skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Rapid characterization of the activities of lignin-modifying enzymes based on nanostructure-initiator mass spectrometry (NIMS)

Abstract

Background: Producing valuable fuels and chemicals from lignin is a key factor for making lignocellulosic biomass economically feasible; however, significant roadblocks exist due to our lack of detailed understanding of how lignin is enzymatically depolymerized and of the range of possible lignin fragments that can be produced. Development of suitable enzymatic assays for characterization of putative lignin active enzymes is an important step towards improving our understanding of the catalytic activities of relevant enzymes. Previously, we have successfully built an assay platform based on glycan substrates containing a charged perfluorinated tag and nanostructure-initiator mass spectrometry to study carbohydrate active enzymes, especially various glycosyl hydrolyses. Here, we extend this approach to develop a reliable and rapid assay to study lignin-modifying enzymes. Results: Two β-aryl ether bond containing model lignin dimer substrates, designed to be suitable for studying the activities of lignin-modifying enzymes (LMEs) by nanostructure-initiator mass spectrometry (NIMS), were successful synthesized. Small-angle neutron scattering experiments showed that these substrates form micelles in solution. Two LMEs, laccase from the polypore mushroom Trametes versicolor, and manganese peroxidase (MnP) from white rot fungus Nematoloma frowardii, were tested for catalytic activity against the two model substrates. We show that the reaction of laccase and MnPmore » with phenolic substrate yields products that arise from the cleavage of the carbon-carbon single bond between the α-carbon and the adjacent aryl carbon, consistent with the mechanism for producing phenoxy radical as reaction intermediates. Reactions of the nonphenolic substrate with laccase, on the other hand, adopt a different pathway by producing an α-oxidation product; as well as the cleavage of the β-aryl ether bond. No cleavage of the carbon-carbon bond between the α-carbon and the aryl carbon was observed. To facilitate understanding of reaction kinetics, the reaction time course for laccase activity on the phenolic substrate (I) was generated by the simultaneous measurement of all products at different time points of the reaction. Withdrawal of only a small sample aliquot (0.2 μL at each time point) ensured minimum perturbation of the reaction. The time course can help us to understand the enzyme kinetics. Conclusions: A new assay procedure has been developed for studying lignin-modifying enzymes by nanostructure-initiator mass spectrometry. Enzyme assays of a laccase and a MnP on phenolic and nonphenolic β-aryl ether substrates revealed different primary reaction pathways due to the availability of the phenoxy radical intermediates. Our assay provides a wealth of information on bond cleavage events not available using conventional colorimetric assays and can easily be carried out in microliter volumes and the quantitative analysis of product formation and kinetics is rapidly achieved by NIMS. This is the first time that NIMS technology was applied to study the activities of lignin-modifying enzymes. Unlike other previous works, our use of amphiphilic guaiacylglycerol β-O-4 substrate (I) enables the formation of micelles. This approach helps avoid the re-polymerization of the resulting monomeric product. As a result, our assay can clearly demonstrate the degradation pathways of phenolic guaiacylglycerol β-O-4 type of molecules with laccase and MnP.« less

Authors:
ORCiD logo; ; ; ; ; ; ; ;
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER); USDOE National Nuclear Security Administration (NNSA)
OSTI Identifier:
1618736
Alternate Identifier(s):
OSTI ID: 1506365
Grant/Contract Number:  
AC02-05CH11231; NA0003525
Resource Type:
Journal Article: Published Article
Journal Name:
Biotechnology for Biofuels
Additional Journal Information:
Journal Name: Biotechnology for Biofuels Journal Volume: 11 Journal Issue: 1; Journal ID: ISSN 1754-6834
Publisher:
Springer Science + Business Media
Country of Publication:
Netherlands
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Lignin; β-Aryl ether; Lignin-modifying enzymes; NIMS; Enzyme assays

