[Purification and characterization of ferulic acid esterase from Penicillium citrinum]

Extracellular feruloyl esterase (EC 3.1.1.73) from Penicillium citrinum culture filtrates was studied. The effect of feruloyl esterases on the enzymatic hydrolysis of brewer's spent grain was also investigated.

Feruloyl esterase was purified to homogeneity by ammonium sulfate precipitation, ion-exchange chromatography with a DEAE-Sepharose Fast Flow column, and column chromatography with a Phenyl-Sepharose 6 Fast Flow column.

The purified homogeneous preparation of native feruloyl esterase had a molecular mass of 58 kDa by native-PAGE and a subunit molecular mass of 31 kDa by SDS-PAGE. The optimum enzymatic activity was achieved at pH 6.0 and 45 - 65 degrees C. The enzyme was stable at pH from 5.0 to 6.0 and temperature from 25 to 55 degrees C. The enzymatic activity was slightly enhanced by Mg2+, Fe2+, Mn2+, Ca2+, and Na+, whereas it was slightly inhibited by Zn2+, strongly inhibited by Cu2+, and completely inhibited by Hg2+ and phenylmethanesulfonyl fluoride. EDTA had a slight influence on the enzymatic activity. The determination of k(cat)/K(m) revealed that the enzyme hydrolyzed methyl p-coumarate, methyl sinapate, methyl ferulate, methyl caffeate, and the values were 823, 416, 103, and 0, respectively. The k(cat)/K(m) values showed that the enzyme hydrolyzed MpCA faster and more efficiently than all the other substrates. When the crude feruloyl esterase was used to hydrolyze the brewer's spent grain, about 7.2% of the alkaline-extractable ferulic acid could be released, with the concentration of 5 u feruloyl esterase /g.

A feruloyl esterase was discovered. Its biochemical characteristics were different from what has been reported in literature. This provided an important basis for the exploitation of a feruloyl esterase.