Instituto de Recursos Naturales y Agrobiología de Sevilla (IRNAS), CSIC, Avenida de la Reina Mercedes, 10, E-41012 Sevilla, Spain.
Departments of Forest Biomaterials and Chemical and Biomolecular Engineering, North Carolina State University , Raleigh, North Carolina 27695, United States.
Biomacromolecules. 2017 Apr 10;18(4):1322-1332. doi: 10.1021/acs.biomac.7b00071. Epub 2017 Mar 23.
Lignins were isolated from spruce, wheat straw, and eucalyptus by using the milled wood lignin (MWL) method. Functional groups and compositional analyses were assessed via 2D NMR and P NMR to realize their effect on enzyme binding. Films of the lignins were fabricated and ellipsometry, atomic force microscopy, and water contact angle measurements were used for their characterization and to reveal the changes upon enzyme adsorption. Moreover, lignin thin films were deposited on quartz crystal microgravimetry (QCM) and surface plasmon (SPR) resonance sensors and used to gain further insights into the lignin-cellulase interactions. For this purpose, a commercial multicomponent enzyme system and a monocomponent Trichoderma reesei exoglucanase (CBH-I) were considered. Strong enzyme adsorption was observed on the various lignins but compared to the multicomponent cellulases, CBH-I displayed lower surface affinity and higher binding reversibility. This resolved prevalent questions related to the affinity of this enzyme with lignin. Remarkably, a strong correlation between enzyme binding and the syringyl/guaiacyl (S/G) ratio was found for the lignins, which presented a similar hydroxyl group content (P NMR): higher protein affinity was determined on isolated spruce lignin (99% G units), while the lowest adsorption occurred on isolated eucalyptus lignin (70% S units). The effect of electrostatic interactions in enzyme adsorption was investigated by SPR, which clearly indicated that the screening of charges allowed more extensive protein adsorption. Overall, this work furthers our understanding of lignin-cellulase interactions relevant to biomass that has been subjected to no or little pretreatment and highlights the widely contrasting effects of the nature of lignin, which gives guidance to improve lignocellulosic saccharification and related processes.
通过使用磨木木质素（MWL）方法，从云杉、麦草和桉树中分离木质素。通过二维 NMR 和 P NMR 评估功能基团和组成分析，以了解它们对酶结合的影响。制备木质素薄膜，并使用椭圆光度法、原子力显微镜和水接触角测量对其进行表征，并揭示酶吸附后的变化。此外，将木质素薄膜沉积在石英晶体微天平（QCM）和表面等离子体（SPR）共振传感器上，以进一步深入了解木质素-纤维素酶相互作用。为此，考虑了一种商业多组分酶系统和一种单组分里氏木霉外切葡聚糖酶（CBH-I）。在各种木质素上观察到强烈的酶吸附，但与多组分纤维素酶相比，CBH-I 显示出较低的表面亲和力和较高的结合可逆性。这解决了与该酶与木质素亲和力相关的普遍问题。值得注意的是，在木质素之间发现了酶结合与丁香基/愈创木基（S/G）比之间的强相关性，其具有相似的羟基含量（P NMR）：在分离的云杉木质素（99% G 单元）上确定了更高的蛋白质亲和力，而在分离的桉树木质素（70% S 单元）上的吸附最低。通过 SPR 研究了酶吸附中静电相互作用的影响，这清楚地表明，电荷屏蔽允许更广泛的蛋白质吸附。总的来说，这项工作增进了我们对生物质中木质素-纤维素酶相互作用的理解，这些生物质未经预处理或预处理很少，并强调了木质素性质的广泛对比影响，为提高木质纤维素糖化和相关过程提供了指导。
