Abstract
Biocatalytic transformations generally rely on purified enzymes or whole cells to perform complex transformations that are used on industrial scale for chemical, drug, and biofuel synthesis, pesticide decontamination, and water purification. However, both of these systems have inherent disadvantages related to the costs associated with enzyme purification, the long-term stability of immobilized enzymes, catalyst recovery, and compatibility with harsh reaction conditions. We developed a novel strategy for producing rationally designed biocatalytic surfaces based on Biofilm Integrated Nanofiber Display (BIND), which exploits the curli system of E. coli to create a functional nanofiber network capable of covalent immobilization of enzymes. This approach is attractive because it is scalable, represents a modular strategy for site-specific enzyme immobilization, and has the potential to stabilize enzymes under denaturing environmental conditions. We site-specifically immobilized a recombinant α-amylase, fused to the SpyCatcher attachment domain, onto E. coli curli fibers displaying complementary SpyTag capture domains. We characterized the effectiveness of this immobilization technique on the biofilms and tested the stability of immobilized α-amylase in unfavorable conditions. This enzyme-modified biofilm maintained its activity when exposed to a wide range of pH and organic solvent conditions. In contrast to other biofilm-based catalysts, which rely on high cellular metabolism, the modified curli-based biofilm remained active even after cell death due to organic solvent exposure. This work lays the foundation for a new and versatile method of using the extracellular polymeric matrix of E. coli for creating novel biocatalytic surfaces.
Highlights
Biocatalysis provides an environmentally friendly alternative to chemical synthesis with its ability to perform complex chemical transformations in a scalable manner (Wohlgemuth, 2007)
While multiple proteins have been successfully attached to SpyCatcher (Fairhead et al, 2014; Nguyen et al, 2014; Schoene et al, 2014; Zakeri et al, 2012), this paper represents the first example of using the SpyTag and SpyCatcher fusions to immobilize an industry-relevant enzyme onto curli fibers
We demonstrated a novel platform for the immobilization of enzymes onto the extracellular matrix of an engineered biofilm
Summary
Biocatalysis provides an environmentally friendly alternative to chemical synthesis with its ability to perform complex chemical transformations in a scalable manner (Wohlgemuth, 2007). Enzymes are inherently attractive as catalysts due to their ability to perform chemo-, regio- and stereo-selective catalysis even on large, complex molecules This fuels their use in the pharmaceutical industry and elsewhere, as alternatives to less selective synthetic chemical transformations (Murphy, 2012; Pollard and Woodley, 2007). Enzymes can be used in purified form, in crude cell lysates, encased in synthetic protective materials such as a polymer matrix or lipid vesicle, or within whole cells The attributes of these biocatalytic approaches have been extensively reviewed in the literature (Halan et al, 2012; Krishna, 2002; Pollard and Woodley, 2007; Rosche et al, 2009; Zhou and Hartmann, 2012). An approach that optimally combines the criteria of high surface area, enhanced enzyme stability, rapid mass transport, and modularity remains elusive
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