Enhancing cellulase activity through single-molecule imaging and protein engineering as a testbed for understanding and improving enzymatic deconstruction of insoluble substrates

通过单分子成像和蛋白质工程增强纤维素酶活性，作为理解和改进不溶性底物酶解构的测试平台

• 批准号: 2301377
• 负责人: William Hancock
• 金额: $ 63万
• 依托单位: Pennsylvania State Univ University Park
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-15 至 2026-03-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2301377&HistoricalAwards=false
• 关键词: Enhancing; cellulase; activity; through; single

Plant-derived cellulose is the most abundant biopolymer on Earth. Cellulose is used to make paper, clothing, and building materials. It is composed of chains of glucose molecules. Cellulases are enzymes that cut these chains into their sugar building blocks. One approach for reducing our reliance on fossil fuels is to digest abundant, non-food plant materials like switchgrass and discarded corn stalks into sugar molecules. These can be fermented into renewable biofuels. The objective of this project is to use cutting-edge light microscopy to better understand how cellulases work. The knowledge generated would be used to design and test enzymes that may be more efficient, lowering the cost and increasing the availability of renewable energy. In parallel with research, educational materials to expose plant biology students to single-molecule biophysics and biophysics students to plant biology will be developed and delivered. To strengthen and increase the diversity of the bioeconomy workforce, students from underrepresented populations will be actively recruited to participate in the research project.The ability of cellulases to break down plant cell walls is hindered by the crystallinity of cellulose. The presence of other polymers such as lignin and xylans are thought to coat cellulose and thus block access by cellulase enzymes. This project will combine single-molecule fluorescence tracking, computational modeling, and protein engineering to investigate cellulase mechanisms. The proposed experiments build on ongoing work in which the investigators constructed a custom multimodal microscope and used it to track cellulases moving along cellulose with nanometer-scale precision. The work is divided into three Aims that explore specific aspects of cellulase activity. The goal of Aim 1 is to uncover the key principles that regulate substrate binding and threading of the cellulose polymer strand into the catalytic tunnel of the cellulase enzyme to initiate cellulose deconstruction. The goal of Aim 2 is to uncover how the substrate affinity and turnover rate of cellulases are tuned to maximize the enzyme’s catalytic activity while minimizing product inhibition and premature substrate release. The goal of Aim 3 is to identify mechanisms by which cellulases navigate complex mixtures of biopolymers, while achieving specific digestion of their cellulase substrate, avoiding off-target binding, and overcoming roadblocks imposed by cell wall complexity that hamper cellulose degradation.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

植物性的纤维素是最丰富的生物聚合物。诸如开关的材料和将玉米茎放入糖分子中。这是可再生能源的效率。在纤维素中，纤维素的纤维素是纤维素的结晶性，例如木质素和xylans的存在。蛋白质工程机制1是要揭示纤维素聚合物链的底物结合到纤维素酶的催化性调节中。同时，通过纤维素酶的复杂混合物来最大程度地减少生物聚合物的复杂混合物，同时实现了其细胞酶底物的特定消化，并克服了由于复杂性而刺激的障碍，以阻碍纤维素降解利用基金会的知识分子和更广泛的影响审查标准。

项目成果

Xylan inhibition of cellulase binding and processivity observed at single-molecule resolution

• DOI: 10.1039/d4su00006d
• 发表时间: 2024-04-04
• 期刊: RSC SUSTAINABILITY
• 作者: Zexer，Nerya;Paradiso，Alec;Anderson，Charles T.
• 通讯作者: Anderson，Charles T.