Enhancement of biocatalyst activity and protection against stressors using a microbial exoskeleton

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.