Proteolytic ceramic capillary membranes for the preparative production of bioactive peptides

用于制备生物活性肽的蛋白水解陶瓷毛细管膜

• 批准号: 278836263
• 负责人: Professor Dr. Sascha Beutel
• 金额: --
• 依托单位: Fachgebiet Keramische Werkstoffe und Bauteile
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/278836263?language=en
• 关键词: Proteolytic; ceramic; capillary; membranes; preparative

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.

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水解产生的，并在胃肠道中被吸收。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，抗菌和免疫调节特性。到目前为止，只有少数生物活性肽被系统地鉴定和表征，特别是它们各自蛋白质来源的分配仍然很大程度上未被探索。多种多样的生物活性肽可以从一个单一的蛋白质来源产生，其整体被称为“蛋白溶体”。到目前为止，对蛋白溶体的系统研究只针对最突出的牛奶蛋白，但对从其他相关食品蛋白中提取的普遍存在的生物活性肽知之甚少。这个后续项目旨在对生物活性肽的持续生产进行基础研究，并打算系统地绘制蛋白质水解体，以选择相关的食品蛋白质。为此，研制了一种管状陶瓷毛细管反应器系统。项目第一阶段筛选了多种蛋白酶，并在毛细管表面进行了活性固定化，特别是丝氨酸蛋白酶Alcalase被证明是高效的。开发的反应器系统最重要的优点是高度可重复性和精确可调的生产特定的肽组成（所谓的肽指纹）取决于可定制的反应参数。然后，所产生的多肽在制备规模上通过层析进行分离和纯化。在后续项目中，将通过质谱和硅消化法对蛋白水解体进行表征，并通过生物活性测定法对其生物活性特性进行测试，这些生物活性测定法已在第一个项目阶段建立。进一步研究和优化陶瓷毛细管在孔径和分布、配体密度、表面电荷、弯曲度等方面的性能，既能增加底物与酶的接触时间，又能防止毛细孔膜堵塞。开发的反应器系统将进一步扩大到多毛细管模块，模块的系列组合将被测试以获得更小的肽。第一个资助期揭示了将开发的陶瓷毛细管反应器系统应用于其他蛋白水解工艺的技术平台的可能性。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的可能性，例如his标签，前体蛋白的成熟，例如前胶原到胶原蛋白，或特异性生产抗体片段Fab和Fc。