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-Ill) 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, 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)-', pH and temperature optima of 5 and 55-60 "c, respectively, and a Km of 2-08 mM and a V,,, of 175 pmol min-l (mg protein)-l. 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-Ill on destarched wheat bran was 31 U (mg protein)-' in the presence of Trichoderma Wide xylanase and 3 U (mg protein)-l in the absence. Apparent pH dependent binding of A. niger FAE-Ill to microcrystalline cellulose was also demonstrated.

Efficient Extraction of Ferulic Acid from Sugar Beet Pulp Using the Culture Supernatant of Penicillium chrysogenum

“…Type A enzymes such as A. niger FAE II 37) and FAE III 38) are inactive towards MCA. Type B enzymes such as A. niger FAE I 37) and CinnAE 39) are not able to hydrolyze MSA.…”

Partial Purification of Ferulic Acid Esterase from Malted Barley

“…Microbial feruloyl esterases that have been purified varied in molecular weight from 24kDa (FAE-II from Neocallimastix strain MC-2 7 ) to132kDa (FAE-I from Aspergillus niger 10 ). In the case of Aspergillus niger three feruloyl esterases have been characterised and their estimated molecular weights are 132kDa (FAE-I), 29kDa (FAE-II) and 36kDa (FAE-III) 10,11 . This suggests that such a variation in molecular weights is not unusual.…”

“…It is likely therefore that the removal of the aryl substituents is necessary for the breakdown of the cell wall pentosans. The cell walls of the aleurone layer, the crushed cell layer, the sheaf cells and some parts of the embryo fluoresce strongly in ultraviolet light, while the cell walls of the starchy endosperm fluoresce less strongly [2][3][4]8,11 . The fluorescence is due to the ferulic acid (4-hydroxy-3-methoxy cinnamic acid), which is attached to the C-5 position of some arabinose residues 1,25 .…”

The interactive effects of enriched sources of Aspergillus ferulic acid esterase and Trichoderma xylanase on the quantitative release of hydroxycinnamic acids from oat hulls

“…Faulds and Williamson (1994) found that ferulic acid esterase from Aspergillus niger could release ferulic and sinapic acids (3,5-dimethoxy-4-hydroxycinnamic acid), but not p-coumaric or caffeic acids (3, 4-dihydroxycinnamic acid). This indicates that this ferulic acid esterase is active only on methyl esters of certain hydroxycinnamic acids, including ferulic and sinapic esters, but not p-coumaric or caffeic esters.…”

“…In contrast, ferulic acid esterase from Pseudomonas fluorescens subsp. Cellulosa is effective on all these methyl hydroxycinnamic esters (Faulds and Williamson 1994), including ferulic acid and p-coumaric acid.…”