Whole cell biocatalysts can perform numerous industrially-relevant chemical reactions. While they are less expensive than purified enzymes, whole cells suffer from inherent reaction rate limitations due to transport resistance imposed by the cell membrane. Furthermore, it is desirable to immobilize the biocatalysts to enable ease of separation from the reaction mixture. In this study, we used a layer-by-layer (LbL) self-assembly process to create a microbial exoskeleton which, simultaneously immobilized, protected, and enhanced the reactivity of a whole cell biocatalyst. As a proof of concept, we used Escherichia coli expressing homoprotocatechuate 2,3-dioxygenase (HPCD) as a model biocatalyst and coated it with up to ten alternating layers of poly(diallyldimethylammonium chloride) (PDADMAC) and silica. The microbial exoskeleton also protected the biocatalyst against a variety of external stressors including: desiccation, freeze/thaw, exposure to high temperatures, osmotic shock, as well as against enzymatic attack by lysozyme, and predation by protozoa. While we observed increased permeability of the outer membrane after exoskeleton deposition, this had a moderate effect on the reaction rate (up to two-fold enhancement). When the exoskeleton construction was followed by detergent treatment to permeabilize the cytoplasmic membrane, up to 15-fold enhancement in the reaction rate was reached. With the exoskeleton, we increased in the reaction rate constants as much as 21-fold by running the biocatalyst at elevated temperatures ranging from 40 °C to 60 °C, a supraphysiologic temperature range not accessible by unprotected bacteria.
Bibliographical noteFunding Information:
This work was funded through a University of Minnesota, BioTechnology Institute MnDRIVE seed grant. Parts of this work were carried out in the Characterization Facility at the University of Minnesota, which receives partial support from NSF through the MRSEC program. The Hitachi SU-8320 SEM preparation system were provided by NSF MRI DMR-1229263. We thank Dr. Melanie Rogers, and Professor John Lipscomb for assistance with the HPCD plasmid, Sara BinAhmed and Professor Santiago Castrillon for assistance with the zeta potential measurements, Fang Wang for TEM sectioning, and Mr. Joey Benson for collecting the TEM images, and Drs Kelly Aukema, Tony Dodge, and James Christenson for their helpful discussions.
© 2019, The Author(s).