Engineered catalytic biofilms: Site‐specific enzyme immobilization onto E. coli curli nanofibers

Abstract

23Biocatalytic transformations generally rely on purified enzymes or whole cells to perform 24 complex transformations that are used on industrial scales for chemical, drug, and biofuel 25 synthesis, pesticide decontamination and water purification. However, both of these systems 26 have inherent disadvantages related to the costs associated with enzyme purification, the long-27 term stability of immobilized enzymes, catalyst recovery and compatibility with harsh reaction 28 conditions. We developed a novel strategy for producing rationally designed biocatalytic 29 surfaces based on Biofilm Integrated Nanofiber Display (BIND), which exploits the curli system 30 of E. coli to create a functional nanofiber network capable of covalent immobilization of 31 enzymes. This approach is attractive because it is scalable, represents a modular strategy for site-32 specific enzyme immobilization, and has the potential to stabilize enzymes under denaturing 33 environmental conditions. We site-specifically immobilized a recombinant α-amylase, fused to 34 the SpyCatcher attachment domain, onto E. coli curli fibers displaying complementary SpyTag 35 capture domains. We characterized the effectiveness of this immobilization technique on the 36 biofilms and tested the stability of immobilized α-amylase in unfavorable conditions. This 37 enzyme-modified biofilm maintained its activity when exposed to a wide range of pH and 38 organic solvent conditions. In contrast to other biofilm-based catalysts, which rely on cellular 39 metabolism to remain active, the modified curli-based biofilm remained active even after cell 40 death due to organic solvent exposure. This work lays the foundation for a new and versatile 41 method of using the extracellular polymeric matrix of E. coli for creating novel biocatalytic 42 surfaces. 43 44 3

Amyloid Structures as Biofilm Matrix Scaffolds

“…3A). Based on this work, Botyanszki et al (99) exploited the curli system of E. coli to create a biocatalytic biofilm in which a network of functional nanofibers is capable of covalently and site specifically immobilizing an industrially relevant enzyme, ␣-amylase.…”

Portable, On-Demand Biomolecular Manufacturing

“…Nanobody and DARPin sequences were cloned into pET15b. The His-YFPSpyCatcher and -Amylase-SpyCatcher plasmids have been previously cloned and described (Botyanszki et al, 2015;Nguyen et al, 2014) …”

B 12 -dependent photoresponsive protein hydrogels for controlled stem cell/protein release

“…Because of its high efficiency and modularity, this chemistry has led to a number of applications, including control of biomacromolecular topology, synthesis of bioactive and "living" materials, and biomolecular imaging (16,18,(27)(28)(29)(30)(31)(32)(33)(34)(35)(36). It has proven to be a powerful method for constructing complex biomolecular architectures both in vitro and in vivo.…”