Citation Formats

Deng, Kai, Zeng, Jijiao, Cheng, Gang, Gao, Jian, Sale, Kenneth L., Simmons, Blake A., Singh, Anup K., Adams, Paul D., and Northen, Trent R. Rapid characterization of the activities of lignin-modifying enzymes based on nanostructure-initiator mass spectrometry (NIMS). Netherlands: N. p., 2018. Web. doi:10.1186/s13068-018-1261-2.
Deng, Kai, Zeng, Jijiao, Cheng, Gang, Gao, Jian, Sale, Kenneth L., Simmons, Blake A., Singh, Anup K., Adams, Paul D., & Northen, Trent R. Rapid characterization of the activities of lignin-modifying enzymes based on nanostructure-initiator mass spectrometry (NIMS). Netherlands. doi:10.1186/s13068-018-1261-2.
Deng, Kai, Zeng, Jijiao, Cheng, Gang, Gao, Jian, Sale, Kenneth L., Simmons, Blake A., Singh, Anup K., Adams, Paul D., and Northen, Trent R. Thu . "Rapid characterization of the activities of lignin-modifying enzymes based on nanostructure-initiator mass spectrometry (NIMS)". Netherlands. doi:10.1186/s13068-018-1261-2.
@article{osti_1618736,
title = {Rapid characterization of the activities of lignin-modifying enzymes based on nanostructure-initiator mass spectrometry (NIMS)},
author = {Deng, Kai and Zeng, Jijiao and Cheng, Gang and Gao, Jian and Sale, Kenneth L. and Simmons, Blake A. and Singh, Anup K. and Adams, Paul D. and Northen, Trent R.},
abstractNote = {Background: Producing valuable fuels and chemicals from lignin is a key factor for making lignocellulosic biomass economically feasible; however, significant roadblocks exist due to our lack of detailed understanding of how lignin is enzymatically depolymerized and of the range of possible lignin fragments that can be produced. Development of suitable enzymatic assays for characterization of putative lignin active enzymes is an important step towards improving our understanding of the catalytic activities of relevant enzymes. Previously, we have successfully built an assay platform based on glycan substrates containing a charged perfluorinated tag and nanostructure-initiator mass spectrometry to study carbohydrate active enzymes, especially various glycosyl hydrolyses. Here, we extend this approach to develop a reliable and rapid assay to study lignin-modifying enzymes. Results: Two β-aryl ether bond containing model lignin dimer substrates, designed to be suitable for studying the activities of lignin-modifying enzymes (LMEs) by nanostructure-initiator mass spectrometry (NIMS), were successful synthesized. Small-angle neutron scattering experiments showed that these substrates form micelles in solution. Two LMEs, laccase from the polypore mushroom Trametes versicolor, and manganese peroxidase (MnP) from white rot fungus Nematoloma frowardii, were tested for catalytic activity against the two model substrates. We show that the reaction of laccase and MnP with phenolic substrate yields products that arise from the cleavage of the carbon-carbon single bond between the α-carbon and the adjacent aryl carbon, consistent with the mechanism for producing phenoxy radical as reaction intermediates. Reactions of the nonphenolic substrate with laccase, on the other hand, adopt a different pathway by producing an α-oxidation product; as well as the cleavage of the β-aryl ether bond. No cleavage of the carbon-carbon bond between the α-carbon and the aryl carbon was observed. To facilitate understanding of reaction kinetics, the reaction time course for laccase activity on the phenolic substrate (I) was generated by the simultaneous measurement of all products at different time points of the reaction. Withdrawal of only a small sample aliquot (0.2 μL at each time point) ensured minimum perturbation of the reaction. The time course can help us to understand the enzyme kinetics. Conclusions: A new assay procedure has been developed for studying lignin-modifying enzymes by nanostructure-initiator mass spectrometry. Enzyme assays of a laccase and a MnP on phenolic and nonphenolic β-aryl ether substrates revealed different primary reaction pathways due to the availability of the phenoxy radical intermediates. Our assay provides a wealth of information on bond cleavage events not available using conventional colorimetric assays and can easily be carried out in microliter volumes and the quantitative analysis of product formation and kinetics is rapidly achieved by NIMS. This is the first time that NIMS technology was applied to study the activities of lignin-modifying enzymes. Unlike other previous works, our use of amphiphilic guaiacylglycerol β-O-4 substrate (I) enables the formation of micelles. This approach helps avoid the re-polymerization of the resulting monomeric product. As a result, our assay can clearly demonstrate the degradation pathways of phenolic guaiacylglycerol β-O-4 type of molecules with laccase and MnP.},
doi = {10.1186/s13068-018-1261-2},
journal = {Biotechnology for Biofuels},
issn = {1754-6834},
number = 1,
volume = 11,
place = {Netherlands},
year = {2018},
month = {9}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record at 10.1186/s13068-018-1261-2

Figures / Tables:

Fig. 1 Fig. 1: Structures of phenolic substrate I and nonphenolic substrate XII

Save / Share:

Works referenced in this record:

Lignin Valorization: Improving Lignin Processing in the Biorefinery
journal, May 2014

  • Ragauskas, A. J.; Beckham, G. T.; Biddy, M. J.
  • Science, Vol. 344, Issue 6185, p. 1246843-1246843
  • DOI: 10.1126/science.1246843

