PURIFICATION AND CHARACTERIZATION OF A FERULIC ACID ESTERASE (FAE-III) FROM ASPERGILLUS-NIGER - SPECIFICITY FOR THE PHENOLIC MOIETY AND BINDING TO MICROCRYSTALLINE CELLULOSE

An inducible ferulic acid esterase (FAE-III) has been isolated, purifi ed and partially characterized from Aspergillus niger after growth on oat spelt xylan. The purification procedure utilized ammonium sulphate precipitation, hydrophobic interaction and anion-exchange chromatogra phy. The purified enzyme appeared almost pure by SDS-PACE, with an app arent M(r) of 36000. A single band, corresponding to a pl of 3.3 was o bserved on isoelectric focusing. With methyl ferulate as substrate, th e enzyme had a specific activity of 67 IU (mg protein)(-1), pH and tem perature optima of 5 and 55-60 degrees C, respectively, and a K-m of 2 .08 mM and a V-max of 175 mu mol 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, bu t at a level 420-fold less (on methyl ferulate) than the A. niger este rase. 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 unsatu rated. The specific activity of FAE-III on destarched wheat bran was 3 1 U (mg protein)(-1) in the presence of Trichoderma viride xylanase an d 3 U (mg protein)(-1) in the absence. Apparent pH dependent binding o f A. niger FAE-III to microcrystalline cellulose was also demonstrated .