Fungal Feruloyl Esterases Research Articles

Synergistic enzyme assisted release of ferulic acid from brewer´s spent grain

Brewer’s spent grain (BSG), the most abundant by-product of the beer industry, can be considered a sustainable source of ferulic acid (4-hydroxy-3-methoxycinnamic acid) (FA). FA is esterified to the arabinoxylan in the BSG. FA exhibits a variety of biological activities and is a target for valorization of BSG. We present a novel enzyme-assisted approach that utilizes the synergistic action of a specific bacterial endo-1,4-β-xylanase and a fungal ferulic acid esterase activity to selectively release high yields of FA from BSG. The study compares the efficacy of three different fungal feruloyl esterases (E.C. 3.1.1.73) of carbohydrate esterase family 1 (CE1), HiFae, AoFae1, and AnFae1, acting separately or in combination with two different endo-1,4-β-xylanases (EC 3.2.1.8), a bacterial GH30 enzyme from Dickeya chrysanthemi, and Shearzyme®, a commercial GH10 enzyme from Aspergillus aculeatus, respectively. On an equimolar dosage basis, the AnFae1-GH30 was the most efficient combination in releasing FA from BSG. This combination showed significant synergy in a dose-dependent manner, enabling up to 85% FA recovery from BSG within 30 min. of treatment. The results demonstrate a novel enzymatic approach for valorization of BSG as FA is considered a high-value natural compound for use in e.g. anti-aging skin care products.

Fungal feruloyl esterases can catalyze release of diferulic acids from complex arabinoxylan

Feruloyl esterases (FAEs, EC 3.1.1.73) catalyze the hydrolytic cleavage of ester bonds between feruloyl and arabinosyl moieties in arabinoxylans. Recently, we discovered that two bacterial FAEs could catalyze release of diferulic acids (diFAs) from highly substituted, cross-linked corn bran arabinoxylan. Here, we show that several fungal FAEs, notably AnFae1 (Aspergillus niger), AoFae1 (A. oryzae), and MgFae1 (Magnaporthe oryzae (also known as M. grisae)) also catalyze liberation of diFAs from complex arabinoxylan. By comparing the enzyme kinetics of diFA release to feruloyl esterase activity of the enzymes on methyl- and arabinosyl-ferulate substrates we demonstrate that the diFA release activity cannot be predicted from the activity of the enzymes on these synthetic substrates. A detailed structure-function analysis, based on AlphaFold2 modeled enzyme structures and docking with the relevant di-feruloyl ligands, reveal how distinct differences in the active site topology and surroundings may explain the diFA releasing action of the enzymes. Interestingly, the analysis also unveils that the carbohydrate binding module of the MgFae1 may play a key role in the diFA releasing ability of this enzyme. The findings contribute further understanding of the function of FAEs in the deconstruction of complex arabinoxylans and provide new opportunities for enzyme assisted upgrading of complex bran arabinoxylans.

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

Twenty-eight fungal feruloyl esterases (FAEs) were evaluated for their synthetic abilities in a ternary system of n-hexane: t-butanol: 100 mM MOPS-NaOH pH 6.0 forming detergentless microemulsions. Five main derivatives were synthesized, namely prenyl ferulate, prenyl caffeate, butyl ferulate, glyceryl ferulate, and l-arabinose ferulate, offering, in general, higher yields when more hydrophilic alcohol substitutions were used. Acetyl xylan esterase-related FAEs belonging to phylogenetic subfamilies (SF) 5 and 6 showed increased synthetic yields among tested enzymes. In particular, it was shown that FAEs belonging to SF6 generally transesterified aliphatic alcohols more efficiently while SF5 members preferred bulkier l-arabinose. Predicted surface properties and structural characteristics were correlated with the synthetic potential of selected tannase-related, acetyl-xylan-related, and lipase-related FAEs (SF1-2, -6, -7 members) based on homology modeling and small molecular docking simulations.

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

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.

Occurrence, properties, and applications of feruloyl esterases

Feruloyl esterases hydrolyze the ester linkages of ferulic and diferulic acids present in plant cell walls. This interesting group of enzymes also has a potentially broad range of applications in the pharmaceutical and agri-food industries. An overview of the current knowledge of fungal feruloyl esterases focusing on the diverse of substrate specificity and potential applications is presented in this review. Furthermore, biological functions of ferulic acid are discussed.

