Enzymatic cleavage of model lignin dimers depends on pH, enzyme, and bond type

Onley, Jenny R.; Gupta, Kshitiz; de Raad, Markus; Bowen, Benjamin P.; Tan, Stephen; Yoder, Sam; Sale, Kenneth L.; Singh, Anup K.; Simmons, Blake A.; Adams, Paul D.; Northen, Trent R.; Deng, Kai

doi:10.1038/s41598-025-88571-7

Enzymatic cleavage of model lignin dimers depends on pH, enzyme, and bond type

Journal Article · Tue Mar 25 00:00:00 EDT 2025 · Scientific Reports

DOI:https://doi.org/10.1038/s41598-025-88571-7· OSTI ID:2563696

Onley, Jenny R. ^[1]; Gupta, Kshitiz ^[2]; de Raad, Markus ^[3]; Bowen, Benjamin P. ^[3]; Tan, Stephen ^[4]; Yoder, Sam ^[4]; Sale, Kenneth L. ^[5]; Singh, Anup K. ^[2]; Simmons, Blake A. ^[6]; Adams, Paul D. ^[7]; Northen, Trent R. ^[4]; Deng, Kai ^[1]

Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Sandia National Laboratories (SNL-NM), Albuquerque, NM (United States)
Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Livermore National Laboratory (LLNL), Livermore, CA (United States)
Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
Sandia National Laboratories (SNL-NM), Albuquerque, NM (United States); Joint BioEnergy Institute (JBEI), Emeryville, CA (United States)
Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States); Joint BioEnergy Institute (JBEI), Emeryville, CA (United States)
Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States); University of California, Berkeley, CA (United States)

Lignin is composed of phenylpropanoid monomers linked by ether and carbon-carbon bonds to form a complex heterogeneous structure. Bond-specific studies of lignin-modifying enzymes (LMEs; e.g., laccases and peroxidases) are limited by the polymerization of model lignin substrates and repolymerization of cleavage products. Here we present a high throughput platform to screen LME activities on four tagged model lignin compounds that represent the β-O-4’, β-β’, 5–5’, and 4-O-5’ linkages in lignin. We utilized nanostructure-initiator mass spectrometry (NIMS) and model lignin compounds with tags containing perfluorinated and cationic moieties, which effectively limit polymerization and condensation of the substrates and their degrading products. Sub-microliter sample droplets were printed on the NIMS chip with a novel robotics method. This rapid platform enabled characterization of LMEs across a range of pH 3–10 and relative quantification of modified (typically oxidized), cleaved, and polymerized products. All tested enzymes oxidized the four substrates and cleaved the β-O-4’ and β-β’ substrates to monomeric products. We discovered that the active pH range depended on both the substrate and the enzyme type. This has important applications for biomass conversion to biofuels and bioproducts, where the relative percentages of different bond types in lignin varies depending on feedstock and chemical pretreatment methods.

View Accepted Manuscript (DOE)

Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 2563696

Journal Information:: Scientific Reports, Journal Name: Scientific Reports Journal Issue: 1 Vol. 15; ISSN 2045-2322

Publisher:: Nature Publishing GroupCopyright Statement

Country of Publication:: United States

Language:: English

References (43)

