Purification, characterization and mass spectrometric sequencing of a thermophilic glucuronoyl esterase from Sporotrichum thermophile

FEMS Microbiol Lett. 2009 Jun;296(2):178-84. doi: 10.1111/j.1574-6968.2009.01631.x. Epub 2009 May 18.

Abstract

The cellulolytic system of the thermophilic fungus Sporotrichum thermophile contains a recently discovered esterase that may hydrolyze the ester linkage between the 4-O-methyl-D-glucuronic acid of glucuronoxylan and lignin alcohols. The glucuronoyl esterase named StGE1 was purified to homogeneity with a molecular mass of M(r) 58 kDa and pI 6.7. The enzyme activity was optimal at pH 6.0 and 60 degrees C. The esterase displayed a narrow pH range stability at pH 8.0 and retained 50% of its activity after 430 and 286 min at 50 and 55 degrees C, respectively. The enzyme was active on substrates containing glucuronic acid methyl ester, showing a lower catalytic efficiency on 4-nitrophenyl 2-O-(methyl-4-O-methyl-alpha-d-glucopyranosyluronate)-beta-D-xylopyranoside than its mesophilic counterparts reported in the literature, which is typical of thermophilic enzymes. StGE1 was proved to be a modular enzyme containing a noncatalytic carbohydrate-binding module. LC-MS/MS analysis provided peptide mass and sequence information that facilitated the identification and classification of StGE1 as a family 15 glucuronoyl esterase that showed the highest homology with the hypothetical glucuronoyl esterase CHGG_10774 of Chaetomium globosum CBS 148.51. This work represents the first example of the purification and identification of a thermophilic glucuronoyl esterase from S. thermophile.

MeSH terms

  • Amino Acid Sequence
  • Enzyme Stability
  • Fungal Proteins / chemistry*
  • Fungal Proteins / isolation & purification
  • Fungal Proteins / metabolism*
  • Glucuronates / metabolism
  • Glucuronidase / chemistry*
  • Glucuronidase / isolation & purification
  • Glucuronidase / metabolism*
  • Hydrogen-Ion Concentration
  • Isoelectric Point
  • Lignin / metabolism
  • Mass Spectrometry
  • Molecular Sequence Data
  • Molecular Weight
  • Sequence Homology, Amino Acid
  • Sporothrix / enzymology*
  • Substrate Specificity
  • Temperature
  • Xylans / metabolism

Substances

  • Fungal Proteins
  • Glucuronates
  • Xylans
  • 4-O-methylglucuronic acid
  • glucuronoxylan
  • Lignin
  • alpha-(4-O-methyl)-D-glucuronidase
  • Glucuronidase