Artificial cell synthesis using biocatalytic polymerization-induced self-assembly.

Artificial cells are biomimetic microstructures that mimic functions of natural cells, can be applied as building blocks for molecular systems engineering, and host synthetic biology pathways. Here we report enzymatically synthesized polymer-based artificial cells with the ability to express proteins. Artificial cells were synthesized using biocatalytic atom transfer radical polymerization-induced self-assembly, in which myoglobin synthesizes amphiphilic block co-polymers that self-assemble into structures such as micelles, worm-like micelles, polymersomes and giant unilamellar vesicles (GUVs). The GUVs encapsulate cargo during the polymerization, including enzymes, nanoparticles, microparticles, plasmids and cell lysate. The resulting artificial cells act as microreactors for enzymatic reactions and for osteoblast-inspired biomineralization. Moreover, they can express proteins such as a fluorescent protein and actin when fed with amino acids. Actin polymerizes in the vesicles and alters the artificial cells' internal structure by creating internal compartments. Thus, biocatalytic atom transfer radical polymerization-induced self-assembly-derived GUVs can mimic bacteria as they are composed of a microscopic reaction compartment that contains genetic information for protein expression upon induction.

Engineered myoglobin as a catalyst for atom transfer radical cyclisation.

Myoglobin was subjected to site-directed mutagenesis and transformed into a catalyst able to perform atom transfer radical cyclisation reactions, i.e. intramolecular atom transfer radical additions. Replacing the iron-coordinating histidine with serine, or introducing small changes inside or at the entrance of the active site, transformed the completely inactive wild-type myoglobin into an artificial metalloenzyme able to catalyse the 5-exo cyclisation of halogenated unsaturated compounds for the synthesis of γ-lactams. This new-to-nature activity was achieved not only with purified protein but also in crude cell lysate and in whole cells.

Peroxidase Activity of Myoglobin Variants Reconstituted with Artificial Cofactors.

Myoglobin (Mb) can react with hydrogen peroxide (H2 O2 ) to form a highly active intermediate compound and catalyse oxidation reactions. To enhance this activity, known as pseudo-peroxidase activity, previous studies have focused on the modification of key amino acid residues of Mb or the heme cofactor. In this work, the Mb scaffold (apo-Mb) was systematically reconstituted with a set of cofactors based on six metal ions and two ligands. These Mb variants were fully characterised by UV-Vis spectroscopy, circular dichroism (CD) spectroscopy, inductively coupled plasma mass spectrometry (ICP-MS) and native mass spectrometry (nMS). The steady-state kinetics of guaiacol oxidation and 2,4,6-trichlorophenol (TCP) dehalogenation catalysed by Mb variants were determined. Mb variants with iron chlorin e6 (Fe-Ce6) and manganese chlorin e6 (Mn-Ce6) cofactors were found to have improved catalytic efficiency for both guaiacol and TCP substrates in comparison with wild-type Mb, i. e. Fe-protoporphyrin IX-Mb. Furthermore, the selected cofactors were incorporated into the scaffold of a Mb mutant, swMb H64D. Enhanced peroxidase activity for both substrates were found via the reconstitution of Fe-Ce6 into the mutant scaffold.

A Methodology for Remote Microwave Sterilization Applicable to the Coronavirus and Other Pathogens Using Retrodirective Antenna Arrays.

This paper describes an innovative remote surface sterilization approach applicable to the new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The process is based on the application of a liquid film on the surface or object under sterilization (OUS). A beacon signal is used to self-steer the transmitted power from the designed retrodirective antenna array (RDA) towards the OUS using circularly polarized fields; then, the sterilization is completed by raising and maintaining the required temperature for a certain time. Results suggest that the process takes 5 minutes or less for an angular coverage range over 60 degrees whilst abiding by the relevant safety protocols. This paper also models the power incident onto the OUS, providing consistent results with full-wave simulations. A practical RDA system is developed using a 2 × 1 microstrip patch array operating at 2.5 GHz and tested through the positioning of a representative target surface. Measurements, developed by sampling the power transmitted by the heterodyne RDA, are reported for various distances and angles, operating in the near-field of the system. To further validate the methodology, an additional experiment investigating virus deactivation through microwave heating was also developed. Measurements have been performed with an open cavity microwave oven on the Coronavirus (strain 229E) and egg white protein in a cuvette. This demonstrates that the temperature increases of aqueous films up to 70 [Formula: see text]C by remote microwave-induced heat can denature proteins and deactivate viruses. Possible applications of the method include sterilization of ambulances, medical equipment, and internet of things (IoT) devices.

Enzyme-initiated free radical polymerizations of vinyl monomers using horseradish peroxidase.

In this chapter, we highlight the use of horseradish peroxidase (HRP) as a catalyst to initiate free radical polymerizations of vinyl monomers under benign reaction conditions. A variety of vinyl monomers, including 4-acryloylmorpholine (AM), 2-hydroxyethyl methacrylate (HEMA), and poly(ethylene glycol) methyl ether acrylate (PEGA) were polymerized. The enzyme converts exogenous hydrogen peroxide into a usable radical source, which when coupled with a ß-diketone, yields a radical that initiates chain growth in the presence of monomers. The resulting polymers were characterized using nuclear magnetic resonance (NMR) spectroscopy and gel permeation chromatography (GPC). By using enzymatic free radical polymerizations, polymers can be generated in a sustainable, environmentally-friendly, and scalable fashion.