Hidayat Budi J, Weisskopf Carmen, Felby Claus, Johansen Katja S, Thygesen Lisbeth G
UniBio a/s, Billedskaerervej 8, 5230, Odense M, Denmark.
Department of Biomaterials, Max Planck Institute of Colloids and Interfaces, Am Műhlenberg 1, Potsdam, 14476, Germany.
AMB Express. 2015 Dec;5(1):76. doi: 10.1186/s13568-015-0165-9. Epub 2015 Dec 1.
Binding of enzymes to the substrate is the first step in enzymatic hydrolysis of lignocellulose, a key process within biorefining. During this process elongated plant cells such as fibers and tracheids have been found to break into segments at irregular cell wall regions known as dislocations or slip planes. Here we study whether cellulases bind to dislocations to a higher extent than to the surrounding cell wall. The binding of fluorescently labelled cellobiohydrolases and endoglucanases to filter paper fibers was investigated using confocal laser scanning microscopy and a ratiometric method was developed to assess and quantify the abundance of the binding of cellulases to dislocations as compared to the surrounding cell wall. Only Humicola insolens EGV was found to have stronger binding preference to dislocations than to the surrounding cell wall, while no difference in binding affinity was seen for any of the other cellulose variants included in the study (H. insolens EGV variants, Trichoderma reesei CBHI, CBHII and EGII). This result favours the hypothesis that fibers break at dislocations during the initial phase of hydrolysis mostly due to mechanical failure rather than as a result of faster degradation at these locations.
酶与底物的结合是木质纤维素酶促水解的第一步，而木质纤维素酶促水解是生物精炼中的关键过程。在此过程中，已发现诸如纤维和管胞等细长的植物细胞会在称为位错或滑移面的不规则细胞壁区域断裂成片段。在这里，我们研究纤维素酶与位错的结合程度是否高于与周围细胞壁的结合程度。使用共聚焦激光扫描显微镜研究了荧光标记的纤维二糖水解酶和内切葡聚糖酶与滤纸纤维的结合，并开发了一种比率法来评估和量化纤维素酶与位错的结合丰度，并与周围细胞壁进行比较。仅发现特异腐质霉的外切葡聚糖酶EGV对位错的结合偏好强于对周围细胞壁的结合偏好，而该研究中包含的任何其他纤维素变体（特异腐质霉EGV变体、里氏木霉CBHI、CBHII和EGII）在结合亲和力上均未观察到差异。这一结果支持了这样的假设，即在水解初始阶段，纤维在位错处断裂主要是由于机械故障，而非这些位置降解更快的结果。
