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
Technology Division, Joint BioEnergy Institute, Emeryville, CA, USA.
Biomaterials and Biomanufacturing Department, Sandia National Laboratories, Livermore, CA, USA.
Sci Rep. 2025 Mar 25;15(1):10296. doi: 10.1038/s41598-025-88571-7.
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.
木质素由通过醚键和碳 - 碳键连接的苯丙烷单体组成，形成复杂的异质结构。对木质素修饰酶（LMEs，如漆酶和过氧化物酶）的键特异性研究受到模型木质素底物聚合和裂解产物再聚合的限制。在此，我们展示了一个高通量平台，用于筛选LMEs对四种标记的模型木质素化合物的活性，这些化合物代表了木质素中的β - O - 4'、β - β'、5 - 5'和4 - O - 5'键。我们利用了纳米结构引发剂质谱（NIMS）以及带有含全氟和阳离子部分标签的模型木质素化合物，这有效地限制了底物及其降解产物的聚合和缩合。用一种新颖的机器人方法将亚微升样品液滴打印在NIMS芯片上。这个快速平台能够在pH 3 - 10范围内对LMEs进行表征，并对修饰（通常是氧化）、裂解和聚合产物进行相对定量。所有测试的酶都氧化了这四种底物，并将β - O - 4'和β - β'底物裂解为单体产物。我们发现活性pH范围取决于底物和酶的类型。这对于生物质转化为生物燃料和生物产品具有重要应用，因为木质素中不同键类型的相对百分比会因原料和化学预处理方法而异。
