Substrate Report for: NPh-4-Fe-Xylp

A feruloyl esterase (StFAE-A) produced by Sporotrichum thermophile was purified to homogeneity. The purified homogeneous preparation of native StFAE-A exhibited a molecular mass of 57.0+/-1.5 kDa, with a mass of 33+/-1 kDa on SDS-PAGE. The pI of the enzyme was estimated by cation-exchange chromatofocusing to be at pH 3.1. The enzyme activity was optimal at pH 6.0 and 55-60 degrees C. The purified esterase was stable at the pH range 5.0-7.0. The enzyme retained 70% of activity after 7 h at 50 degrees C and lost 50% of its activity after 45 min at 55 degrees C and after 12 min at 60 degrees C. Determination of k(cat)/ K(m) revealed that the enzyme hydrolyzed methyl p-coumarate 2.5- and 12-fold more efficiently than methyl caffeate and methyl ferulate, respectively. No activity on methyl sinapinate was detected. The enzyme was active on substrates containing ferulic acid ester linked to the C-5 and C-2 linkages of arabinofuranose and it hydrolyzed 4-nitrophenyl 5- O- trans-feruloyl-alpha- l-arabinofuranoside (NPh-5-Fe-Ara f) 2-fold more efficiently than NPh-2-Fe-Ara f. Ferulic acid (FA) was efficiently released from destarched wheat bran when the esterase was incubated together with xylanase from S. thermophile (a maximum of 34% total ferulic acid released after 1 h incubation). StFAE-A by itself could release FA, but at a level almost 47-fold lower than that obtained in the presence of xylanase. The potential of StFAE-A for the synthesis of various phenolic acid esters was tested using a ternary water-organic mixture consisting of n-hexane, 1-butanol and water as a reaction system.

The lignocellulolytic fungus Aureobasidium pullulans NRRL Y 2311-1 produces feruloyl esterase activity when grown on birchwood xylan. Feruloyl esterase was purified from culture supernatant by ultrafiltration and anion-exchange, hydrophobic interaction, and gel filtration chromatography. The pure enzyme is a monomer with an estimated molecular mass of 210 kDa in both native and denatured forms and has an apparent degree of glycosylation of 48%. The enzyme has a pI of 6.5, and maximum activity is observed at pH 6.7 and 60 degrees C. Specific activities for methyl ferulate, methyl p-coumarate, methyl sinapate, and methyl caffeate are 21.6, 35.3, 12.9, and 30.4 micro mol/min/mg, respectively. The pure feruloyl esterase transforms both 2-O and 5-O arabinofuranosidase-linked ferulate equally well and also shows high activity on the substrates 4-O-trans-feruloyl-xylopyranoside, O-[5-O-[(E)-feruloyl]-alpha-L-arabinofuranosyl]-(1,3)-O-beta-D-xylopyranosyl-(1,4)-D-xylopyranose, and p-nitrophenyl-acetate but reveals only low activity on p-nitrophenyl-butyrate. The catalytic efficiency (k(cat)/K(m)) of the enzyme was highest on methyl p-coumarate of all the substrates tested. Sequencing revealed the following eight N-terminal amino acids: AVYTLDGD.

4-Nitrophenyl 5-O-trans-feruloyl-alpha-L-arabinofuranoside and 4-nitrophenyl 2-O-trans-feruloyl-alpha-L-arabinofuranoside, synthesized by our group (M. Mastihubov, J. Szemesov, and P. Biely), were found to be suitable substrates for determination of activity of feruloyl esterases (FeEs) exhibiting affinity for 5-O- and 2-O-feruloylated alpha-L-arabinofuranosyl residues. One assay is based on coupling the FeE-catalyzed formation of 4-nitrophenyl alpha-L-arabinofuranoside with its efficient hydrolysis by alpha-L-arabinofuranosidase to release 4-nitrophenol. An alternative assay explores the difference in the molar absorbances at 340 nm of the substrate (ferulic acid esters) and the reaction products, which are (1) free ferulic acid and 4-nitrophenyl alpha-L-arabinofuranoside in samples free of alpha-L-arabinofuranosidase and (2) ferulic acid, 4-nitrophenyl alpha-L-arabinofuranoside, and/or 4-nitrophenol in samples containing alpha-L-arabinofuranosidase. The new substrates represent convenient tools to differentiate FeEs on the basis of substrate specificity.