Functional Validation of Two Fungal Subfamilies in Carbohydrate Esterase Family 1 by Biochemical Characterization of Esterases From Uncharacterized Branches

Abstract

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.

Fungal Treatment for the Valorization of Technical Soda Lignin

“…The protein sequences of the detected proteins were shared between 82 and 90% identity between each other. According to the CAZy classification system, the CE1 family contain most of the characterized fungal acetyl xylan esterases, feruloyl esterases and p -coumaroyl esterases [ 53 , 71 ]. These enzymes are involved in breaking down ester cross-links of lignin and hemicelluloses and in lignin solubilization [ 72 , 73 ].…”

Fungal glycoside hydrolase family 44 xyloglucanases are restricted to the phylum Basidiomycota and show a distinct xyloglucan cleavage pattern

“…The plasmids pPicZαA- xegA Ds and pPicZαA- xegA Po were extracted and linearized with PmeI (New England Biolabs, Ipswich, MA) and subsequently transformed into P. pastoris strain X-33 by electroporation. The positive colonies were selected by colony Western Blot using 6x-His Tag Monoclonal Antibody conjugated to alkaline phosphatase (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA) and detected with a chromogenic substrate 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) ( Li et al., 2020 ).…”

Bioconversion of Lignocellulosic Biomass into Value Added Products under Anaerobic Conditions: Insight into Proteomic Studies

“…Cellulose, one of the major components of lignocellulose, is a homopolysaccharide with amorphous and crystalline regions [44] made of glucose monomers linked by β-1,4-Dglucan. Hemicellulose however is a heterogenous polysaccharide conformed by a xylan backbone that contains xylose, arabinose, mannose, glucose, galactose and sugar acids in different proportions depending on the source [43,45].…”

“…Hemicellulolytic carbohydrate esterases remove the ester group from carbohydrates and facilitates the access to GHs [64]. Esterases are classified into 18 families according to the CAZy database (www.CAZy.org; accessed on 23 September 2021), these include feruloyl esterases (EC 3.1.1.73) from the CE1 family which catalyze the cleavage of the ester bond at the O-5 position between a ferulic acid and arabinose liberating hydroxycinnamic acids; as well as acetyl xylan esterases (EC 3.1.1.72), which in turn catalyzes the cleavage of ester linkages on the position O-2 and O-3 between an acetyl group and xylose [45,65]. Various studies reported higher expression of hemicellulases than cellulases, as hemicellulose is more exposed than cellulose [52,53].…”