Purification, characterization and mass spectrometric sequencing of a thermophilic glucuronoyl esterase from Sporotrichum thermophile.
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.
Vafiadi
Christina
C
BIOtechMASS Unit, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, Athens, Greece.
Topakas
Evangelos
E
Biely
Peter
P
Christakopoulos
Paul
P
D000595:Amino Acid Sequence; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D005965:Glucuronates / Q000378:metabolism; D005966:Glucuronidase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008031:Lignin / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017386:Sequence Homology, Amino Acid; D013175:Sporothrix / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature; D014990:Xylans / Q000378:metabolism
D005656:Fungal Proteins; D005965:Glucuronates; D014990:Xylans; C036035:4-O-methylglucuronic acid; C038910:glucuronoxylan; D008031:Lignin; C098344:alpha-(4-O-methyl)-D-glucuronidase; D005966:Glucuronidase