Agger JW

References (9)

Title : Efficient activity screening of new glucuronoyl esterases using a pNP-based assay - Madsen_2024_Enzyme.Microb.Technol_178_110444
Author(s) : Madsen MS , Martins PA , Agger JW
Ref : Enzyme Microb Technol , 178 :110444 , 2024
Abstract : Glucuronoyl esterases (CE15, EC 3.1.1.117) catalyze the hydrolysis of ester bonds between lignin and carbohydrates in lignocellulose. They are widespread within fungi and bacteria, and are subjects to research interest due to their potential applicability in lignocellulose processing. Identifying new and relevant glucuronoyl esterase candidates is challenging because available model substrates poorly represent the natural substrate, which leads to inefficient screening for the activity. In this study, we demonstrate how fifteen novel, fungal, putative glucuronoyl esterases from family CE15 were expressed and screened for activity towards a commercially available, colorimetric assay based on the methyl-ester of 4-O-methyl-aldotriuronic acid linked to para-nitrophenol (methyl ester-UX-beta-pNP) and coupled with the activity of GH67 (alpha-glucuronidase) and GH43 (beta-xylosidase) activity. The assay provides easy means for accurately establishing activity and determining specific activity of glucuronoyl esterases. Out of the fifteen expressed CE15 proteins, seven are active and were purified to determine their specific activity. The seven active enzymes originate from Auricularia subglabra (3 proteins), Ganoderma sinensis (2 proteins) and Neocallimastix californiae (2 proteins). Among the CE15 proteins not active towards the screening substrate (methyl ester-UX-beta-pNP) were proteins originating from Schizophyllum commune, Podospora anserina, Trametes versicolor, and Coprinopsis cinerea. It is unexpected that CE15 proteins from such canonical lignocellulose degraders do not have the anticipated activity, and these observations call for deeper investigations.
ESTHER : Madsen_2024_Enzyme.Microb.Technol_178_110444
PubMedSearch : Madsen_2024_Enzyme.Microb.Technol_178_110444
PubMedID: 38581869
Gene_locus related to this paper: 9aphy-a0a2g8sv16 , 9aphy-a0a2g8sk56 , 9fung-ORY47566 , 9fung-KAG4101299 , 9fung-KAG4087973 , aurst-EJD51299 , aurst-EJD54138 , aurst-EJD54133 , cerui-gce

Title : Fungal feruloyl esterases can catalyze release of diferulic acids from complex arabinoxylan - Lin_2023_Int.J.Biol.Macromol__123365
Author(s) : Lin S , Hunt CJ , Holck J , Brask J , Krogh K , Meyer AS , Wilkens C , Agger JW
Ref : Int J Biol Macromol , :123365 , 2023
Abstract : Feruloyl esterases (FAEs, EC 3.1.1.73) catalyze the hydrolytic cleavage of ester bonds between feruloyl and arabinosyl moieties in arabinoxylans. Recently, we discovered that two bacterial FAEs could catalyze release of diferulic acid moieties (diFAs) from highly substituted, cross-linked corn bran arabinoxylan. Here, we show that several fungal FAEs, notably AnFae1 (Aspergillus niger), AoFae1 (A. oryzae), and MgFae1 (Magnaporthe oryzae (also known as M. grisae)) also catalyze liberation of diFAs from complex arabinoxylan. By comparing the enzyme kinetics of diFA release to feruloyl esterase activity of the enzymes on methyl- and arabinosyl-ferulate substrates we demonstrate that the diFA release activity cannot be predicted from the activity of the enzymes on these synthetic substrates. A detailed structure-function analysis, based on AlphaFold2 modeled enzyme structures and docking with the relevant di-feruloyl ligands, reveal how distinct differences in the active site topology and surroundings may explain the diFA releasing action of the enzymes. Interestingly, the analysis also unveils that the carbohydrate binding module of the MgFae1 may play a key role in the diFA releasing ability of this enzyme. The findings contribute further understanding of the function of FAEs in the deconstruction of complex arabinoxylans and provide new opportunities for enzyme assisted upgrading of complex bran arabinoxylans.
ESTHER : Lin_2023_Int.J.Biol.Macromol__123365
PubMedSearch : Lin_2023_Int.J.Biol.Macromol__123365
PubMedID: 36690236
Gene_locus related to this paper: humin-HiFae1 , malci-McFae1 , 9zzzz-CE1.6RZN , 9zzzz-DAC80243 , 9pezi-a0a481sy08 , aspni-FAEA , aspor-q2uh24 , aspor-q2umx6 , aspor-q2unw5 , aspor-q2up89 , neucr-faeb , magoy-l7ic25

