Thermostability enhancement of polyethylene terephthalate degrading PETase using self- and nonself-ligating protein scaffolding approaches.

Polyethylene terephthalate (PET) hydrolase enzymes show promise for enzymatic PET degradation and green recycling of single-use PET vessels representing a major source of global pollution. Their full potential can be unlocked with enzyme engineering to render activities on recalcitrant PET substrates commensurate with cost-effective recycling at scale. Thermostability is a highly desirable property in industrial enzymes, often imparting increased robustness and significantly reducing quantities required. To date, most engineered PET hydrolases show improved thermostability over their parental enzymes. Here, we report engineered thermostable variants of Ideonella sakaiensis PET hydrolase enzyme (IsPETase) developed using two scaffolding strategies. The first employed SpyCatcher-SpyTag technology to covalently cyclize IsPETase, resulting in increased thermostability that was concomitant with reduced turnover of PET substrates compared to native IsPETase. The second approach using a GFP-nanobody fusion protein (vGFP) as a scaffold yielded a construct with a melting temperature of 80°C. This was further increased to 85°C when a thermostable PETase variant (FAST PETase) was scaffolded into vGFP, the highest reported so far for an engineered PET hydrolase derived from IsPETase. Thermostability enhancement using the vGFP scaffold did not compromise activity on PET compared to IsPETase. These contrasting results highlight potential topological and dynamic constraints imposed by scaffold choice as determinants of enzyme activity.

The Role of Structural Order in the Mechanism of Charge Transport across Tunnel Junctions with Various Iron-Storing Proteins.

In biomolecular electronics, the role of structural order in charge transport (CT) is poorly understood. It has been reported that the metal oxide cores of protein cages (e.g., iron oxide and ferrihydrite nanoparticles (NPs) present in ferritin and E2-LFtn, which is E2 protein engineered with an iron-binding sequence) play an important role in the mechanism of CT. At the same time, the NP core also plays a major role in the structural integrity of the proteins. This paper describes the role of structural order in CT across tunnel junctions by comparing three iron-storing proteins. They are (1) DNA binding protein from starved cells (Dps, diameter (∅) = 9 nm); (2) engineered archaeal ferritin (AfFtn-AA, ∅ = 12 nm); and (3) engineered E2 of pyruvate dehydrogenase enzyme complex (E2-LFtn, ∅ = 25 nm). Both holo-Dps and apo-Dps proteins undergo CT by coherent tunneling because their globular architecture and relative structural stability provide a coherent conduction pathway. In contrast, apo-AfFtn-AA forms a disordered structure across which charges have to tunnel incoherently, but holo-AfFtn-AA retains its globular structure and supports coherent tunneling. The large E2-LFtn always forms disordered structures across which charges incoherently tunnel regardless of the presence of the NP core. These findings highlight the importance of structural order in the mechanism of CT across biomolecular tunnel junctions.

Temperature-Dependent Coherent Tunneling across Graphene-Ferritin Biomolecular Junctions.

Understanding the mechanisms of charge transport (CT) across biomolecules in solid-state devices is imperative to realize biomolecular electronic devices in a predictive manner. Although it is well-accepted that biomolecule-electrode interactions play an essential role, it is often overlooked. This paper reveals the prominent role of graphene interfaces with Fe-storing proteins in the net CT across their tunnel junctions. Here, ferritin (AfFtn-AA) is adsorbed on the graphene by noncovalent amine-graphene interactions confirmed with Raman spectroscopy. In contrast to junctions with metal electrodes, graphene has a vanishing density of states toward its intrinsic Fermi level ("Dirac point"), which increases away from the Fermi level. Therefore, the amount of charge carriers is highly sensitive to temperature and electrostatic charging (induced doping), as deduced from a detailed analysis of CT as a function of temperature and iron loading. Remarkably, the temperature dependence can be fully explained within the coherent tunneling regime due to excitation of hot carriers. Graphene is not only demonstrated as an alternative platform to study CT across biomolecular tunnel junctions, but it also opens rich possibilities in employing interface electrostatics in tuning CT behavior.

Biomolecular control over local gating in bilayer graphene induced by ferritin.