Biotechnological applications and potential of fungal feruloyl esterases based on prevalence, classification and biochemical diversity

Feruloyl esterases are part of the enzymatic spectrum employed by fungi and other microorganisms to degrade plant polysaccharides. They release ferulic acid and other aromatic acids from these polymeric structures and have received an increasing interest in industrial applications such as in the food, pulp and paper and bio-fuel industries. This review provides an overview of the current knowledge on fungal feruloyl esterases focussing in particular on the differences in substrate specificity, regulation of their production, prevalence of these enzymes in fungal genomes and industrial applications.

Microencapsulated bacterial cells can be used to produce the enzyme feruloyl esterase: preparation and in-vitro analysis

Biotechnological production of ferulic acid, a precursor of vanillin, is an attractive alternative for various industries due to the high price and demand for natural ferulic acid. Feruloyl esterase has been identified as a key enzyme involved in microbial transformations of ferulic acid to vanillin. Several fungal feruloyl esterases have been purified and characterized for their use in the production of ferulic acid. This paper, for the first time, discusses the use of lactic acid bacteria for the production of ferulic acid. Specifically, we have used Lactobacillus cells and microencapsulation so that ferulic acid can be produced continuously using various types of fermentation systems. Bacteria were encapsulated in alginate-poly-L-lysine-alginate (APA) microcapsules, and the production of ferulic acid by lactobacilli was detected using a real-time high-performance liquid chromatography (HPLC)-based assay. Results show that ferulic acid can be produced using microencapsulated Lactobacillus fermentum (ATCC 11976) with significant levels of biological feruloyl esterase activity.

Mono- and dimeric ferulic acid release from brewer's spent grain by fungal feruloyl esterases

Ultraflo L, a beta-glucanase preparation from Humicola insolens sold for reducing viscosity problems in the brewing industry, exhibited activity against the methyl esters of ferulic, caffeic, p-coumaric and sinapic acids, displaying mainly type-B feruloyl esterase activity. Ultraflo also contained the ability to release 65% of the available ferulic acid (FA) together with three forms of diferulate from brewer's spent grain (BSG). An "esterase-free" Ultraflo preparation greatly enhanced the ability of a feruloyl esterase from Aspergillus niger, AnFAEA, to release FA (from 23 to 47%) and its dimeric forms, especially the 8,5' benzofuran form, from BSG. While total release of these phenolic acids was not observed, this synergistic enhancement of ferulate release demonstrates that FA and its dimeric forms present in BSG require the addition of more than a xylanase. This suggests either that FA is not solely attached to arabinoxylan in the barley cell wall, or that the cell wall polysaccharides in BSG hinder the accessibility of enzymes to the ferulates, due to processing treatments.

Purification and characterization of an extracellular feruloyl esterase from the thermophilic anaerobe Clostridium stercorarium.

Feruloyl esterases act as accessory enzymes for the complete saccharification of plant cell wall hemicelluloses. Although many fungal feruloyl esterases have been purified and characterized, few bacterial phenolic acid esterases have been characterized. This study shows the extracellular production of a feruloyl esterase by the thermophilic anaerobe Clostridium stercorarium when grown on birchwood xylan. The feruloyl esterase was purified 500-fold in successive steps involving ultrafiltration, preparative isoelectric focusing and column chromatography by anion exchange, gel filtration and hydrophobic interaction. The purified enzyme released ferulic, rho-coumaric, caffeic and sinapinic acid from the respective methyl esters. The purified enzyme also released ferulic acid from a de-starched wheat bran preparation. At pH 8.0 and 65 degrees C, the Km and Vmax values for the hydrolysis of methyl ferulate were 0.04 mmol l-l and 131 micromol min-1 mg-1, respectively; the respective values for methyl coumarate were 0.86 mmol l-l and 18 micromol min-1 mg-1. The purified feruloyl esterase had an apparent mass of 33 kDa under denaturing conditions and showed optimum activity at pH 8.0 and 65 degrees C. At a concentration of 5 mmol l-l, the ions Ca2+, Cu2+, Co2+ and Mn2+ reduced the activity by 70-80%.