structure, and function of MopR, the regulator of phenol degradation in Acinetobacter calcoaceticus NCIB8250. J Bacteriol 179:1329–1336PubMedGoogle Scholar
  16. Schmidt-Dannert C (1999) Recombinant microbial lipases for biotechnological applications. Bioorg Med Chem 7:2123–2130Google Scholar
  17. Sutherland IW, Kennedy L (1996) Polysaccharide lyases from gellan-producing Sphingomonas spp. Microbiology 142:867–872PubMedGoogle Scholar
  18. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680 PubMedGoogle Scholar
  19. Videira PA, Cortes LL, Fialho AM, Sá-Correia I (2000) Identification of the pgmG gene, encoding a bifuntional protein with phosphoglucomutase and phosphomannomutase activities, in the gellan gum-producing strain Sphingomonas paucimobilis ATCC 31461. Appl Environ Microbiol 66:2252–2258CrossRefPubMedGoogle Scholar
  20. Videira PA, Fialho AM, Geremia RA, Breton C, Sá-Correia I (2001) Biochemical characterization of the beta-1,4-glucuronosyltransferase GelK in the gellan gum-producing strain Sphingomonas paucimobilis ATCC 31461. Biochem J 358:457–464CrossRefPubMedGoogle Scholar
  21. Williamson G, Kroon PA, Faulds CB (1998) Hairy plant polysaccharides: a close shave with microbial esterases. Microbiology 144:2011–2023Google Scholar
  22. Yabuuchi E, Yano I, Oyaizu H, Hashimoto Y, Ezaki T, Yamamoto H (1990) Proposals of Sphingomonas gen. nov. and comb. nov., Sphingomonas parapaucimobilis sp. nov., Sphingomonas yanoikuyae sp. nov., Sphingomonas adhaesiva sp. nov., Sphingomonas capsulata comb. nov., and two genospecies of the genus. Microbiol Immunol 34:99–119PubMedGoogle Scholar
  23. Zock J, Cantwell C, Swartling J, Hodges R, Pohl T, Sutton K, Rosteck P Jr, McGilvray D, Queener S (1994) The Bacillus subtilis pnbA gene encoding p-nitrobenzyl esterase: cloning, sequence and high-level expression in Escherichia coli. Gene 151:37–43CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  • P. A. Videira
    • 1
  • A. M. Fialho
    • 1
  • A. R. Marques
    • 1
  • P. M. Coutinho
    • 1
  • I. Sá-Correia
    • 1
    Email author
  1. 1.Centro de Engenharia Biológica e QuímicaInstituto Superior TécnicoLisboaPortugal

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