Proteolytic ceramic capillary membranes for the preparative production of bioactive peptides

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水解产生的，并在胃肠道中被再吸收。这些生物活性肽的功能范围从抗高血压到抗氧化、血液稀释、降低胆固醇、抗微生物和免疫调节特性。到目前为止，只有少数生物活性肽已被系统地鉴定和表征，特别是它们各自的蛋白质来源的分配仍然在很大程度上未被探索。多种多样的生物活性肽可以潜在地从一个单一的蛋白质来源产生，其整体被称为“蛋白酶体”。到目前为止，对蛋白酶体的系统研究仅针对最主要的乳蛋白进行，但是，人们对从其他相关食物蛋白中提取的普遍存在的生物活性肽知之甚少。up项目，旨在对生物活性肽的连续生产进行基础研究，并打算系统地绘制蛋白酶体图谱，以选择相关食品。proteins.为此，开发了一种管式陶瓷毛细管反应器系统。在第一个项目阶段，筛选了各种蛋白酶并将其主动固定在毛细管表面上，特别是丝氨酸蛋白酶Alcalase被证明是非常有效的。所开发的反应器系统的最重要的优点是根据可定制的反应参数高度可再现和精确可调节的特定肽组合物（所谓的肽指纹）的生产。然后，将产生的肽分级分离并通过色谱法以制备规模纯化。在后续项目中，蛋白酶体将通过质谱和计算机消化进行表征，并通过在第一个项目阶段建立的生物活性测定来测试其生物活性。此外，将进一步研究和优化陶瓷毛细管关于孔径和分布、配体密度、表面电荷以及弯曲度的性质，以增加底物和酶的接触时间，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，以获得更小的肽。第一个资助期揭示了开发的陶瓷毛细管反应器系统作为其他蛋白水解过程技术平台的可能性。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（例如His标签）、前体蛋白（例如前胶原至胶原）的成熟或抗体片段Fab和Fc的特异性产生的可能性。

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.