Electrical field-induced charge modulation in graphene-based devices at the nanoscale with ultrahigh density carrier accumulation is important for various practical applications. In bilayer graphene (BLG), inversion symmetry can simply be broken by an external electric field. However, control over charge carrier density at the nanometer scale is a challenging task. We demonstrate local gating of BLG in the nanometer range by adsorption of AfFtnAA (which is a bioengineered ferritin, an iron-storing globular protein with ∅ = 12 nm). Low-temperature electrical transport measurements with field-effect transistors with these AfFtnAA/BLG surfaces show hysteresis with two Dirac peaks. One peak at a gate voltage V BG = 35 V is associated with pristine BLG, while the second peak at V BG = 5 V results from local doping by ferritin. This charge trapping at the biomolecular length scale offers a straightforward and non-destructive method to alter the local electronic structure of BLG.

The influences of substrates' physical properties on enzymatic PET hydrolysis: Implications for PET hydrolase engineering.

Plastic pollution in diverse terrestrial and marine environments is a widely recognised and growing problem. Bio-recycling and upcycling of plastic waste is a potential solution to plastic pollution, as these processes convert plastic waste into useful materials. Polyethylene terephthalate (PET) is the most abundant plastic waste, and this material can be degraded by a class of recently discovered bacterial esterase enzymes known as PET hydrolases (PETase). Investigations of the enzymatic hydrolysis of diverse PET molecules have clearly revealed that the biodegradability of various PET substrates depends on both their chemical structure and physical properties, including polymer length, crystallinity, glass transition temperature, surface area, and surface charge. This review summarises the known impacts of crystallinity and other physical properties on enzymatic PET hydrolysis.

Solid-State Protein Junctions: Cross- Laboratory Study Shows Preservation of Mechanism at Varying Electronic Coupling.

Successful integration of proteins in solid-state electronics requires contacting them in a non-invasive fashion, with a solid conducting surface for immobilization as one such contact. The contacts can affect and even dominate the measured electronic transport. Often substrates, substrate treatments, protein immobilization, and device geometries differ between laboratories. Thus the question arises how far results from different laboratories and platforms are comparable and how to distinguish genuine protein electronic transport properties from platform-induced ones. We report a systematic comparison of electronic transport measurements between different laboratories, using all commonly used large-area schemes to contact a set of three proteins of largely different types. Altogether we study eight different combinations of molecular junction configurations, designed so that Ageoof junctions varies from 105 to 10-3 µm2. Although for the same protein, measured with similar device geometry, results compare reasonably well, there are significant differences in current densities (an intensive variable) between different device geometries. Likely, these originate in the critical contact-protein coupling (∼contact resistance), in addition to the actual number of proteins involved, because the effective junction contact area depends on the nanometric roughness of the electrodes and at times, even the proteins may increase this roughness. On the positive side, our results show that understanding what controls the coupling can make the coupling a design knob. In terms of extensive variables, such as temperature, our comparison unanimously shows the transport to be independent of temperature for all studied configurations and proteins. Our study places coupling and lack of temperature activation as key aspects to be considered in both modeling and practice of protein electronic transport experiments.

Production of Hollow Bacterial Cellulose Microspheres Using Microfluidics to Form an Injectable Porous Scaffold for Wound Healing.

Bacterial cellulose (BC) is a biocompatible material with high purity and robust mechanical strength used to fabricate desirable scaffolds for 3D cell culture and wound healing. However, the chemical resistance of BC and its insolubility in the majority of solutions make it difficult to manipulate using standard chemical methods. In this study, a microfluidic process is developed to produce hollow BC microspheres with desirable internal structures and morphology. Microfluidics is used to generate a core-shell structured microparticle with an alginate core and agarose shell as a template to encapsulate Gluconacetobacter xylinus for long-term static culture. G. xylinus then secretes BC, which becomes entangled within the shell of the structured hydrogel microparticles and forms BC microspheres. The removal of the hydrogel template via thermal-chemical treatments yields robust BC microspheres exhibiting a hollow morphology. These hollow microspheres spontaneously assemble as functional units to form a novel injectable scaffold. In vitro, a highly porous scaffold is created to enable effective 3D cell culture with a high cell proliferation rate and better depth distribution. In vivo, this injectable scaffold facilitates tissue regeneration, resulting in rapid wound-healing in a Sprague Dawley rat skin model.

Long-Range Tunneling Processes across Ferritin-Based Junctions.

The mechanism of long-range charge transport across tunneling junctions with monolayers of ferritin is investigated. It is shown that the mechanism can be switched between coherent tunneling, sequential tunneling, and hopping by changing the iron content inside the ferritin. This study shows that ferritins are an interesting class of biomolecules to control charge transport.