Immobilization of trypsin on a 3D printed support

Carriers bearing immobilized enzymes are the key components in enzyme bioreactors such as packed bed bioreactors. In this work, carriers have been 3D printed with complex geometries to achieve a monolithic bed with perfectly ordered structure – as opposed to current randomly packed beds. Two different material compositions have been 3D printed in an easy one-step process using a digital light processing (DLP) printer. The materials were obtained by copolymerisation of an acrylate crosslinker to impart mechanical resistance and the bifunctional monomer 2-carboxyethyl acrylate (CEA) which displays both a carboxylic group for enzyme immobilization and an acrylate group for its incorporation in the polymeric network. Pore forming agents were also introduced to increase the active surface area for binding the substrate. A photoinitiator and a photoabsorber were also part of the resin composition to trigger the polymerisation and to enhance the resolution of the 3D printed objects, respectively. Covalent bonding of trypsin was successfully demonstrated using BCA assay, while the catalytic activity of the immobilised trypsin was confirmed by monitoring the hydrolysis reaction of BAEE as test substrate. Hollow cylinders were initially printed to test the materials in batch experiments. More complex structures based on Schoen’s gyroid were also produced and tested in dynamic experiments as monolithic bioreactor under continuous flow. The bioreactors were operated at steady state conditions and product formation was observed at all flow rates tested. Although the concentration of the product in the outlet stream decreased at higher flow rates, the productivity expressed in terms of amount of product formed per unit time rose with flow rate. This work demonstrates, for the first time, the feasibility of 3D printed bioreactors with immobilized enzymes. The present work was carried out at the Institute for Bioengineering at the University of Edinburgh, UK.