Proteolytic ceramic capillary membranes for the preparative production of bioactive peptides

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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.