Perspective on Lignin Conversion Strategies That Enable Next Generation Biorefineries Shrestha, Shilva; Goswami, Shubhasish; Banerjee, Deepanwita ChemSusChem, Vol. 17, Issue 16 https://doi.org/10.1002/cssc.202301460	journal	April 2024
Advances in Catalytic Depolymerization of Lignin Wan, Zhouyuanye; Zhang, Hongjie; Guo, Yanzhu ChemistrySelect, Vol. 7, Issue 40 https://doi.org/10.1002/slct.202202582	journal	October 2022
Degradation of d,l-syringaresinol, a ?-?? linked lignin model compound, by Fusarium solani M-13-1 Kamaya, Yasushi; Nakatsubo, Fumiaki; Higuchi, Takayoshi Archives of Microbiology, Vol. 129, Issue 4 https://doi.org/10.1007/BF00414702	journal	June 1981
Laccases for biorefinery applications: a critical review on challenges and perspectives Roth, Simon; Spiess, Antje C. Bioprocess and Biosystems Engineering, Vol. 38, Issue 12 https://doi.org/10.1007/s00449-015-1475-7	journal	October 2015
Laccase and horseradish peroxidase for green treatment of phenolic micropollutants in real drinking water and wastewater Maryskova, Milena; Linhartova, Lucie; Novotny, Vit Environmental Science and Pollution Research, Vol. 28, Issue 24 https://doi.org/10.1007/s11356-021-12910-0	journal	February 2021
Chemicals From Lignin Luo, Hao; Abu-Omar, Mahdi M. Encyclopedia of Sustainable Technologies https://doi.org/10.1016/B978-0-12-409548-9.10235-0	book	January 2017
Lignocellulosic biomass: Acid and alkaline pretreatments and their effects on biomass recalcitrance – Conventional processing and recent advances Lorenci Woiciechowski, Adenise; Dalmas Neto, Carlos José; Porto de Souza Vandenberghe, Luciana Bioresource Technology, Vol. 304 https://doi.org/10.1016/j.biortech.2020.122848	journal	May 2020
Can laccases catalyze bond cleavage in lignin? Munk, Line; Sitarz, Anna K.; Kalyani, Dayanand C. Biotechnology Advances, Vol. 33, Issue 1 https://doi.org/10.1016/j.biotechadv.2014.12.008	journal	January 2015
Review of advances in the development of laccases for the valorization of lignin to enable the production of lignocellulosic biofuels and bioproducts Leynaud Kieffer Curran, Laure M. C.; Pham, Le Thanh Mai; Sale, Kenneth L. Biotechnology Advances, Vol. 54 https://doi.org/10.1016/j.biotechadv.2021.107809	journal	January 2022
Lignin structure and its engineering Ralph, John; Lapierre, Catherine; Boerjan, Wout Current Opinion in Biotechnology, Vol. 56 https://doi.org/10.1016/j.copbio.2019.02.019	journal	April 2019
Recent advances in laccase activity assays: A crucial challenge for applications on complex substrates Martin, Elise; Dubessay, Pascal; Record, Eric Enzyme and Microbial Technology, Vol. 173 https://doi.org/10.1016/j.enzmictec.2023.110373	journal	February 2024
Purification and biochemical characterization of two isolated laccase isoforms from Agaricus bisporus CU13 and their potency in dye decolorization Othman, Abdelmageed M.; Elsayed, Maysa A.; Elshafei, Ali M. International Journal of Biological Macromolecules, Vol. 113 https://doi.org/10.1016/j.ijbiomac.2018.03.043	journal	July 2018
OpenMSI Arrayed Analysis Toolkit: Analyzing Spatially Defined Samples Using Mass Spectrometry Imaging de Raad, Markus; de Rond, Tristan; Rübel, Oliver Analytical Chemistry, Vol. 89, Issue 11 https://doi.org/10.1021/acs.analchem.6b05004	journal	May 2017
Inhibitors in Commercially Available 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonate) Affect Enzymatic Assays Zhang, Yifei; Hess, Henry Analytical Chemistry, Vol. 92, Issue 1 https://doi.org/10.1021/acs.analchem.9b04751	journal	December 2019
Insight into Depolymerization Mechanism of Bacterial Laccase for Lignin Zhu, Daochen; Liang, Nian; Zhang, Rongxian ACS Sustainable Chemistry & Engineering, Vol. 8, Issue 34 https://doi.org/10.1021/acssuschemeng.0c03457	journal	August 2020
