The combination of protein scaffolding with immobilization on silica-surfaces has potential to generate a novel in vitro reactor type for degrading polymeric waste. The concept is based on mimicking known organelles with high membrane-protein activity and surface area like the mitochondrium or endoplasmatic reticulum. Like the surface area of these organelles, we also tried to maximize the surface area of the reactor. Also we wanted to simplify the purification of degradation products as well as generating separated reaction areas inside of the reactor.
Figure 1 Possible design of a in vitro bioreactor. Two separated reaction chambers are filled with a surface enlarging steel structure covered with silica and divided by a selective permeable barrier. The reaction chambers are filled with enzyme solution containing the Scaffold with Si4-tag and Degradation-Ligand fusion proteins. The reaction chambers are provided with fresh enzymes with pipes on top of the reactor. Through pipes on the left the xylan or any other polymer of interest enters the first reaction chamber, after degradation small molecules diffuse through the barrier and can be used for reactions in the second chamber or leave the system through the pipe on the right. The red circle shows a zoom in into one module of the steel structure. The module shows a lamellar design. The red circle shows the scheme of Scaffold immobilisation on the silica surface and boundry of degradation enzymes via ligand domain interactions.
In this example we use two different reaction rooms separated by an Ultrafiltration membrane (pore size 0,22 μL). The solved polymer is transported into the reactor via the pipe on the left hand and is then degraded by the three enzymes Ruxyn1, aes and xynA, attached to the silica-tagged protein scaffold. As the titer of monomers rises the more flux is oriented into the second reaction chamber past the membrane, while the polymeric structure remains in the first due to the small pore size. Also enzymes could not pass the membrane. Different Scaffold / Enzyme mixtures with the same basic protein-scaffold domains are usable with separated pipes for fresh enzyme supplies. Finally, this concept could offer the possibility of high yield surface reactions and simple handling of reaction conditions while avoiding known disadvantages of in vivo production.
(Dueber et al., 2009)
