Proteolytic ceramic capillary membranes for the preparative production of bioactive peptides

多肽是生物体中用途最广泛的一类物质。它们是由传统食物蛋白质的水解产生的，并在胃肠道中被吸收。这些生物活性多肽的功能范围从降高血压到抗氧化、血液稀释、降低胆固醇、抗菌和免疫调节特性。到目前为止，只有很少的生物活性多肽被系统地鉴定和表征，特别是它们各自的蛋白质来源的归属在很大程度上仍然没有被探索。一种单一的蛋白质来源可以潜在地产生多种生物活性多肽，整个蛋白质来源被称为“蛋白水解体”。到目前为止，仅对最重要的乳蛋白进行了系统的研究，但对从其他相关食品蛋白中提取的无处不在的生物活性多肽知之甚少。该后续项目旨在为生物活性多肽的持续生产奠定基础，并打算系统地定位蛋白聚合体，以选择相关的食品蛋白。为此，研制了一种管式陶瓷毛细管反应器系统。在第一期工程中，我们筛选了多种酶，并在毛细管表面进行了活性固定化，其中丝氨酸蛋白酶Alcalase被证明是高效的。开发的反应器系统最重要的优点是根据可定制的反应参数，高度可重复性和精确可调地生产特定的多肽成分(所谓的多肽指纹图谱)。然后，通过层析的方法在制备规模上对所产生的多肽进行分级和纯化。在后续项目中，蛋白质水解体将通过质谱学和硅胶消化进行表征，并通过在第一个项目阶段建立的生物活性分析来测试其生物活性。此外，还将进一步研究和优化陶瓷毛细管的孔径大小和分布、配体密度、表面电荷以及弯曲度等性能，以增加底物与酶的接触时间，同时防止毛细管膜的孔堵塞。开发的反应器系统将进一步扩大到多毛细管模块，并将测试模块的串联组合以获得更小的多肽。第一个资助期表明，开发的陶瓷毛细管反应器系统可能应用于其他蛋白质降解过程的技术平台。因此，后续项目还旨在评估使用反应器系统连续切割融合结构域的可能性，例如His-tag，前体蛋白的成熟，例如前胶原到胶原，或特异性产生抗体片段Fab和Fc。

Peptides are one of the most versatile classes of substances in living organisms. They are generated by hydrolysis of conventional food proteins and are resorbed in the gastrointestinal tract. The functionality of these bioactive peptides ranges from antihypertensive to antioxidative, blood-thinning, cholesterol-lowering, antimicrobial and immunomodulating properties. As of now, only few bioactive peptides have been systematically identified and characterized and especially the assignment to their respective protein source is still largely unexplored. A wide variety of bioactive peptides can be potentially generated from one single protein source, the entirety of which is called the “proteolysome”. So far, a systematic investigation of the proteolysome has been carried out only for the most prominent milk proteins, but little is known about the ubiquitous bioactive peptides which can be derived from other relevant food proteins.This is addressed by this follow-up project which aims at the fundamental investigation of the continuous production of bioactive peptides and intends to systematically map the proteolysome for a selection of relevant food proteins. For this purpose, a tubular ceramic capillary reactor system was developed. In the first project phase various proteases were screened and actively immobilized on the capillary surface, especially the serine protease Alcalase proved to be highly effective. The most important advantage of the developed reactor system is the highly reproducible and exactly adjustable production of specific peptide compositions (so called peptide fingerprints) depending on tailorable reaction parameters. Afterwards, the produced peptides are fractionated and purified at preparative scale by means of chromatography. In the follow-up project, the proteolysome will be characterized by mass spectroscopy and in silico digestion and tested regarding their bioactive properties with bioactivity assays, which were established in the first project phase. Furthermore, the properties of the ceramic capillaries regarding pore size and distribution, ligand density, surface charge, as well as tortuosity will be further studied and optimized in order to both increase contact time of substrate and enzyme and at the same time to prevent blocking of the pores of the capillary membrane. The developed reactor system will be further scaled up to a multi-capillary module and the serial combination of modules will be tested to obtain even smaller peptides.The first funding period revealed the possibility to apply the developed ceramic capillary reactor system as a technology platform for other proteolytic processes. Therefore, the follow-up project also aims to evaluate the possibility to use the reactor system for a continuous cleavage of fusion domains, such as e.g. His-tags, a maturation of precursor proteins, such as e.g. pro-collagen to collagen, or the specific production of antibody fragments Fab and Fc.