Applied Microbiology and Biotechnology

, Volume 87, Issue 5, pp 1765–1772

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

DOI: 10.1007/s00253-010-2655-7

Cite this article as:
Topakas, E., Moukouli, M., Dimarogona, M. et al. Appl Microbiol Biotechnol (2010) 87: 1765. doi:10.1007/s00253-010-2655-7

Abstract

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.

Keywords

Glucuronoyl esteraseNucleophilic serineActive siteLignin–carbohydrate complexSporotrichum thermophilePichia pastoris

Supplementary material

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

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