Applied Microbiology and Biotechnology

, Volume 87, Issue 5, pp 1765–1772 | Cite as

Functional expression of a thermophilic glucuronoyl esterase from Sporotrichum thermophile: identification of the nucleophilic serine

  • Evangelos Topakas
  • Maria Moukouli
  • Maria Dimarogona
  • Christina Vafiadi
  • Paul Christakopoulos
Biotechnologically Relevant Enzymes and Proteins


A glucuronoyl esterase (GE) from the thermophilic fungus Sporotrichum thermophile, belonging to the carbohydrate esterase family 15 (CE-15), was functionally expressed in the methylotrophic yeast Pichia pastoris. The putative GE gene ge2 from the genomic DNA was successfully cloned in frame with the sequence for the Saccharomyces cerevisiae α-factor secretion signal under the transcriptional control of the alcohol oxidase (AOX1) promoter and integrated in P. pastoris X-33 to confirm that the encoded enzyme StGE2 exhibits esterase activity. The enzyme was active on substrates containing glucuronic acid methyl ester, showing optimal activity at pH 7.0 and 55°C. The esterase displayed broad pH range stability between 4–10 and temperature stability up to 50°C, rendering StGE2 a strong candidate for future biotechnological applications that require robust biocatalysts. ClustalW alignment of StGE2 with characterized GEs and selected homologous sequences, members of CE-15 family, revealed a novel consensus sequence G-C-S-R-X-G that features the characteristic serine residue involved in the generally conserved catalytic mechanism of the esterase family. The putative serine has been mutated, and the corresponding enzyme has been expressed in P. pastoris to prove that the candidate nucleophilic residue is responsible for catalyzing the enzymatic reaction.


Glucuronoyl esterase Nucleophilic serine Active site Lignin–carbohydrate complex Sporotrichum thermophile Pichia pastoris 



The authors thank Dr. Peter Biely from Institute of Chemistry, Slovak Academy of Sciences (Bratislava, Slovakia) who supplied the synthetic substrates for assaying GE activity.

Supplementary material

253_2010_2655_MOESM1_ESM.pdf (46 kb)
ESM 1 (PDF 45 kb)


