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

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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.