Purification and characterization of a ferulic acid esterase (FAE-III) from Aspergillus niger: specificity for the phenolic moiety and binding to microcrystalline cellulose

Abstract

An inducible ferulic acid esterase (FAE-III) has been isolated, purified and partially characterized from Aspergillus niger after growth on oat spelt xylan. The purification procedure utilized ammonium sulphate precipitation, hydrophobic interaction and anion-exchange chromatography. The purified enzyme appeared almost pure by SDS-PAGE, with an apparent M_r of 36000. A single band, corresponding to a pl of 3·3 was observed on isoelectric focusing. With methyl ferulate as substrate, the enzyme had a specific activity of 67 IU (mg protein)^-1, pH and temperature optima of 5 and 55–60 °C, respectively, and a Km of 2·08 mM and a V_max of 175 μmol min^-1 (mg protein)^-1. The enzyme was also active upon methyl sinapinate, methyl-3,4-dimethoxy cinnamate and methyl p-coumarate, but not benzoic acid methyl esters or methyl caffeate. Similarly, Streptomyces olivochromogenes FAE showed activity against methyl ferulate, methyl sinapinate and methyl p-coumarate, but at a level 420-fold less (on methyl ferulate) than the A. niger esterase. No activity was detected against the benzoate methyl esters. For both enzymes, this shows the necessity for C-3 on the phenol ring to be methoxylated and the aliphatic region of the substrate to be unsaturated. The specific activity of FAE-III on destarched wheat bran was 31 U (mg protein)^-1 in the presence of Trichoderma viride xylanase and 3 U (mg protein)^-1 in the absence. Apparent pH dependent binding of A. niger FAE-III to microcrystalline cellulose was also demonstrated.