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

在当今的化学合成中，酶催化的反应是必不可少的，这是因为酶在环境反应条件下能够以无与伦比的选择性执行困难的转化。然而，大多数商业化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。目前的固定化方法面临着难以预测的固定化成功、酶在各自载体上的滞留以及固定化对酶活性的影响等问题。因此，对扩大现有固定化策略组合的新方法的需求很高。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动固定化方法来应对这些挑战。它是基于生物技术主力枯草芽孢杆菌的内孢子壳，最近被描述为一种新型的蛋白质展示平台(所谓的SporoBeads)。应建立和优化它，以便在技术介质中进行要求苛刻的酶催化反应。此外，应将其作为直接在技术(固定化)条件下筛选酶变体催化性能的设备进行评估。为了具有实际意义，这项研究将集中在目前很少被研究，但合成上非常有吸引力的脂肪酶催化硅基醚键转化作为实现这些目标的模型系统，需要结合脂肪酶介导的技术生物催化的专业知识和应用内孢子分化的知识以及有效地对革兰氏阳性模式生物枯草杆菌进行基因工程的能力，这反映在这项联合计划的两个PI的共同努力中。将探索用于硅醚转化的脂肪酶组合，并将SporoBeads建立为不同结构复杂性脂肪酶的自动固定化平台。所获得的知识将结合起来评估满足脂肪酶功能化SporoBead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，将首先实施用于硅醚裂解和形成的脂肪酶的结构指导改进，并将评估SporoBeads作为遗传筛选工具的潜力。一旦这些前提条件得到满足，展示改进的脂肪酶的SporoBeads将被优化，以在苛刻的生物催化条件下发挥作用。最终，该项目中获得的经验还将提供一种工作流程和策略，用于交互和有效地优化和调整反应条件和孢子显示，当应用SporoBeads与其他酶进行苛刻的生物催化和筛选时。

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.