Oxidation of phenolic arylglycerol .beta.-aryl ether lignin model compounds by manganese peroxidase from Phanerochaete chrysosporium: oxidative cleavage of an .alpha.-carbonyl model compound Tuor, Urs; Wariishi, Hiroyuki; Schoemaker, Hans E. Biochemistry, Vol. 31, Issue 21 https://doi.org/10.1021/bi00136a011	journal	June 1992
The Catalytic Valorization of Lignin for the Production of Renewable Chemicals Zakzeski, Joseph; Bruijnincx, Pieter C. A.; Jongerius, Anna L. Chemical Reviews, Vol. 110, Issue 6, p. 3552-3599 https://doi.org/10.1021/cr900354u	journal	June 2010
Nanostructure-initiator mass spectrometry: a protocol for preparing and applying NIMS surfaces for high-sensitivity mass analysis Woo, Hin-Koon; Northen, Trent R.; Yanes, Oscar Nature Protocols, Vol. 3, Issue 8 https://doi.org/10.1038/nprot.2008.110	journal	July 2008
Author Correction: SciPy 1.0: fundamental algorithms for scientific computing in Python Virtanen, Pauli; Gommers, Ralf; Oliphant, Travis E. Nature Methods, Vol. 17, Issue 3 https://doi.org/10.1038/s41592-020-0772-5	journal	February 2020
A multiplexed nanostructure-initiator mass spectrometry (NIMS) assay for simultaneously detecting glycosyl hydrolase and lignin modifying enzyme activities Ing, Nicole; Deng, Kai; Chen, Yan Scientific Reports, Vol. 11, Issue 1 https://doi.org/10.1038/s41598-021-91181-8	journal	June 2021
Understanding factors controlling depolymerization and polymerization in catalytic degradation of β-ether linked model lignin compounds by versatile peroxidase Zeng, Jijiao; Mills, Matthew J. L.; Simmons, Blake A. Green Chemistry, Vol. 19, Issue 9 https://doi.org/10.1039/C6GC03379B	journal	January 2017
Identification and quantification of lignin monomers and oligomers from reductive catalytic fractionation of pine wood with GC × GC – FID/MS Dao Thi, Hang; Van Aelst, Korneel; Van den Bosch, Sander Green Chemistry, Vol. 24, Issue 1 https://doi.org/10.1039/D1GC03822B	journal	January 2022
A combinatorial droplet microfluidic device integrated with mass spectrometry for enzyme screening Ha, Noel S.; Onley, Jenny R.; Deng, Kai Lab on a Chip, Vol. 23, Issue 15 https://doi.org/10.1039/D2LC00980C	journal	January 2023
Pathways for degradation of lignin in bacteria and fungi Bugg, Timothy D. H.; Ahmad, Mark; Hardiman, Elizabeth M. Natural Product Reports, Vol. 28, Issue 12, p. 1883-1896 https://doi.org/10.1039/c1np00042j	journal	January 2011
Perspective on oligomeric products from lignin depolymerization: their generation, identification, and further valorization Han, Yinglei; Simmons, Blake A.; Singh, Seema Industrial Chemistry & Materials, Vol. 1, Issue 2 https://doi.org/10.1039/d2im00059h	journal	January 2023
Outer membrane vesicles catabolize lignin-derived aromatic compounds in Pseudomonas putida KT2440 Salvachúa, Davinia; Werner, Allison Z.; Pardo, Isabel Proceedings of the National Academy of Sciences, Vol. 117, Issue 17 https://doi.org/10.1073/pnas.1921073117	journal	April 2020
Intracellular pathways for lignin catabolism in white-rot fungi del Cerro, Carlos; Erickson, Erika; Dong, Tao Proceedings of the National Academy of Sciences, Vol. 118, Issue 9 https://doi.org/10.1073/pnas.2017381118	journal	February 2021
Phenol Oxidases in some Wood-rotting Fungi Law, Kathleen Annals of Botany, Vol. 14, Issue 1 https://doi.org/10.1093/oxfordjournals.aob.a083235	journal	January 1950
Matplotlib: A 2D Graphics Environment Hunter, John D. Computing in Science & Engineering, Vol. 9, Issue 3 https://doi.org/10.1109/MCSE.2007.55	journal	January 2007
Fungal laccases – occurrence and properties Baldrian, Petr FEMS Microbiology Reviews, Vol. 30, Issue 2 https://doi.org/10.1111/j.1574-4976.2005.00010.x	journal	March 2006
Lignin-Degrading Enzyme from the Hymenomycete Phanerochaete chrysosporium Burds Tien, M.; Kirk, T. K. Science, Vol. 221, Issue 4611 https://doi.org/10.1126/science.221.4611.661	journal	August 1983
