Kahn Amaranta, Galanopoulou Anastasia P, Hatzinikolaou Dimitris G, Moraïs Sarah, Bayer Edward A
Department of Biomolecular Sciences, The Weizmann Institute of Science, Rehovot, Israel.
Microbiology Group, Department of Biology, National and Kapodistrian University of Athens, Athens, Greece.
Methods Mol Biol. 2018;1796:153-166. doi: 10.1007/978-1-4939-7877-9_12.
Enzymatic breakdown of plant biomass is an essential step for its utilization in biorefinery applications, and the products could serve as substrates for the sustainable and environmentally friendly production of fuels and chemicals. Toward this end, the incorporation of enzymes into polyenzymatic cellulosome complexes-able to specifically bind to and hydrolyze crystalline cellulosic materials, such as plant biomass-is known to increase the efficiency and the overall hydrolysis performance of a cellulase system. Despite their relative abundance in various mesophilic anaerobic cellulolytic bacteria, there are only a few reports of cellulosomes of thermophilic origin. However, since various biorefinery processes are favored by elevated temperatures, the development of thermophilic designer cellulosomes could be of great importance. Owing to the limited number of thermophilic cellulosomes, designer cellulosomes, composed of mixtures of mesophilic and thermophilic components, have been constructed. As a result, the overall thermal profile of the individual parts and the resulting complex has to be extensively evaluated. Here, we describe a practical guide for the determination of temperature stability for cellulases in the cellulosome complexes. The approach is also appropriate for other related enzymes, notably xylanases as well as other glycoside hydrolases. We provide detailed experimental procedures for the evaluation of the thermal stability of the individual designer cellulosome components and their complexes as well as protocols for the assessment of complex integrity at elevated temperatures.
植物生物质的酶促分解是其在生物炼制应用中得以利用的关键步骤，其产物可作为可持续且环保的燃料和化学品生产的底物。为此，将酶整合到能够特异性结合并水解结晶纤维素材料（如植物生物质）的多酶纤维小体复合物中，已知可提高纤维素酶系统的效率和整体水解性能。尽管嗜温厌氧纤维素分解菌中纤维小体相对丰富，但关于嗜热来源纤维小体的报道却很少。然而，由于各种生物炼制过程在高温下更具优势，嗜热设计型纤维小体的开发可能具有重要意义。由于嗜热纤维小体数量有限，已构建了由嗜温和嗜热成分混合物组成的设计型纤维小体。因此，必须对各个部分以及所得复合物的整体热特性进行广泛评估。在此，我们描述了一种用于测定纤维小体复合物中纤维素酶温度稳定性的实用指南。该方法也适用于其他相关酶，特别是木聚糖酶以及其他糖苷水解酶。我们提供了评估各个设计型纤维小体成分及其复合物热稳定性的详细实验步骤，以及在高温下评估复合物完整性的方案。
