The fungal members of Carbohydrate Esterase family 1 (CE1) from the CAZy database include both acetyl xylan esterases (AXEs) and feruloyl esterases (FAEs). AXEs and FAEs are essential auxiliary enzymes to unlock the full potential of feedstock. They are being used in many biotechnology applications including food and feed, pulp and paper, and biomass valorization. AXEs catalyze the hydrolysis of acetyl group from xylan, while FAEs release ferulic and other hydroxycinnamic acids from xylan and pectin. Previously, we reported a phylogenetic analysis for the fungal members of CE1, establishing five subfamilies (CE1_SF1-SF5). Currently, the characterized AXEs are in the subfamily CE1_SF1, whereas CE1_SF2 contains mainly characterized FAEs. These two subfamilies are more related to each other than to the other subfamilies and are predicted to have evolved from a common ancestor, but target substrates with a different molecular structure. In this study, four ascomycete enzymes from CE1_SF1 and SF2 were heterologously produced in Pichia pastoris and characterized with respect to their biochemical properties and substrate preference toward different model and plant biomass substrates. The selected enzymes from CE1_SF1 only exhibited AXE activity, whereas the one from CE1_SF2 possessed dual FAE/AXE activity. This dual activity enzyme also showed broad substrate specificity toward model substrates for FAE activity and efficiently released both acetic acid and ferulic acid (∼50%) from wheat arabinoxylan and wheat bran which was pre-treated with a commercial xylanase. These fungal AXEs and FAEs also showed promising biochemical properties, e.g., high stability over a wide pH range and retaining more than 80% of their residual activity at pH 6.0-9.0. These newly characterized fungal AXEs and FAEs from CE1 have high potential for biotechnological applications. In particular as an additional ingredient for enzyme cocktails to remove the ester-linked decorations which enables access for the backbone degrading enzymes. Among these novel enzymes, the dual FAE/AXE activity enzyme also supports the evolutionary relationship of CE1_SF1 and SF2.
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Feruloyl esterases (FAEs) and acetyl xylan esterases (AXEs) are important enzymes for plant biomass degradation and are both present in Carbohydrate Esterase family 1 (CE1) of the Carbohydrate‐Active enZymes database. In this study, ten novel fungal CE1 enzymes from different subfamilies were heterologously produced and screened for their activity towards model and complex plant biomass substrates. CE1_1 enzymes possess AXE activity, while CE1_5 enzymes showed FAE activity. Two enzymes from CE1_2 and one from CE1_5 possess dual feruloyl/acetyl xylan esterase (FXE) activity, showing expansion of substrate specificity. The new FXEs from CE1 can efficiently release both feruloyl and acetyl residues from feruloylated xylan, making them particularly interesting novel components of industrial enzyme cocktails for plant biomass degradation.
The synthesis of two glucuronoxylans is described, which both consist of a pentaxylan backbone and a glucuronic acid linked to the 2 position in the fourth xylose residue from the reducing end. The two target molecules differ in the 4 position of the glucuronic acid where one is unsubstituted while the other contains a methyl ether. The pentaxylan backbone is assembled in four glycosylation reactions with phenyl thioglycoside donors. The couplings are performed by preactivation of [a] Dr.
Background Feruloyl esterases (FAEs) and acetyl xylan esterases (AXEs) are important accessory enzymes in the deconstruction of plant biomass. Carbohydrate Esterase family 1 (CE1) of the Carbohydrate-Active enZymes database contains both fungal FAEs and AXEs, sharing a high amino acid sequence similarity, even though they target different structural molecules on plant cell wall polysaccharides. Results We recently classified fungal CE1 into five subfamilies (CE1_SF1-5). In this study, ten novel fungal CE1 enzymes from different subfamilies were heterologously produced in Aspergillus niger and characterized to gain insight on relationships among these esterases. The enzymes from CE1_SF1 possess AXE activity, as they hydrolyzed p NP-acetate and released acetic acid from wheat arabinoxylan, but were not active towards FAE substrates. CE1_SF5 showed FAE activity as they hydrolyzed methyl ferulate and other FAE related substrates, and release ferulic acid from wheat arabinoxylan. These FAEs preferred feruloylated arabinoxylan over pectin. Two CE1_SF2, sharing over 70% amino acid sequence identity, possessed the opposite activity. Interestingly, one enzyme from CE1_SF1 and one from CE1_SF5 possess dual feruloyl/acetyl xylan esterase (FXE) activity. These dual activity enzymes showed expansion of substrate specificity. Conclusions The new FXEs from CE1 can efficiently release both ferulic acid and acetic acid from feruloylated xylan, making them particularly interesting novel components of industrial enzyme cocktails for plant biomass degradation.
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