Institut für Molekulare Enzymtechnologie, Heinrich-Heine-Universität Düsseldorf, Forschungszentrum Jülich, 52425, Jülich, Germany.
Bioeconomy Science Center (BioSC), c/o, Forschungszentrum Jülich, 52425, Jülich, Germany.
Microb Cell Fact. 2019 Feb 7;18(1):33. doi: 10.1186/s12934-019-1081-5.
Immobilization is an appropriate tool to ease the handling and recycling of enzymes in biocatalytic processes and to increase their stability. Most of the established immobilization methods require case-to-case optimization, which is laborious and time-consuming. Often, (chromatographic) enzyme purification is required and stable immobilization usually includes additional cross-linking or adsorption steps. We have previously shown in a few case studies that the molecular biological fusion of an aggregation-inducing tag to a target protein induces the intracellular formation of protein aggregates, so called inclusion bodies (IBs), which to a certain degree retain their (catalytic) function. This enables the combination of protein production and immobilization in one step. Hence, those biologically-produced immobilizates were named catalytically-active inclusion bodies (CatIBs) or, in case of proteins without catalytic activity, functional IBs (FIBs). While this strategy has been proven successful, the efficiency, the potential for optimization and important CatIB/FIB properties like yield, activity and morphology have not been investigated systematically.
We here evaluated a CatIB/FIB toolbox of different enzymes and proteins. Different optimization strategies, like linker deletion, C- versus N-terminal fusion and the fusion of alternative aggregation-inducing tags were evaluated. The obtained CatIBs/FIBs varied with respect to formation efficiency, yield, composition and residual activity, which could be correlated to differences in their morphology; as revealed by (electron) microscopy. Last but not least, we demonstrate that the CatIB/FIB formation efficiency appears to be correlated to the solvent-accessible hydrophobic surface area of the target protein, providing a structure-based rationale for our strategy and opening up the possibility to predict its efficiency for any given target protein.
We here provide evidence for the general applicability, predictability and flexibility of the CatIB/FIB immobilization strategy, highlighting the application potential of CatIB-based enzyme immobilizates for synthetic chemistry, biocatalysis and industry.
固定化是一种在生物催化过程中缓解酶的处理和回收并提高其稳定性的合适工具。大多数已建立的固定化方法需要针对具体情况进行优化，这既费力又费时。通常需要（色谱）酶纯化，并且稳定的固定化通常包括额外的交联或吸附步骤。我们之前在一些案例研究中表明，将聚集诱导标签与目标蛋白进行分子生物学融合会导致细胞内形成蛋白质聚集体，即所谓的包涵体（IB），在一定程度上保留其（催化）功能。这使得蛋白质生产和固定化可以一步完成。因此，这些生物产生的固定化剂被命名为催化活性包涵体（CatIB），或者在没有催化活性的蛋白质的情况下，功能 IB（FIB）。虽然该策略已被证明是成功的，但效率、优化潜力以及重要的 CatIB/FIB 性质，如产率、活性和形态，尚未得到系统研究。
我们在这里评估了不同酶和蛋白质的 CatIB/FIB 工具箱。评估了不同的优化策略，例如接头缺失、C 端与 N 端融合以及替代聚集诱导标签的融合。获得的 CatIB/FIB 在形成效率、产率、组成和残余活性方面存在差异，这可以与它们形态上的差异相关联；如电子显微镜所示。最后但同样重要的是，我们证明 CatIB/FIB 的形成效率似乎与目标蛋白的溶剂可及疏水面积相关，为我们的策略提供了基于结构的原理，并为预测任何给定目标蛋白的效率提供了可能性。
我们在这里提供了 CatIB/FIB 固定化策略的一般适用性、可预测性和灵活性的证据，突出了 CatIB 基酶固定化剂在合成化学、生物催化和工业中的应用潜力。