Title : Transesterification with CE15 glucuronoyl esterase from Cerrena unicolor reveals substrate preferences - Perna_2023_Biotechnol.Lett__
Author(s) : Perna V , Agger JW
Ref : Biotechnol Lett , : , 2023
Abstract : 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 alpha-benzyl or the gamma-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 gamma-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.
ESTHER : Perna_2023_Biotechnol.Lett__
PubMedSearch : Perna_2023_Biotechnol.Lett__
PubMedID: 38150097

Title : New insights to diversity and enzyme-substrate interactions of fungal glucuronoyl esterases - Agger_2023_Appl.Microbiol.Biotechnol__
Author(s) : Agger JW , Madsen MS , Martinsen LK , Martins PA , Barrett K , Meyer AS
Ref : Applied Microbiology & Biotechnology , : , 2023
Abstract : Glucuronoyl esterases (GEs) (EC 3.1.1.117) catalyze the cleavage of ester-linked lignin-carbohydrate complexes that has high impact on the plant cell wall integrity. The GEs are among the very few known types of hydrolytic enzymes that act at the interface of lignin, or which may potentially interact with lignin itself. In this review, we provide the latest update of the current knowledge on GEs with a special focus on the fungal variants. In addition, we have established the phylogenetic relationship between all GEs and this reveals that the fungal enzymes largely fall into one major branch, together with only a minor subset of bacterial enzymes. About 22% of the fungal proteins carry an additional domain, which is almost exclusively a CBM1 binding domain. We address how GEs may interact with the lignin-side of their substrate by molecular docking experiments based on the known structure of the Cerrena unicolor GE (CuGE). The docking studies indicate that there are no direct interactions between the enzyme and the lignin polymer, that the lignin-moiety is facing away from the protein surface and that an elongated carbon-chain between the ester-linkage and the first phenyl of lignin is preferable. Much basic research on these enzymes has been done over the past 15 years, but the next big step forward for these enzymes is connected to application and how these enzymes can facilitate the use of lignocellulose as a renewable resource. KEY POINTS: Fungal GEs are closely related and are sometimes linked to a binding module Molecular docking suggests good accommodation of lignin-like substructures GEs could be among the first expressed enzymes during fungal growth on biomass.
ESTHER : Agger_2023_Appl.Microbiol.Biotechnol__
PubMedSearch : Agger_2023_Appl.Microbiol.Biotechnol__
PubMedID: 37256329

Title : Enzymatic Cleavage of Diferuloyl Cross-Links in Corn Bran Arabinoxylan by Two Bacterial Feruloyl Esterases - Lin_2022_J.Agric.Food.Chem_70_13349
Author(s) : Lin S , Brask J , Munk L , Holck J , KBRM , Meyer AS , Agger JW , Wilkens C
Ref : Journal of Agricultural and Food Chemistry , 70 :133349 , 2022
Abstract : Corn bran is an abundant coprocessing stream of corn-starch processing, rich in highly substituted, diferuloyl-cross-linked glucurono-arabinoxylan. The diferuloyl cross-links make the glucurono-arabinoxylan recalcitrant to enzymatic conversion and constitute a hindrance for designing selective enzymatic upgrading of corn glucurono-arabinoxylan. Here, we show that two bacterial feruloyl esterases, wtsFae1A and wtsFae1B, each having a carbohydrate-binding module of family 48, are capable of cleaving the ester bonds of the cross-linkages and releasing 5-5', 8-5', 8-5' benzofuran, and 8-O-4' diferulate from soluble and insoluble corn bran glucurono-arabinoxylan. All four diferulic acids were released at similar efficiency, indicating nondiscriminatory enzymatic selectivity for the esterified dimer linkages, the only exception being that wtsFae1B had a surprisingly high propensity for releasing the dimers, especially 8-5' benzofuran diferulate, indicating a potential, unique catalytic selectivity. The data provide evidence of direct enzymatic release of diferulic acids from corn bran by newly discovered feruloyl esterases, i.e., a new enzyme activity. The findings yield new insight and create new opportunities for enzymatic opening of diferuloyl cross-linkages to pave the way for upgrading of recalcitrant arabinoxylans.
ESTHER : Lin_2022_J.Agric.Food.Chem_70_13349
PubMedSearch : Lin_2022_J.Agric.Food.Chem_70_13349
PubMedID: 36205442
Gene_locus related to this paper: 9zzzz-CE1.6RZN , 9zzzz-DAC80243

