Purification and characterization of an extracellular feruloyl esterase from the thermophilic anaerobe Clostridium stercorarium.
Feruloyl esterases act as accessory enzymes for the complete saccharification of plant cell wall hemicelluloses. Although many fungal feruloyl esterases have been purified and characterized, few bacterial phenolic acid esterases have been characterized. This study shows the extracellular production of a feruloyl esterase by the thermophilic anaerobe Clostridium stercorarium when grown on birchwood xylan. The feruloyl esterase was purified 500-fold in successive steps involving ultrafiltration, preparative isoelectric focusing and column chromatography by anion exchange, gel filtration and hydrophobic interaction. The purified enzyme released ferulic, rho-coumaric, caffeic and sinapinic acid from the respective methyl esters. The purified enzyme also released ferulic acid from a de-starched wheat bran preparation. At pH 8.0 and 65 degrees C, the Km and Vmax values for the hydrolysis of methyl ferulate were 0.04 mmol l-l and 131 micromol min-1 mg-1, respectively; the respective values for methyl coumarate were 0.86 mmol l-l and 18 micromol min-1 mg-1. The purified feruloyl esterase had an apparent mass of 33 kDa under denaturing conditions and showed optimum activity at pH 8.0 and 65 degrees C. At a concentration of 5 mmol l-l, the ions Ca2+, Cu2+, Co2+ and Mn2+ reduced the activity by 70-80%.
Donaghy
J A
JA
Food Science Division (Microbiology), Department of Agriculture for Northern Ireland, Belfast, UK. John.donaghy@dani.gov.uk
Bronnenmeier
K
K
Soto-Kelly
P F
PF
McKay
A M
AM
D000693:Anaerobiosis; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D003013:Clostridium / Q000201:enzymology* / Q000254:growth & development; D003470:Culture Media; D013379:Substrate Specificity; D014990:Xylans / Q000378:metabolism
D003470:Culture Media; D014990:Xylans; D002265:Carboxylic Ester Hydrolases; C070196:feruloyl esterase