Abstract
Cereal feedstocks have high arabinoxylan content as their main hemicellulose, which is linked to lignin by hydroxycinnamic acids such as ferulic acid. The ferulic acid is linked to arabinoxylan by ester bonds, and generally, the high substitution of ferulic acid leads to a loss of activity of xylanases targeting the arabinoxylan. In the current study, a feruloyl esterase (FAE-1) from a termite hindgut bacteria was functionally characterised and used in synergy with xylanases during xylan hydrolysis. The FAE-1 displayed temperature and pH optima of 60 ℃ and 7.0, respectively. FAE-1 did not release reducing sugars from beechwood xylan (BWX), wheat arabinoxylan (WAX) and oat spelt xylan (OX), however, displayed high activity of 164.74 U/mg protein on p-nitrophenyl-acetate (pNPA). In contrast, the GH10 xylanases; Xyn10 and XT6, and a GH11 xylanase, Xyn2A, showed more than two-fold increased activity on xylan substrates with low sidechain substitutions; BWX and OX, compared to the highly branched substrate, WAX. Interestingly, the FAE-1 and GH10 xylanases (Xyn10D and XT6) displayed a degree of synergy (DS) that was higher than 1 in all enzyme loading combinations during WAX hydrolysis. The 75%XT6:25%FAE-1 synergistic enzyme combination increased the release of reducing sugars by 1.34-fold from WAX compared to the control, while 25%Xyn10D:75%FAE-1 synergistic combination released about 2.1-fold of reducing sugars from WAX compared to controls. These findings suggest that FAE-1 can be used in concert with xylanases, particularly those from GH10, to efficiently degrade arabinoxylans contained in cereal feedstocks for various industrial settings such as in animal feeds and baking.
Highlights
IntroductionXylan consists of a linear backbone chain of β-(1,4)-linked xylose units substituted with acetyl, D-glucuronic acid (GlcA) and/or 4-O-methyl-D-glucuronic acid (Me-GlcA), and L-arabinose residues
Ferulic acid cross-linking of xylan to lignin restricts Carbohydrate-Active Enzymes (CAZymes) such as xylanases and cellulases from degrading structural polysaccharides efficiently and, as a result, limits the utilisation of agricultural crop residues as (1) feeds for animals by shielding the non-starch polysaccharide from the xylanolytic hydrolytic activity; (2) it limits feedstocks uses for biofuel by hindering the biomass access to holo-cellulolytic enzymes; and (3) affects the production of value-added chemical products suchIn general, xylan consists of a linear backbone chain of β-(1,4)-linked xylose units substituted with acetyl, D-glucuronic acid (GlcA) and/or 4-O-methyl-D-glucuronic acid (Me-GlcA), and L-arabinose residues
Specific activity and characterization feruloyl esterases (FAE)‐1 specific activity and biochemical properties The focus of the current study was to (1) demonstrate that the FAE-1 derived from a termite hindgut bacteria is active; (2) biochemically characterise the FAE-1; and (3) formulate binary FAE-1 to xylanase synergistic enzyme mixtures to improve the release of reducing sugars during xylan hydrolysis
Summary
Xylan consists of a linear backbone chain of β-(1,4)-linked xylose units substituted with acetyl, D-glucuronic acid (GlcA) and/or 4-O-methyl-D-glucuronic acid (Me-GlcA), and L-arabinose residues. Agricultural feedstock sources such as maize, wheat, oat, rice and barley consist of arabinoxylans (AX) and glucuronoarabinoxylans (GAX) that are linked to. AX or GAX are linked to ferulic acid, ferulic acid dimers, or coumaric acid via ester bonds which are formed at the fifth carbon hydroxy group (C(O)5-hydroxy group) of α-L-arabinosyl residues (Grabber et al 2000; Faulds et al 2003 and Mnich et al 2020). We propose that it is important to establish how the synergy between different xylanases and feruloyl esterases (FAE) may improve the degradation of these xylans contained in agricultural feedstocks
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