Enzymes for selective decoupling of woody biomass: From fundamentals to industrial potential

The need for an economy based on renewable materials has resulted in growing interest in the use of woody biomass in a wider field of application. However, the chemical complexity of lignocellulose and the dense structure of wood pose challenges in its processing. The aim in a materials biorefinery is to extract the individual wood-components in as native and intact form as possible; and highly specific enzymes could be used for this. In this work, lignocellulosic enzymes with the potential of selectively decoupling and decomposing wood polymers were investigated and characterised. \ua0One of the studies was devoted to assessing enzyme accessibility in wood structures and evaluating the importance of pre-treatment to open up the dense wood structure, in order to improve enzyme performance. It was shown that the physical contribution during steam explosion is crucial for enhanced enzymatic hydrolysis of wood. Moreover, the performance of two endo-mannanases produced by the bacterium Cellvibrio japonicus(CjMan5A and CjMan26A) were compared on mannan polymers with dissimilar backbone structures and decorations, including the industrially relevant spruce galactoglucomannan. The enzymes were shown to be differently affected by the backbone heterogeneity and the presence of side groups on the substrates, demonstrating the variation in substrate preferences among mannanases. It was further shown that chemical acetylation of mannans reduced substrate hydrolysis significantly. Acetylation was therefore suggested as a tool to limit the biodegradation of mannan-based material. \ua0The major part of the work described in this thesis was dedicated to investigating the role and function of glucuronoyl esterases (GEs), which are enzymes that hydrolyse the ester linkages between lignin and hemicelluloses that contribute to the recalcitrance of woody biomass. Extensive structure-function studies of bacterial GE candidates contributed to our understanding of the diversity of this relatively unexplored enzyme family. Both similarities and differences in substrate preferences among the GEs studied revealed enzymes more promiscuous than their characterised fungal counterparts. GE activity was further assayed on lignin-carbohydrate complexes isolated from woody biomass, and GE-mediated ester cleavage was demonstrated with advanced and complementary tools, including size-exclusion chromatography, 31P NMR and 2D NMR. These findings not only confirm the suggested biological role of GEs, but also demonstrate the potential of these enzymes in decoupling lignin from hemicelluloses in industrial settings

Structure-function analysis of two closely related cutinases from Thermobifida cellulosilytica

Cutinases can play a significant role in a biotechnology-based circular economy. However, relatively little is known about the structure–function relationship of these enzymes, knowledge that is vital to advance optimized, engineered enzyme candidates. Here, two almost identical cutinases from Thermobifida cellulosilytica DSM44535 (Thc_Cut1 and Thc_Cut2) with only 18 amino acids difference were used for a rigorous biochemical characterization of their ability to hydrolyze poly(ethylene terephthalate) (PET), PET-model substrates, and cutin-model substrates. Kinetic parameters were compared with detailed in silico docking studies of enzyme-ligand interactions. The two enzymes interacted with, and hydrolyzed PET differently, with Thc_Cut1 generating smaller PET-degradation products. Thc_Cut1 also showed higher catalytic efficiency on long-chain aliphatic substrates, an effect likely caused by small changes in the binding architecture. Thc_Cut2, in contrast, showed improved binding and catalytic efficiency when approaching the glass transition temperature of PET, an effect likely caused by longer amino acid residues in one area at the enzyme\u27s surface. Finally, the position of the single residue Q93 close to the active site, rotated out in Thc_Cut2, influenced the ligand position of a trimeric PET-model substrate. In conclusion, we illustrate that even minor sequence differences in cutinases can affect their substrate binding, substrate specificity, and catalytic efficiency drastically

Biochemical and structural features of diverse bacterial glucuronoyl esterases facilitating recalcitrant biomass conversion

BackgroundLignocellulose is highly recalcitrant to enzymatic deconstruction, where the recalcitrance primarily results from chemical linkages between lignin and carbohydrates. Glucuronoyl esterases (GEs) from carbohydrate esterase family 15 (CE15) have been suggested to play key roles in reducing lignocellulose recalcitrance by cleaving covalent ester bonds found between lignin and glucuronoxylan. However, only a limited number of GEs have been biochemically characterized and structurally determined to date, limiting our understanding of these enzymes and their potential exploration.ResultsTen CE15 enzymes from three bacterial species, sharing as little as 20% sequence identity, were characterized on a range of model substrates; two protein structures were solved, and insights into their regulation and biological roles were gained through gene expression analysis and enzymatic assays on complex biomass. Several enzymes with higher catalytic efficiencies on a wider range of model substrates than previously characterized fungal GEs were identified. Similarities and differences regarding substrate specificity between the investigated GEs were observed and putatively linked to their positioning in the CE15 phylogenetic tree. The bacterial GEs were able to utilize substrates lacking 4-OH methyl substitutions, known to be important for fungal enzymes. In addition, certain bacterial GEs were able to efficiently cleave esters of galacturonate, a functionality not previously described within the family. The two solved structures revealed similar overall folds to known structures, but also indicated active site regions allowing for more promiscuous substrate specificities. The gene expression analysis demonstrated that bacterial GE-encoding genes were differentially expressed as response to different carbon sources. Further, improved enzymatic saccharification of milled corn cob by a commercial lignocellulolytic enzyme cocktail when supplemented with GEs showcased their synergistic potential with other enzyme types on native biomass.ConclusionsBacterial GEs exhibit much larger diversity than fungal counterparts. In this study, we significantly expanded the existing knowledge on CE15 with the in-depth characterization of ten bacterial GEs broadly spanning the phylogenetic tree, and also presented two novel enzyme structures. Variations in transcriptional responses of CE15-encoding genes under different growth conditions suggest nonredundant functions for enzymes found in species with multiple CE15 genes and further illuminate the importance of GEs in native lignin–carbohydrate disassembly

