Cleavage of Lignin-Carbohydrate Complexes (LCCs) with Glucuronoyl Esterases for Enzymatic Lignin Biorefining

A transition towards new sustainable alternatives to fossil based materials is crucial to facilitate a future competitive bioeconomy. This had led to a growing interest for utilization of lignocellulosic waste material accumulating from numerous sources such as the agricultural or forestry industry.

Lignocellulose is an abundant feedstock composed of primarily three polymeric structures; lignin, hemicellulose and cellulose. Woods and plants have evolved to resist microbial degradation, and their cell wall components are arranged in a highly complex recalcitrant matrix creating challenges in industrial isolation and utilization of the polymers.

Biorefinery processes have primarily been focused on recovery and conversion of carbohydrates whereas lignin has mainly been considered as a by-product. Lignin, however, is a potential high value product since it represents a major source of aromatic compounds that have several applications, such as materials and platform chemicals.

Yet valorization of lignin requires recovery of pure lignin in as native and intact a form as possible, and highly specific enzymes are required to accomplish this recovery. Glucuronoyl esterases (GEs, CAZy CE15) are proposed to enable targeted cleavage of lignin-carbohydrate complexes (LCCs) specifically ester LCCs formed between 4-O-methyl-glucuronyl substitutions on glucuronoxylan and lignin alcohols and they are presumed to be apparent in biomasses rich in glucuronoxylan such as hardwood.

A limitation to the understanding of the function of GEs is the lack of natural substrates. In this study, a natural substrate containing an enriched fraction of LCCs from birch wood suitable for CE15 characterization was prepared. This substrate enabled characterization of several fungal GEs, including one from Cerrena unicolor (CuGE).

Expected reaction products were detected after hydrolysis of the LCC substrate with CuGE and significant increase of product release was observed with a combination of CuGE and a GH10 endo-xylanase compared to the action of GH10 alone. This demonstrates the enzyme’s unique ability to remove all glucuronoxylan associated with lignin via ester linkages.

This thesis presents the first ever example in literature of identified reaction products by CE15s on real biomass and is a great contribution to the understanding of the biological function of GEs. In bioprocess perspectives, this enzyme would be a clear candidate to achieve a pure lignin fraction after minimal pretreatment of the biomass.

In addition, structural complexes of CuGE and several different glucuronoyl xylo-oligosaccharides were solved. This is the first enzyme complex with a substrate mimicking ligand larger than a monosaccharide, and it provides significant insight into the mechanism of how CE15s recognize of LCCs.