Structural and Biochemical Characterization of the Early and Late Enzymes in the Lignin β-Aryl Ether Cleavage Pathway from Sphingobium sp. SYK-6
journal, March 2016

  • Pereira, Jose Henrique; Heins, Richard A.; Gall, Daniel L.
  • Journal of Biological Chemistry, Vol. 291, Issue 19
  • DOI: 10.1074/jbc.M115.700427

Exploring bacterial lignin degradation
journal, April 2014


Encoding substrates with mass tags to resolve stereospecific reactions using Nimzyme: Encoding substrates with mass tags using Nimzyme
journal, February 2012

  • Deng, Kai; George, Kevin W.; Reindl, Wolfgang
  • Rapid Communications in Mass Spectrometry, Vol. 26, Issue 6
  • DOI: 10.1002/rcm.6134

Qualitative and Quantitative Methods for Isolation and Characterization of Lignin-Modifying Enzymes Secreted by Microorganisms
journal, August 2016


Investigations on the laccase-catalyzed polymerization of lignin model compounds using size-exclusion HPLC
journal, September 2002


Rapid Kinetic Characterization of Glycosyl Hydrolases Based on Oxime Derivatization and Nanostructure-Initiator Mass Spectrometry (NIMS)
journal, May 2014

  • Deng, Kai; Takasuka, Taichi E.; Heins, Richard
  • ACS Chemical Biology, Vol. 9, Issue 7
  • DOI: 10.1021/cb5000289

Structural Basis of Stereospecificity in the Bacterial Enzymatic Cleavage of β-Aryl Ether Bonds in Lignin
journal, December 2015

  • Helmich, Kate E.; Pereira, Jose Henrique; Gall, Daniel L.
  • Journal of Biological Chemistry, Vol. 291, Issue 10
  • DOI: 10.1074/jbc.M115.694307

A nanostructure-initiator mass spectrometry-based enzyme activity assay
journal, March 2008

  • Northen, T. R.; Lee, J.-C.; Hoang, L.
  • Proceedings of the National Academy of Sciences, Vol. 105, Issue 10, p. 3678-3683
  • DOI: 10.1073/pnas.0712332105

Effect of Fluorocarbon and Hydrocarbon Chain Lengths in Hybrid Surfactants for Supercritical CO 2
journal, June 2015


Paving the Way for Lignin Valorisation: Recent Advances in Bioengineering, Biorefining and Catalysis
journal, June 2016

  • Rinaldi, Roberto; Jastrzebski, Robin; Clough, Matthew T.
  • Angewandte Chemie International Edition, Vol. 55, Issue 29
  • DOI: 10.1002/anie.201510351

Identification of 4–O–5-Units in Softwood Lignins via Definitive Lignin Models and NMR
journal, May 2016


Enzymatic hydroxylation of aromatic compounds
journal, January 2007


Lignin Biodegradation with Laccase-Mediator Systems
journal, March 2014


Peroxidative Oxidation of Lignin and a Lignin Model Compound by a Manganese SALEN Derivative
journal, April 2016


Understanding factors controlling depolymerization and polymerization in catalytic degradation of β-ether linked model lignin compounds by versatile peroxidase
journal, January 2017

  • Zeng, Jijiao; Mills, Matthew J. L.; Simmons, Blake A.
  • Green Chemistry, Vol. 19, Issue 9
  • DOI: 10.1039/C6GC03379B

Lignin-modifying enzymes from selected white-rot fungi: production and role from in lignin degradation
journal, March 1994


Clathrate nanostructures for mass spectrometry
journal, October 2007

  • Northen, Trent R.; Yanes, Oscar; Northen, Michael T.
  • Nature, Vol. 449, Issue 7165
  • DOI: 10.1038/nature06195

Acoustic deposition with NIMS as a high-throughput enzyme activity assay
journal, March 2012

  • Greving, Matthew; Cheng, Xiaoliang; Reindl, Wolfgang
  • Analytical and Bioanalytical Chemistry, Vol. 403, Issue 3
  • DOI: 10.1007/s00216-012-5908-8

Lignin Valorization through Catalytic Lignocellulose Fractionation: A Fundamental Platform for the Future Biorefinery
journal, June 2016


    Works referencing / citing this record:

    A method for high-throughput screening hydrolase of lignin β-aryl ether linkage from directed evolution by glutathione (GSH) assay
    journal, May 2020


      Figures/Tables have been extracted from DOE-funded journal article accepted manuscripts.