Isolation of the (+)-Pinoresinol-Mineralizing Pseudomonas sp. Strain SG-MS2 and Elucidation of Its Catabolic Pathway Shettigar, Madhura; Balotra, Sahil; Cahill, David Applied and Environmental Microbiology, Vol. 84, Issue 4 https://doi.org/10.1128/AEM.02531-17	journal	February 2018
Characterization of the gene encoding an extracellular laccase of Myceliophthora thermophila and analysis of the recombinant enzyme expressed in Aspergillus oryzae Berka, R. M.; Schneider, P.; Golightly, E. J. Applied and Environmental Microbiology, Vol. 63, Issue 8 https://doi.org/10.1128/aem.63.8.3151-3157.1997	journal	August 1997
Rapid characterization of the activities of lignin-modifying enzymes based on nanostructure-initiator mass spectrometry (NIMS) Deng, Kai; Zeng, Jijiao; Cheng, Gang Biotechnology for Biofuels, Vol. 11, Issue 1 https://doi.org/10.1186/s13068-018-1261-2	journal	September 2018
Pretreatment for biorefineries: a review of common methods for efficient utilisation of lignocellulosic materials Galbe, Mats; Wallberg, Ola Biotechnology for Biofuels, Vol. 12, Issue 1 https://doi.org/10.1186/s13068-019-1634-1	journal	December 2019
Enzymatic degradation of maize shoots: monitoring of chemical and physical changes reveals different saccharification behaviors Barron, Cécile; Devaux, Marie-Françoise; Foucat, Loïc Biotechnology for Biofuels, Vol. 14, Issue 1 https://doi.org/10.1186/s13068-020-01854-1	journal	January 2021
Tailor-made alkaliphilic and thermostable fungal laccases for industrial wood processing Rodríguez-Escribano, David; Pliego-Magán, Rocío; de Salas, Felipe Biotechnology for Biofuels and Bioproducts, Vol. 15, Issue 1 https://doi.org/10.1186/s13068-022-02247-2	journal	December 2022
Heterologous Expression, Characterization, and Comparison of Laccases from the White Rot Causing Basidiomycete Cerrena Unicolor Mai Pham, Le Thanh; Deng, Kai; Northen, Trent Catalysis Research, Vol. 02, Issue 03 https://doi.org/10.21926/cr.2203028	journal	September 2022
Data Structures for Statistical Computing in Python McKinney, Wes Proceedings of the Python in Science Conference https://doi.org/10.25080/Majora-92bf1922-00a	conference	January 2010
Enzymatic Processes to Unlock the Lignin Value Hämäläinen, Veera; Grönroos, Toni; Suonpää, Anu Frontiers in Bioengineering and Biotechnology, Vol. 6 https://doi.org/10.3389/fbioe.2018.00020	journal	March 2018
An Engineered Laccase from Fomitiporia mediterranea Accelerates Lignocellulose Degradation Pham, Le Thanh Mai; Deng, Kai; Choudhary, Hemant Biomolecules, Vol. 14, Issue 3 https://doi.org/10.3390/biom14030324	journal	March 2024
Laccase Properties, Physiological Functions, and Evolution Janusz, Grzegorz; Pawlik, Anna; Świderska-Burek, Urszula International Journal of Molecular Sciences, Vol. 21, Issue 3 https://doi.org/10.3390/ijms21030966	journal	January 2020
Studies on the Enzymatic Degradation of Lignin. The Action of Peroxidase and Laccase on Monomeric and Dimeric Model Compounds. Gierer, Josef; Opara, Augustine E.; Mjöberg, Johan Acta Chemica Scandinavica, Vol. 27 https://doi.org/10.3891/acta.chem.scand.27-2909	journal	January 1973

Similar Records

In Vitro Enzymatic Depolymerization of Lignin with Release of Syringyl, Guaiacyl, and Tricin Units

Journal Article · Sun Nov 26 19:00:00 EST 2017 · Applied and Environmental Microbiology · OSTI ID:1499013

High Throughput expression and characterization of laccases in Saccharomyces cerevisiae

Conference · Mon Apr 26 00:00:00 EDT 2021 · OSTI ID:1866049

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
Laccase
Lignin
Mass spectrometry
Peroxidase

Enzymatic cleavage of model lignin dimers depends on pH, enzyme, and bond type

Citation Formats

References (43)

Similar Records

Related Subjects