Auto-immobilization for demanding enzyme catalysis using lipase-functionalized Bacillus endospores

酶催化反应在当今的化学合成中是不可缺少的，因为酶在环境反应条件下以无与伦比的选择性进行困难的转化。然而，大多数商业上开发的酶需要固定化这些生物催化剂来提高它们的活性、稳健性和长期稳定性。目前的固定化方法受到不可预测的固定化成功、酶在各自载体上的保留以及固定化对酶活性的影响的挑战。因此，扩大目前固定策略组合的新方法需求量很大。该项目旨在通过开发一种新的、可持续的基于生物的催化活性酶的自固定方法来解决这些挑战。它是基于生物技术骨干枯草芽孢杆菌的孢子内外壳，它最近被描述为一种新的蛋白质展示平台（所谓的SporoBeads）。它的建立和优化是为了在技术介质中进行高要求的酶催化反应。此外，它应被评估为在技术（固定化）条件下直接筛选酶变体催化性能的装置。为了适当的实际意义，本研究将重点放在目前很少被研究，但合成非常有吸引力的脂肪酶催化的硅醚键转化作为模型系统。要实现这些目标，需要将脂肪酶介导的技术生物催化的专业知识与应用内孢子分化的知识和有效地基因工程改造革兰氏阳性模式生物枯草芽孢杆菌的能力相结合。正如这一联合建议的两位PI的共同努力所反映的那样。最初，将探索和建立该项目的使能技术。将探索用于转化硅醚的脂肪酶组合，并将SporoBeads建立为不同结构复杂性的脂肪酶的自动固定平台。所获得的知识将被结合起来评估满足脂肪酶功能化SporoBeads合成应用的特定要求的潜力。为了建立SporoBeads作为蛋白质设计的筛选平台，首先将实施结构导向的脂肪酶对硅醚裂解和形成的改进，并评估SporoBeads作为遗传筛选工具的潜力。一旦这些先决条件得到满足，SporoBeads将在苛刻的生物催化条件下显示改进的脂肪酶性能。最终，本项目所获得的经验也将为SporoBeads应用于与其他酶的生物催化和筛选时，对反应条件和孢子展示进行交互、高效的优化和调整提供工作流程和策略。

Enzyme-catalyzed reactions are indispensible in nowadays chemical synthesis, due to the ability of enzymes to perform difficult conversions with unrivaled selectivity at ambient reaction conditions. However, most commercially exploited enzymes require immobilization of these biocatalysts to enhance their activity, robustness and long-term stability. Current immobilization methods are challenged by the unpredictable success of immobilization, the retention of the enzymes on their respective carrier, and the influence that immobilization has on the enzymatic activity. Novel approaches that expand the current portfolio of immobilization strategies are therefore in high demand.This project aims at addressing these challenges by developing a novel and sustainable biology-based auto-immobilization approach for catalytically active enzymes. It is based on the endospore crust of the biotechnological workhorse Bacillus subtilis, which has recently been described as a novel protein display platform (so-called SporoBeads). It shall be established and optimized for conducting demanding enzyme-catalyzed reactions in technical media. Additionally, it shall be evaluated as a device for screening the catalytic performance of enzyme variants directly under technical (immobilized) conditions. For proper practical relevance, this study will focus on the currently hardly investigated, but synthetically very attractive lipase-catalyzed conversion of silyl ether bonds as a model system Reaching these goals requires combining expertise in lipase-mediated technical biocatalysis with the knowledge on applying endospore differentiation and the ability to efficiently genetically engineering the Gram-positive model organism B. subtilis, as reflected by combined efforts of the two PI’s of this joint proposal.Initially, enabling technologies for this project will be explored and established. The lipase portfolio for conversion of silyl ethers will be explored and SporoBeads will be established as an auto-immobilization platform for lipases of varying structural complexities. The knowledge gained will be combined to evaluate the potential for meeting specific requirements for synthetic application of lipase-functionalized SporoBeads. Towards establishment of SporoBeads as a screening platform for protein design, structure-guided improvements of lipases for silyl ether cleavage and formation will first be implemented, and the potential of applying SporoBeads as genetic screening tools will be evaluated. Once these prerequisites have been met, SporoBeads displaying improved lipases for performance under demanding biocatalysis conditions will be optimized.Ultimately, the experiences gained in this project shall also provide a workflow and strategy for interactively and efficiently optimizing and adjusting both the reaction conditions and the spore-display, when applying SporoBeads for demanding biocatalysis with other enzymes and for screening.