Person: Botyanszki, Zsofia
Loading...
Email Address
AA Acceptance Date
Birth Date
Research Projects
Organizational Units
Job Title
Last Name
Botyanszki
First Name
Zsofia
Name
Botyanszki, Zsofia
3 results
Search Results
Now showing 1 - 3 of 3
Publication Engineered Curli-Expressing Biofilms as a Platform for Biocatalysis(2015-12-14) Botyanszki, Zsofia; Joshi, Neel S.; Aizenberg, Joanna; Hochschild, AnnResearch 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 α-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.Publication Programmable biofilm-based materials from engineered curli nanofibres(Nature Publishing Group, 2014) Nguyen, Peter; Botyanszki, Zsofia; Tay, Pei Kun Richie Richie; Joshi, NeelThe significant role of biofilms in pathogenicity has spurred research into preventing their formation and promoting their disruption, resulting in overlooked opportunities to develop biofilms as a synthetic biological platform for self-assembling functional materials. Here we present Biofilm-Integrated Nanofiber Display (BIND) as a strategy for the molecular programming of the bacterial extracellular matrix material by genetically appending peptide domains to the amyloid protein CsgA, the dominant proteinaceous component in Escherichia coli biofilms. These engineered CsgA fusion proteins are successfully secreted and extracellularly self-assemble into amyloid nanofibre networks that retain the functions of the displayed peptide domains. We show the use of BIND to confer diverse artificial functions to the biofilm matrix, such as nanoparticle biotemplating, substrate adhesion, covalent immobilization of proteins or a combination thereof. BIND is a versatile nanobiotechnological platform for developing robust materials with programmable functions, demonstrating the potential of utilizing biofilms as large-scale designable biomaterials.Publication Engineered catalytic biofilms: Site-specific enzyme immobilization onto E. coli curli nanofibers(Wiley-Blackwell, 2015) Botyanszki, Zsofia; Tay, Pei Kun Richie Richie; Nguyen, Peter; Nussbaumer, Martin; Joshi, NeelBiocatalytic 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. Biotechnol. Bioeng. 2015;112: 2016–2024. © 2015 Wiley Periodicals, Inc.