  1. Altschul SF, Madden TL, Schäffer 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 25:3389–3402CrossRefGoogle Scholar
  2. Bendtsen JD, Nielsen H, von Heijne G, Brunak S (2004) Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 340:783–95CrossRefGoogle Scholar
  3. Blom N, Sicheritz-Ponten T, Gupta R, Gammeltoft S, Brunak S (2004) Prediction of post-translational glycosylation and phosphorylation of proteins from the amino acid sequence. Proteomics 4:1633–49CrossRefGoogle Scholar
  4. Brenner S (1988) The molecular evolution of genes and proteins: a tale of two serines. Nature 334:528–530CrossRefGoogle Scholar
  5. Bresler MM, Rosser SJ, Basran A, Bruce NC (2000) Gene cloning and nucleotide sequencing and properties of a cocaine esterase from Rhodococcus sp. strain MB1. Appl Environ Microbiol 66:904–908CrossRefGoogle Scholar
  6. Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B (2009) The carbohydrate-active enzymes database (CAZy): an expert resource for glycogenomics. Nucleic Acids Res 37:D233–238CrossRefGoogle Scholar
  7. Chich J-F, Chapot-Chartier M-P, Ribadeau-Dumas B, Gripon J-C (1992) Identification of the active site serine of the X-propyl dipeptidyl aminopeptidase from Lactococcus lactis. FEBS Lett 314:139–142CrossRefGoogle Scholar
  8. Ďuranová M, Spanikova S, Wosten HAB, Biely P, de Vries RP (2009a) Two glucuronoyl esterases of Phanerochaete chrysosporium. Arch Microbiol 191:133–140CrossRefGoogle Scholar
  9. Ďuranová M, Hirsch J, Kolenová K, Biely P (2009b) Fungal glucuronoyl esterases and substrate uronic acid recognition. Biosci Biotechnol Biochem 73:2483–2487CrossRefGoogle Scholar
  10. Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins MR, Appel RD, Bairoch A (2005) Protein identification and analysis tools on the ExPASy server. In: Walker JM (ed) The proteomics protocols handbook. Humana Press, New Jersey, Totowa, pp 571–607CrossRefGoogle Scholar
  11. Higgins DR, Busser K, Comiskey J, Whittier PS, Purcell TJ, Hoeffler JP (1998) Small vectors for expression based on dominant drug resistance with direct multicopy selection. In: Higgins DR, Cregg JM (eds) Methods in molecular biology: Pichia protocols. Humana Press, New Jersey, Totowa, pp 28–41Google Scholar
  12. Hirsch J, Kooš M, Kováč P (1998) Improved synthesis of an aldobiouronic acid related to hardwood xylans, and preparation of a derivative thereof suitable for linking to proteins. Carbohydr Res 310:145–149CrossRefGoogle Scholar
  13. Hirsch J, Langer V, Kooš M (2005) Synthesis and molecular structure of methyl 4-O-methyl-α-d-glucopyranuronate. Molecules 10:251–258CrossRefGoogle Scholar
  14. Julenius K, Mølgaard A, Gupta R, Brunak S (2005) Prediction, conservation analysis and structural characterization of mammalian mucin-type O-glycosylation sites. Glycobiology 15:153–64CrossRefGoogle Scholar
  15. Kabashima T, Ito K, Yoshimoto T (1996) Dipeptidyl peptidase IV from Xanthomonas maltophilia: sequencing and expression of the enzyme gene and characterization of the expressed enzyme. J Biochem 120:1111–1117Google Scholar
  16. Koseki T, Miwa Y, Akao T, Akita O, Hashizume K (2006) An Aspergillus oryzae acetyl xylan esterase: molecular cloning and characteristics of recombinant enzyme expressed in Pichia pastoris. J Biotechnol 121:381–389CrossRefGoogle Scholar
  17. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefGoogle Scholar
  18. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) ClustalW and ClustalX version 2. Bioinformatics 23:2947–2948CrossRefGoogle Scholar
  19. Li X-L, Špániková S, de Vries RP, Biely P (2007) Identification of genes encoding microbial glucuronoyl esterases. FEBS Lett 581:4029–4035CrossRefGoogle Scholar
  20. Moukouli M, Topakas E, Christakopoulos P (2008) Cloning, characterization and functional expression of an alkalitolerant type C feruloyl esterase from Fusarium oxysporum. Appl Microbiol Biotechnol 79:245–254CrossRefGoogle Scholar
  21. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, New YorkGoogle Scholar
  22. Špániková S, Biely P (2006) Glucuronoyl esterase—novel carbohydrate esterase produced by Schizophyllum commune. FEBS Lett 580:4597–4601CrossRefGoogle Scholar
  23. Stoscheck CM (1990) Quantification of protein. Meth Enzymol 182:50–68CrossRefGoogle Scholar
  24. Topakas E, Kalogeris E, Kekos D, Macris BJ, Christakopoulos P (2003) Production and partial characterization of feruloyl esterase by Sporotrichum thermophile in solid-state fermentation. Proc Biochem 38:1539–1543CrossRefGoogle Scholar
  25. Vafiadi C, Topakas E, Biely P, Christakopoulos P (2009) Purification, characterization and mass spectrometric sequencing of a thermophilic glucuronoyl esterase from Sporotrichum thermophile. FEMS Microbiol Lett 296:178–184CrossRefGoogle Scholar
  26. Vesanto E, Savijoki K, Rantanen T, Steele JL, Palva A (1995) An X-propyl dipeptidyl aminopeptidase (pepX) gene from Lactobacillus helveticus. Microbiology 141:3067–3075CrossRefGoogle Scholar
  27. Yoshpe-Besancon I, Gripon JC, Ribadeau-Dumas B (1994) Xaa-Pro dipeptidyl aminopeptidase from Lactococcus lactis catalyzes kinetically controlled synthesis of peptide bonds involving proline. Biotechnol Appl Biochem 20:131–140Google Scholar
  28. Wood SJ, Li X-L, Cotta MA, Biely P, Duke NEC, Schiffer M, Pokkuluri PR (2008) Crystallization and preliminary X-ray diffraction analysis of the glucuronoyl esterase catalytic domain from Hypocrea jecorina. Acta Cryst F64:255–257Google Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Evangelos Topakas
    • 1
  • Maria Moukouli
    • 1
  • Maria Dimarogona
    • 1
  • Christina Vafiadi
    • 1
  • Paul Christakopoulos
    • 1
  1. 1.BIOtechMASS Unit, Biotechnology Laboratory, School of Chemical EngineeringNational Technical University of AthensAthensGreece

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