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

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 Mr 58 kDa and pI 6.7. The enzyme activity was optimal at pH 6.0 and 60 °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 °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-α-d-glucopyranosyluronate)-β-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. © 2009 Federation of European Microbiological Societies.