Arabinoxylans of commelinid monocots are characterized by high contents of ferulic acid that is incorporated into arabinose-bearing side-chains of varying complexity. Species-related differences in the feruloylated side-chain profiles of grain arabinoxylans are observed and lead to differences in arabinoxylan functionality. Here, a semi-quantitative assay based on (1)H-(13)C-correlation NMR spectroscopy (HSQC experiment) was developed to profile feruloylated side-chains of cereal grain arabinoxylans. Following acidic liberation of the feruloylated side-chains from the xylan backbone and a clean-up step using C18 solid phase extraction, the feruloylated oligosaccharides FA (5-O-trans-feruloyl-L-arabinofuranose), FAX (beta-d-xylopyranosyl-(1 2)-5-O-trans-feruloyl-l-arabinofuranose) and FAXG (alpha-l-galactopyranosyl-(1 2)-beta-d-xylopyranosyl-(1 2)-5-O-trans-feruloyl-l-arabinofuranose) were analyzed by HSQC-NMR. Marker signals were identified for each compound, and experimental conditions such as solvent and internal standard as well as measurement and processing conditions were optimized for a semi-quantitative determination. The approach was validated with respect to accuracy, precision, limit of detection, and limit of quantification. The newly developed approach was applied to several cereal samples including oats, popcorn maize, wheat, and wild rice. Data were compared to an HPLC-DAD/MS approach published earlier by our group, demonstrating that the results of the HSQC approach were comparable to the more time-consuming and technically more challenging HPLC-DAD/MS method.
A type D ferulic acid esterase (FAE) was identified in the culture supernatant of Streptomyces werraensis, purified, sequenced, and heterologously produced in E. coli BL21(DE3)Star by co-expressing chaperones groES-groEL (69 U L(-1)). The unique enzyme with a mass of about 48 kDa showed no similarity to other FAEs, and only moderate homology (78.5%) to a Streptomycete beta-xylosidase. The purified reSwFAED exhibited a temperature optimum of 40 degreesC, a pH optimum in the range from pH seven to eight and a clear preference for bulky natural substrates, such as 5-O-trans-feruloyl-L-arabinofuranose (FA) and beta-D-xylopyranosyl-(1->2)-5-O-trans-feruloyl-L-arabinofuranose (FAX), compared to the synthetic standard substrate methyl ferulate. Treatment of wheat dough with as little as 0.03 U or 0.3 U kg(-1) reSwFAED activity resulted in a significant increase of the bun volume (8.0 or 9.7%, resp.) after baking when combined with polysaccharide-degrading enzymes from Aspergillus. For the first time, the long-standing, but rarely proven positive effect of a FAE in baking was confirmed.
Title: Feruloyl esterase: a key enzyme in biomass degradation Wong DWS Ref: Appl Biochem Biotechnol, 133:87, 2006 : PubMed
Feruloyl esterase forms a part of the enzyme complex that acts collectively and synergistically to completely hydrolyze xylan to its monomers. The enzyme has found potential uses in a wide variety of applications of interest to the agrifood and pharmaceutical industries. This review describes the enzymology of feruloyl esterases involved in xylan degradation. The occurrence of feruloyl esterases in various microorganisms and their physiochemical properties are presented. The nature of the enzyme substrates and products, the role of synergistic interactions with xylanases and other accessory enzymes, as well as the sequence-structure relating to the reaction mechanism are emphasized.
