Xylan inhibition of cellulase binding and processivity observed at single-molecule resolution
Xylan inhibition of cellulase binding and processivity observed at single-molecule resolution
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DOI：
10.1039/d4su00006d
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发表时间：
2024-04-04
期刊：
影响因子：
--
通讯作者：
Anderson，Charles T.
中科院分区：
文献类型：
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作者：
Zexer，Nerya;Paradiso，Alec;Anderson，Charles T.
纤维素酶对纤维素的高效降解是利用木质纤维素生物质生产生物能源的关键。在植物细胞壁中，纤维素与木质素和半纤维素结合，是植物生物质降解的关键因素。已知这些非纤维素细胞壁组分干扰纤维素分解酶的功能。虽然木质素的作用已被广泛研究，但木聚糖（植物次生细胞壁中的主要半纤维素）的贡献往往被忽视。为了研究这些影响，我们通过生长细菌纤维素并补充不同浓度的纯化木聚糖来生成模型细胞壁复合物。我们使用单分子显微镜成像和跟踪荧光标记的TrCel7A，一种常用的模型纤维素酶，因为它结合和水解这些合成复合材料中的纤维素。我们发现微量的木聚糖足以显著抑制Cel7A与纤维素的结合。木聚糖的加入也大大降低了移动酶分子的比例，而不影响它们的速度和运行长度。我们建议，当在低浓度下，木聚糖薄涂层纤维素原纤维，并纳入连续补丁时，在较高的浓度。未发现Cel7A与木聚糖的非生产性结合是主要的抑制机制。我们的研究结果强调了在生物质加工过程中靶向木聚糖去除的重要性，并证明了使用单分子成像来研究纤维素分解酶的活性和局限性的潜力。
Efficient cellulose degradation by cellulase enzymes is crucial for using lignocellulosic biomass in bioenergy production. In the cell wall of plants, cellulose is bound by lignin and hemicellulose, which are key factors contributing to the recalcitrance of plant biomass. These non-cellulosic cell wall components are known to interfere with the function of cellulolytic enzymes. While the effects of lignin have been studied extensively, the contribution of xylan, the major hemicellulose in the secondary cell walls of plants, is often overlooked. To study those effects, we generated model cell wall composites by growing bacterial cellulose supplemented with varying concentrations of purified xylan. We used single-molecule microscopy to image and track fluorescently labeled TrCel7A, a commonly used model cellulase, as it binds and hydrolyses cellulose in these synthetic composites. We found that minute amounts of xylan are sufficient to significantly inhibit the binding of Cel7A to cellulose. The inclusion of xylan also reduced considerably the proportion of moving enzyme molecules, without affecting their velocity and run length. We suggest that, when available at low concentrations, xylan thinly coats cellulose fibrils, and incorporates as continuous patches when available at higher concentrations. Non-productive binding of Cel7A to xylan was not found to be a major inhibition mechanism. Our results highlight the importance of targeting xylan removal during biomass processing and demonstrate the potential of using single-molecule imaging to study the activity and limitations of cellulolytic enzymes.