Enzymatic degradation of lignin‐carbohydrate complexes (LCCs): Model studies using a fungal glucuronoyl esterase from Cerrena unicolor

Abstract

Lignin-carbohydrate complexes (LCCs) are believed to influence the recalcitrance of lignocellulosic plant material preventing optimal utilization of biomass in e.g. forestry, feed and biofuel applications. The recently emerged carbohydrate esterase (CE) 15 family of glucuronoyl esterases (GEs) has been proposed to degrade ester LCC bonds between glucuronic acids in xylans and lignin alcohols thereby potentially improving delignification of lignocellulosic biomass when applied in conjunction with other cellulases, hemicellulases and oxidoreductases. Herein, we report the synthesis of four new GE model substrates comprising α- and ɣ-arylalkyl esters representative of the lignin part of naturally occurring ester LCCs as well as the cloning and purification of a novel GE from Cerrena unicolor (CuGE). Together with a known GE from Schizophyllum commune (ScGE), CuGE was biochemically characterized by means of Michaelis-Menten kinetics with respect to substrate specificity using the synthesized compounds. For both enzymes, a strong preference for 4-O-methyl glucuronoyl esters rather than unsubstituted glucuronoyl esters was observed. Moreover, we found that α-arylalkyl esters of methyl α-D-glucuronic acid are more easily cleaved by GEs than their corresponding ɣ-arylalkyl esters. Furthermore, our results suggest a preference of CuGE for glucuronoyl esters of bulky alcohols supporting the suggested biological action of GEs on LCCs. The synthesis of relevant GE model substrates presented here may provide a valuable tool for the screening, selection and development of industrially relevant GEs for delignification of biomass.

Characterisation of three fungal glucuronoyl esterases on glucuronic acid ester model compounds

“…7b). This observation supports the conclusion that GEs might have a preference for glucuronic acids esterified to bulkier side groups (D’Errico et al 2015; D’Errico et al 2016), which is enabled by the fact that the active site of GEs is located at the surface and thus accessible to larger substrates (Pokkuluri et al 2011). Nevertheless, the activity of WcGE1 towards allylGlcA reached 84% of its activity on BnGlcA, which was by far the highest among the tested enzymes.…”

“…Most studies of GEs to date have been conducted using synthetic substrates obtained through organic synthesis (Biely et al 2015; D’Errico et al 2015; Ďuranová et al 2009a; Katsimpouras et al 2014; Li et al 2007; Špániková et al 2007). The production of these compounds is laborious and requires expertise in organic chemistry, and the compounds often do not allow for high-throughput screening due to their requirement for HPLC or NMR analyses.…”

“…GEs have been characterised on a variety of synthetic substrates that mimic the structure of lignin-carbohydrate complexes (LCCs) (Biely et al 2015; D’Errico et al 2015, 2016; Nylander et al 2015; Ďuranová et al 2009a, b; Katsimpouras et al 2014; Li et al 2007). Variations of the overall structure of these substrates (Fig.…”

Microbial Glucuronoyl Esterases: 10 Years after Discovery

“…Many synthetic substrates, such as alkyl and alkyl aryl alcohol esters of MeGlcA and GlcA or their glycosides used earlier (5,12,21,(33)(34)(35), are not commercially available. Natural sub- strates derived from plant cell wall material, such as suitable lignin-carbohydrate complexes, await their introduction.…”

“…The substrates enabled us to examine for the first time the presence of GE in commercial cellulolytic and hemicellulolytic preparations (38). However, as in the similar case of benzyl glucuronate, the lack of the 4-O-methyl group in GlcA decreases the affinity of the enzymes for the substrates (5,12,(33)(34)(35). It is obvious that methyl esters of analogous glycosides of MeGlcA could serve as ideal substrates.…”