Title : The structural basis of fungal glucuronoyl esterase activity on natural substrates - Ernst_2020_Nat.Commun_11_1026
Author(s) : Ernst HA , Mosbech C , Langkilde AE , Westh P , Meyer AS , Agger JW , Larsen S
Ref : Nat Commun , 11 :1026 , 2020
Abstract : Structural and functional studies were conducted of the glucuronoyl esterase (GE) from Cerrena unicolor (CuGE), an enzyme catalyzing cleavage of lignin-carbohydrate ester bonds. CuGE is an alpha/beta-hydrolase belonging to carbohydrate esterase family 15 (CE15). The enzyme is modular, comprised of a catalytic and a carbohydrate-binding domain. SAXS data show CuGE as an elongated rigid molecule where the two domains are connected by a rigid linker. Detailed structural information of the catalytic domain in its apo- and inactivated form and complexes with aldouronic acids reveal well-defined binding of the 4-O-methyl-a-D-glucuronoyl moiety, not influenced by the nature of the attached xylo-oligosaccharide. Structural and sequence comparisons within CE15 enzymes reveal two distinct structural subgroups. CuGE belongs to the group of fungal CE15-B enzymes with an open and flat substrate-binding site. The interactions between CuGE and its natural substrates are explained and rationalized by the structural results, microscale thermophoresis and isothermal calorimetry.
ESTHER : Ernst_2020_Nat.Commun_11_1026
PubMedSearch : Ernst_2020_Nat.Commun_11_1026
PubMedID: 32094331
Gene_locus related to this paper: cerui-gce

Title : Enzyme kinetics of fungal glucuronoyl esterases on natural lignin-carbohydrate complexes - Mosbech_2019_Appl.Microbiol.Biotechnol_103_4065
Author(s) : Mosbech C , Holck J , Meyer A , Agger JW
Ref : Applied Microbiology & Biotechnology , 103 :4065 , 2019
Abstract : Glucuronoyl esterases (CE15 family) enable targeted cleavage of ester linkages in lignin-carbohydrate complexes (LCCs), particularly those linking lignin and glucuronoyl residues in xylan. A substantial challenge in characterization and kinetic analysis of CE15 enzymes has been the lack of proper substrates. Here, we present an assay using an insoluble LCC-rich lignin fraction from birch; lignin-rich pellet (LRP). The assay employs quantification of enzyme reaction products by LC-MS. The kinetics of four fungal CE15 enzymes, PsGE, CuGE, TtGE, and AfuGE originating from lignocellulose-degrading fungi Punctularia strigosozonata, Cerrena unicolor, Thielavia terrestris, and Armillaria fuscipes respectively were characterized and compared using this new assay. All four enzymes had activity on LRP and showed a clear preference for the insoluble substrate compared with smaller soluble LCC mimicking esters. End-product profiles were near identical for the four enzymes but differences in kinetic parameters were observed. TtGE possesses an alternative active site compared with the three other enzymes as it has the position of the catalytic glutamic acid occupied by a serine. TtGE performed poorly compared with the other enzymes. We speculate that glucuronoyl LCCs are not the preferred substrate of TtGE. Removal of an N-terminal CBM on CuGE affected the catalytic efficiently of the enzyme by reducing K(cat) by more than 30%. Reaction products were detected from all four CE15s on a similar substrate from spruce indicating a more generic GE activity not limited to the hardwood. The assay with natural substrate represents a novel tool to study the natural function and kinetics of CE15s.
ESTHER : Mosbech_2019_Appl.Microbiol.Biotechnol_103_4065
PubMedSearch : Mosbech_2019_Appl.Microbiol.Biotechnol_103_4065
PubMedID: 30949809

Title : The natural catalytic function of CuGE glucuronoyl esterase in hydrolysis of genuine lignin-carbohydrate complexes from birch - Mosbech_2018_Biotechnol.Biofuels_11_71
Author(s) : Mosbech C , Holck J , Meyer AS , Agger JW
Ref : Biotechnol Biofuels , 11 :71 , 2018
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.
ESTHER : Mosbech_2018_Biotechnol.Biofuels_11_71
PubMedSearch : Mosbech_2018_Biotechnol.Biofuels_11_71
PubMedID: 29560026
Gene_locus related to this paper: cerui-gce

Title : A New Functional Classification of Glucuronoyl Esterases by Peptide Pattern Recognition - Agger_2017_Front.Microbiol_8_309
Author(s) : Agger JW , Busk PK , Pilgaard B , Meyer AS , Lange L
Ref : Front Microbiol , 8 :309 , 2017
Abstract : 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.
ESTHER : Agger_2017_Front.Microbiol_8_309
PubMedSearch : Agger_2017_Front.Microbiol_8_309
PubMedID: 28293230