| Research and innovation in recent years has led to a paradigm shift in the bioengineering community regarding biofilms. Rather than focusing on their negative impact in disease and materials fouling, we, and other groups, are re-conceptualizing biofilms as an engineering platform. Our group has developed a novel strategy for producing rationally designed biocatalytic surfaces based on Biofilm Integrated Nanofiber Display (BIND). BIND uses the E. coli curli system to create extracellular functional nanofiber networks. In this work, we used protein capture tags displayed on curli fibers to transform biofilms into biocatalytic surfaces. We wanted to address the issues that currently hinder the applicability of surface displayed enzyme catalysts -- although genetically engineered and scalable, the enzymes displayed through genetic fusion to cell surface proteins are often improperly folded and have reduced activity. Our catalytic-BIND platform takes advantage of the scalability of biofilms and contains a modular strategy for site-specific immobilization of pre-folded enzymes. We investigated the adhesion, stability and reproducibility of engineered biofilms displaying a protein immobilization tag, SpyTag, on micro-plates, filter plates, glass slides, and under flow in microfluidic devices and a laboratory-scale bioreactor. We found that while the biofilms grew most robustly under flow, the stability and sample-to-sample reproducibility necessary for characterization of the biocatalytic surface was most optimal in the filter plates. Next, we immobilized a recombinant alpha-amylase, fused to SpyTag's complementary capture domain, SpyCatcher. We showed that Amylase-SpyCatcher was successfully immobilized onto the biofilms, tested the stability of the immobilized enzymes in a variety of harsh conditions, and explored methods for quantifying the amount of curli fibers and enzymes in the biofilms. Finally, we used enzyme activity as a readout for evaluating the immobilization kinetics of Amylase-SpyCatcher onto filtered and suspension biofilms. From this analysis, we found that, currently, SpyTag accessibility in the biofilms is the major barrier to achieving high immobilization efficiencies. This work lays the foundation for a new, versatile method of creating biocatalytic surfaces that has the potential to become an alternative platform for catalyzing reactions of membrane impermeable substrates. |
