The natural catalytic function of <i>CuG</i>E glucuronoyl esterase in hydrolysis of genuine lignin-carbohydrate complexes from birch

Abstract Background Glucuronoyl esterases belong to carbohydrate esterase family 15 and catalyze de-esterification. Their natural function is presumed to be cleavage of ester linkages in lignin–carbohydrate complexes particularly those linking lignin and glucuronoyl residues in xylans in hardwood. Results Here, we show for the first time a detailed product profile of aldouronic acids released from birchwood lignin by a glucuronoyl esterase from the white-rot fungus Cerrena unicolor (CuGE). CuGE releases substrate for GH10 endo-xylanase which results in significantly increased product release compared to the action of endo-xylanase alone. CuGE also releases neutral xylo-oligosaccharides that can be ascribed to the enzymes feruloyl esterase side activity as demonstrated by release of ferulic acid from insoluble wheat arabinoxylan. Conclusion The data verify the enzyme’s unique ability to catalyze removal of all glucuronoxylan associated with lignin and we propose that this is a direct result of enzymatic cleavage of the ester bonds connecting glucuronoxylan to lignin via 4-O-methyl glucuronoyl-ester linkages. This function appears important for the fungal organism’s ability to effectively utilize all available carbohydrates in lignocellulosic substrates. In bioprocess perspectives, this enzyme is a clear candidate for polishing lignin for residual carbohydrates to achieve pure, native lignin fractions after minimal pretreatment

A New Functional Classification of Glucuronoyl Esterases by Peptide Pattern Recognition

Glucuronoyl esterases are a novel type of enzymes believed to catalyze the hydrolysis of ester linkages between lignin and glucuronoxylan in lignocellulosic biomass, linkages known as lignin carbohydrate complexes. These complexes contribute to the recalcitrance of lignocellulose. Glucuronoyl esterases are a part of the microbial machinery for lignocellulose degradation and coupling their role to the occurrence of lignin carbohydrate complexes in biomass is a desired research goal. Glucuronoyl esterases have been assigned to CAZymes family 15 of carbohydrate esterases, but only few examples of characterized enzymes exist and the exact activity is still uncertain. Here peptide pattern recognition is used as a bioinformatic tool to identify and group new CE15 proteins that are likely to have glucuronoyl esterase activity. 1024 CE15-like sequences were drawn from GenBank and grouped into 24 groups. Phylogenetic analysis of these groups made it possible to pinpoint groups of putative fungal and bacterial glucuronoyl esterases and their sequence variation. Moreover, a number of groups included previously undescribed CE15-like sequences that are distinct from the glucuronoyl esterases and may possibly have different esterase activity. Hence, the CE15 family is likely to comprise other enzyme functions than glucuronoyl esterase alone. Gene annotation in a variety of fungal and bacterial microorganisms showed that coprophilic fungi are rich and diverse sources of CE15 proteins. Combined with the lifestyle and habitat of coprophilic fungi, they are predicted to be excellent candidates for finding new glucuronoyl esterase genes

Enzymatic cellulose oxidation is linked to lignin by long-range electron transfer

Enzymatic oxidation of cell wall polysaccharides by lytic polysaccharide monooxygenases (LPMOs) plays a pivotal role in the degradation of plant biomass. While experiments have shown that LPMOs are copper dependent enzymes requiring an electron donor, the mechanism and origin of the electron supply in biological systems are only partly understood. We show here that insoluble high molecular weight lignin functions as a reservoir of electrons facilitating LPMO activity. The electrons are donated to the enzyme by long-range electron transfer involving soluble low molecular weight lignins present in plant cell walls. Electron transfer was confirmed by electron paramagnetic resonance spectroscopy showing that LPMO activity on cellulose changes the level of unpaired electrons in the lignin. The discovery of a long-range electron transfer mechanism links the biodegradation of cellulose and lignin and sheds new light on how oxidative enzymes present in plant degraders may act in concert.info:eu-repo/semantics/publishe

Transesterification with CE15 glucuronoyl esterase from <i>Cerrena unicolor</i> reveals substrate preferences

Purpose Glucuronoyl esterases (GE, family CE15) catalyse the cleavage of ester linkages in lignin-carbohydrate complexes (LCCs), and this study demonstrate how transesterification reactions with a fungal GE from Cerrena unicolor (CuGE) can reveal the enzyme’s preference for the alcohol-part of the ester-bond.Methods This alcohol-preference relates to where the ester-LCCs are located on the lignin molecule, and has consequences for how the enzymes potentially interact with lignin. It is unknown exactly what the enzymes prefer; either the α-benzyl or the γ-benzyl position. By providing the enzyme with a donor substrate (the methyl ester of either glucuronate or 4-O-methyl-glucuronate) and either one of two acceptor molecules (benzyl alcohol or 3-phenyl-1-propanol) we demonstrate that the enzyme can perform transesterification and it serves as a method for assessing the enzyme’s alcohol preferences.Conclusion CuGE preferentially forms the γ-ester from the methyl ester of 4-O-methyl-glucuronate and 3-phenyl-1-propanol and the enzyme’s substrate preferences are primarily dictated by the presence of the 4-O-methylation on the glucuronoyl donor, and secondly on the type of alcohol

Investigating the role of expansins in lignin extraction from plant biomass

Lignin from lignocellulose has remarkable properties to exploit in future applications like batteries, carbon fibers, plastics, and even pharmaceuticals [1]. However, the utilization of lignocellulose is challenging to exploit due to high recalcitrance and current conversion strategies are destructive, which significantly reduces the quality, consistency and therefore potential use of the aromatic polymer. I want to investigate how extraction of lignin can be improved by the use of enzymes and non-catalytic proteins. I hypothesize that microbial expansin-like proteins can disrupt non-covalent interactions within the lignocellulosic matrix and thereby aid the fractionation of high-quality lignin. Expansins are originally found in plant cells where the function is to loosen the compact structure of the wall to make cell elongation possible. Microbial expansins are found in plant associated and plant degrading bacteria and fungi. Since these organisms do not have cellulosic cell walls the expansins are considered related to the plant-microbe interactions. The expansin structure consists of two domains: a double-psi β-barrel (DPBB) and a carbo-hydrate-binding module family 63 (CBM63). The DPBB is structural similar to glycoside hydrolase family 45 (GH45) but without catalytic activity. I will in my project investigate the physical interaction between soluble microbial expansins and insoluble substrates from biomass to achieve milder and more specific processes for high value lignin extraction. I will assess the binding affinity of expansins with pull-down assays and QCM-D and study synergistic effects of microbial expansins and lignocellulose active enzymes