Monschein Mareike, Jurak Edita, Paasela Tanja, Koitto Taru, Lambauer Vera, Pavicic Mirko, Enjalbert Thomas, Dumon Claire, Master Emma R
Department of Bioproducts and Biosystems, Aalto University, Kemistintie 1, 02150, Espoo, Finland.
Department of Bioproduct Engineering, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands.
Biotechnol Biofuels Bioprod. 2022 Mar 16;15(1):30. doi: 10.1186/s13068-022-02128-8.
Substrate accessibility remains a key limitation to the efficient enzymatic deconstruction of lignocellulosic biomass. Limited substrate accessibility is often addressed by increasing enzyme loading, which increases process and product costs. Alternatively, considerable efforts are underway world-wide to identify amorphogenesis-inducing proteins and protein domains that increase the accessibility of carbohydrate-active enzymes to targeted lignocellulose components.
We established a three-dimensional assay, PACER (plant cell wall model for the analysis of non-catalytic and enzymatic responses), that enables analysis of enzyme migration through defined lignocellulose composites. A cellulose/azo-xylan composite was made to demonstrate the PACER concept and then used to test the migration and activity of multiple xylanolytic enzymes. In addition to non-catalytic domains of xylanases, the potential of loosenin-like proteins to boost xylanase migration through cellulose/azo-xylan composites was observed.
The PACER assay is inexpensive and parallelizable, suitable for screening proteins for ability to increase enzyme accessibility to lignocellulose substrates. Using the PACER assay, we visualized the impact of xylan-binding modules and loosenin-like proteins on xylanase mobility and access to targeted substrates. Given the flexibility to use different composite materials, the PACER assay presents a versatile platform to study impacts of lignocellulose components on enzyme access to targeted substrates.
底物可及性仍然是木质纤维素生物质高效酶解的关键限制因素。底物可及性有限通常通过增加酶负载量来解决，这会增加工艺和产品成本。另外，全球正在进行大量努力，以鉴定能提高碳水化合物活性酶对目标木质纤维素成分可及性的无定形诱导蛋白和蛋白结构域。
我们建立了一种三维检测方法，即PACER（用于分析非催化和酶促反应的植物细胞壁模型），该方法能够分析酶在特定木质纤维素复合材料中的迁移情况。制备了一种纤维素/偶氮木聚糖复合材料以证明PACER概念，然后用于测试多种木聚糖酶的迁移和活性。除了木聚糖酶的非催化结构域外，还观察到类疏松蛋白促进木聚糖酶通过纤维素/偶氮木聚糖复合材料迁移的潜力。
PACER检测方法成本低廉且可并行操作，适用于筛选能够提高酶对木质纤维素底物可及性的蛋白质。通过PACER检测，我们可视化了木聚糖结合模块和类疏松蛋白对木聚糖酶迁移率及对目标底物可及性的影响。鉴于可灵活使用不同的复合材料，PACER检测提供了一个通用平台，用于研究木质纤维素成分对酶与目标底物可及性的影响。