Contribution of Structural Modification to Enhanced Enzymatic Hydrolysis and 3-D Structural Analysis of Steam-Exploded Wood using X-Ray Tomography

Steam explosion pretreatment modifies both the chemical and physical structures of a biomass. Chemical modifications are generated during the treatment of biomass with steam at high temperature. Physical modifications are created during the explosion step, which produces disintegrated and defibrillated biomass. In this study, the contribution of each modification to an increase in enzymatic hydrolysis has been studied. It was found that both physical and chemical modifications contributed to an increase in enzymatic hydrolysability. Additionally, high resolution X-ray tomography was performed to identify the structural modification created during the steam explosion process. Comparison of the 3-D microstructure of a steam-exploded wood sample with an untreated wood sample revealed that several kinds of cracks were created during the explosion step, and the micro-structure of the wood sample was vigorously destroyed

A glucuronoyl esterase from Acremonium alcalophilum cleaves native lignin-carbohydrate ester bonds

The Glucuronoyl esterases (GE) have been proposed to target lignin-carbohydrate (LC) ester bonds between lignin moieties and glucuronic acid side groups of xylan, but to date, no direct observations of enzymatic cleavage on native LC ester bonds have been demonstrated. In the present investigation, LCC fractions from spruce and birch were treated with a recombinantly produced GE originating from Acremonium alcalophilum (AaGE1). A combination of size exclusion chromatography and 31P NMR analyses of phosphitylated LCC samples, before and after AaGE1 treatment provided the first evidence for cleavage of the LC ester linkages existing in wood

Understanding enzyme-substrate interactions in Carbohydrate Esterase Family 15

Carbohydrate Esterase Family 15 (CE15) is a rather small family, comprising approximately 200 members, which was established in CAZy (www.cazy.org) in 2006. The family was created following the characterization of a glucuronoyl esterase (GE) from the fungus Schizophyllum commune [1], which was shown to cleave methyl moieties ester-linked to the O6 position of glucuronic acid. CE15 enzymes are proposed to cleave ester linkages between lignin and glucuronoxylan, so-called lignin-carbohydrate complexes (LCCs), which are important features in biomass recalcitrance. We recently characterized ten new GEs from three bacterial species and solved the structures of two of these, essentially doubling both the biochemical and structural data available for the family [2]. An in-depth understanding of how CE15 enzymes interact with their complex substrates is still lacking, as only one structure with a monosaccharide ligand has been solved to date [3]. To address this, we have pursued solving new GE structures and obtaining protein-ligand complex structures. The studies have resulted in a novel structure exhibiting features with prominent inserts surrounding the active site, suggesting different specificities between bacterial and fungal GEs. In addition, we have solved the first structures of a CE15 enzyme with larger ligands, which gives direct evidence of how these enzymes interact with the different parts of its proposed physiological LCC substrates. Combined with kinetic characterizations, these new investigations greatly add to the knowledge of enzyme-substrate interactions in CE15 and enhances how these enzymes may act in natural conditions, which could aid in industrial biomass conversion. ______________[1]\ua0 Španikov\ue1 S.; Biely P. FEBS Lett. 2006, 580, 4597-4601.[2]\ua0 Arnling B\ue5\ue5th J.; Mazurkewich S.; Knudsen R.M.; Poulsen J.N.; Olsson L.; Lo Leggio L.; Larsbrink J. Biotechnol Biofuels. 2018, 11, 213-226.[3]\ua0 Charavgi M.D.; Dimarogona M.; Topakas E.; Christakopoulos P.; Chrysina E.D. Acta Crystallogr. D Biol. Crystallogr. 2013, 69, 63-73

Biochemical and structural investigation of the CE15 family: Glucuronoyl esterases acting on recalcitrant biomass

Glucuronoyl esterases (GEs) are a relatively new class of enzymes which cleave ester linkages between lignin and glucuronoxylan. GEs have been identified in many biomass-degrading microbes and are now classified into the Carbohydrate Esterase Family 15 (CE15). The CE15 family is diverse (as low as 20% sequence identity), however to-date only a few GEs from a small clade of the CE15 phylogenetic tree have been biochemically characterized and only two protein structures have been solved. To investigate the diversity of CE15 members, we have studied a broad range of proteins from across the phylogenetic tree. Enzymes were biochemically characterized and three-dimensional structures for two of the enzymes were solved. Analysis of the structures suggest possible binding sites for lignin fragments and xylooligosaccharides that have not been previously reported. Investigations into the molecular determinants supporting the potential binding sites is being pursued

ACTIVE SITE EVOLUTION IN BIOMASS DEGRADING ENZYMES

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