Fungal feruloyl esterases: Functional validation of genome mining based enzyme discovery including uncharacterized subfamilies

Abstract

Feruloyl esterases (FAEs) are a diverse group of enzymes that specifically catalyze the hydrolysis of ester bonds between a hydroxycinnamic (e.g. ferulic) acid and plant poly- or oligosaccharides. FAEs as auxiliary enzymes significantly assist xylanolytic and pectinolytic enzymes in gaining access to their site of action during biomass saccharification for biofuel and biochemical production. A limited number of FAEs have been functionally characterized compared to over 1000 putative fungal FAEs that were recently predicted by similarity-based genome mining, which divided phylogenetically into different subfamilies (SFs). In this study, 27 putative and six characterized FAEs from both ascomycete and basidiomycete fungi were selected and heterologously expressed in Pichia pastoris and the recombinant proteins biochemically characterized to validate the previous genome mining and phylogenetical grouping and to expand the information on activity of fungal FAEs. As a result, 20 enzymes were shown to possess FAE activity, being active towards pNP-ferulate and/or methyl hydroxycinnamate substrates, and covering 11 subfamilies. Most of the new FAEs showed activities comparable to those of previously characterized fungal FAEs.

The Synthetic Potential of Fungal Feruloyl Esterases: A Correlation with Current Classification Systems and Predicted Structural Properties

“…Two out of three SF9 FAEs have been characterized as enzymes with dual activity (FAE, tannase) according to Dilokpimol et al [21]. They showed synthetic activity when glycerol was used as an acceptor, but not with the other alcohols.…”

“…Following, the FAE content (% w/w FAE/total protein) of each enzymatic sample was estimated by SDS-PAGE and subsequent quantification was done by a densitometric method using JustTLC software (Sweday, Lund, Sweden) as presented in Table 1. Enzymatic assays for the detection of hydrolytic activity were done as described by Dilokpimol et al [21].…”

“…Sequence comparison was done by pBLAST. Phylogenetic analysis was performed according to Dilokpimol et al [18,21]. Predicted structures of FAEs were built by YASARA Structure via homology modeling [40].…”

Characterization of a feruloyl esterase from Aspergillus terreus facilitates the division of fungal enzymes from Carbohydrate Esterase family 1 of the carbohydrate‐active enzymes (CAZy) database

“…Six characterized FAEs are present in both main branches of subfamily 5 of CE1 (Figs and S1). These include N. crassa FaeD (Crepin et al ., ), A. sydowii FaeC and FaeD1 (Dilokpimol et al ., ), A. nidulans FaeC (Debeire et al ., ) and A. niger FaeC (Dilokpimol et al ., ), as well as AtFaeD characterized in this study, thus supporting that CE1 subfamily 5 is also a FAE subfamily. In addition to the artificial FAE substrates, several of the functionally characterized subfamily 5 enzymes have also been shown to release ferulic acid from natural plant biomass substrates, such as wheat arabinoxylan (WAX) and SBP (Crepin et al ., ; Dilokpimol et al ., ), or hydrolyse feruloylated oligosaccharides (Debeire et al ., ).…”

“…Overall, the substrate profile of AtFaeD was highly comparable with another characterized member of CE1 subfamily 5, AnFaeC (Dilokpimol et al ., ). Although data on a smaller set of synthetic hydroxycinnamic acid substrates are available for another CE1 subfamily member, A. sydowii AsFaeC, this suggests that it possibly also has activity towards a broad substrate range, as it is active on methyl ferulate, methyl sinapate, methyl p ‐coumarate and methyl caffeate (Dilokpimol et al ., ).…”