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| Content Provider | Springer Nature : BioMed Central |
|---|---|
| Author | Deng, Kai Zeng, Jijiao Cheng, Gang Gao, Jian Sale, Kenneth L. Simmons, Blake A. Singh, Anup K. Adams, Paul D. Northen, Trent R. |
| 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 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. |
| Related Links | https://biotechnologyforbiofuels.biomedcentral.com/counter/pdf/10.1186/s13068-018-1261-2.pdf |
| Ending Page | 9 |
| Page Count | 9 |
| Starting Page | 1 |
| File Format | HTM / HTML |
| ISSN | 27313654 |
| DOI | 10.1186/s13068-018-1261-2 |
| Journal | Biotechnology for Biofuels and Bioproducts |
| Issue Number | 1 |
| Volume Number | 11 |
| Language | English |
| Publisher | BioMed Central |
| Publisher Date | 2018-09-27 |
| Access Restriction | Open |
| Subject Keyword | Biotechnology Plant Breeding Environmental Engineering Renewable and Green Energy Microbiology Lignin β-Aryl ether Lignin-modifying enzymes NIMS Enzyme assays Plant Breeding/Biotechnology Environmental Engineering/Biotechnology |
| Content Type | Text |
| Resource Type | Article |
| Subject | Energy Management, Monitoring, Policy and Law Renewable Energy, Sustainability and the Environment Applied Microbiology and Biotechnology Biotechnology |
| Journal Impact Factor | 6.1/2023 |
| 5-Year Journal Impact Factor | 6/2023 |
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