Assessment of Enzyme Functionality at Metal-Organic Framework Interfaces Developed through Molecular Simulations.

The catalytic efficiency and unrivaled selectivity with which enzymes convert substrates to products have been tapped for widespread chemical transformations within biomedical technology, biofuel production, gas sensing, and the upgrading of commodity chemicals, just to name a few. However, the feasibility of enzymes implementation is challenged by the lack of reusability and loss of native catalytic activity due to the irreversible biocatalyst denaturation at high temperatures and in the presence of industrial solvents. Enzyme immobilization, a prerequisite for enzyme reusability, offers controllable strategies for increased functional viability of the biocatalyst in a synthetic environment. Herein we used molecular dynamics (MD) simulations and probed the noncovalent interactions between model enzymes of technological interest, i.e., carbonic anhydrase (CA) and myeloperoxidase (MPO), with selected metal-organic frameworks (MOFs; MIL-160 and ZIF-8) of proven industrial implementation. We found that the CA and MPO can bind to MIL-160 at optimal binding energies of 201 and 501 kJ mol-1, respectively, that are strongly influenced by the increased incidence of hydrogen bonding between enzymes and the frameworks. The free energy of binding of enzymes to ZIF-8, on the other hand, was found to be less strongly influenced by hydrogen bonding networks relative to the occurrence of hydrophobic-hydrophobic interactions that yielded 106 kJ mol-1 for CA and 201 kJ mol-1 for MPO.

Hyaluronic Acid Allows Enzyme Immobilization for Applications in Biomedicine.

Enzymes are proteins that control the efficiency and effectiveness of biological reactions and systems, as well as of engineered biomimetic processes. This review highlights current applications of a diverse range of enzymes for biofuel production, plastics, and chemical waste management, as well as for detergent, textile, and food production and preservation industries respectively. Challenges regarding the transposition of enzymes from their natural purpose and environment into synthetic practice are discussed. For example, temperature and pH-induced enzyme fragilities, short shelf life, low-cost efficiency, poor user-controllability, and subsequently insufficient catalytic activity were shown to decrease pertinence and profitability in large-scale production considerations. Enzyme immobilization was shown to improve and expand upon enzyme usage within a profit and impact-oriented commercial world and through enzyme-material and interfaces integration. With particular focus on the growing biomedical market, examples of enzyme immobilization within or onto hyaluronic acid (HA)-based complexes are discussed as a definable way to improve upon and/or make possible the next generation of medical undertakings. As a polysaccharide formed in every living organism, HA has proven beneficial in biomedicine for its high biocompatibility and controllable biodegradability, viscoelasticity, and hydrophilicity. Complexes developed with this molecule have been utilized to selectively deliver drugs to a desired location and at a desired rate, improve the efficiency of tissue regeneration, and serve as a viable platform for biologically accepted sensors. In similar realms of enzyme immobilization, HA's ease in crosslinking allows the molecule to user-controllably enhance the design of a given platform in terms of both chemical and physical characteristics to thus best support successful and sustained enzyme usage. Such examples do not only demonstrate the potential of enzyme-based applications but further, emphasize future market trends and accountability.

Flavonols modulate lateral root emergence by scavenging reactive oxygen species in Arabidopsis thaliana.

Flavonoids are a class of specialized metabolites with subclasses including flavonols and anthocyanins, which have unique properties as antioxidants. Flavonoids modulate plant development, but whether and how they impact lateral root development is unclear. We examined potential roles for flavonols in this process using Arabidopsis thaliana mutants with defects in genes encoding key enzymes in flavonoid biosynthesis. We observed the tt4 and fls1 mutants, which produce no flavonols, have increased lateral root emergence. The tt4 root phenotype was reversed by genetic and chemical complementation. To more specifically define the flavonoids involved, we tested an array of flavonoid biosynthetic mutants, eliminating roles for anthocyanins and the flavonols quercetin and isorhamnetin in modulating lateral root development. Instead, two tt7 mutant alleles, with defects in a branchpoint enzyme blocking quercetin biosynthesis, formed reduced numbers of lateral roots and tt7-2 had elevated levels of kaempferol. Using a flavonol-specific dye, we observed that in the tt7-2 mutant, kaempferol accumulated within lateral root primordia at higher levels than wild-type. These data are consistent with kaempferol, or downstream derivatives, acting as a negative regulator of lateral root emergence. We examined ROS accumulation using ROS-responsive probes and found reduced fluorescence of a superoxide-selective probe within the primordia of tt7-2 compared with wild-type, but not in the tt4 mutant, consistent with opposite effects of these mutants on lateral root emergence. These results support a model in which increased level of kaempferol in the lateral root primordia of tt7-2 reduces superoxide concentration and ROS-stimulated lateral root emergence.

Peer Teaching Increases Knowledge and Changes Perceptions about Genetically Modified Crops in Non-Science Major Undergraduates.

We analyzed effects of peer teaching on non-science major undergraduates' knowledge, perceptions, and opinions about genetically modified (GM) crops and their use in agriculture. Undergraduates enrolled in an introductory nonmajors biology course participated in a service-learning program (SLP) in which they acted as cross-age peer teachers to high school students, teaching about the role of genetics in crop improvement through traditional breeding and GM approaches. Using pre/postassessments, we found that undergraduates' opinions shifted to favor the use of GM organisms (GMOs) in agriculture after SLP participation, rising from 46 to 97%. Perceptions about risks and benefits of GMOs also shifted from 43% stating that GMOs are harmful or suspect to no students describing GMOs in that way. Knowledge about GMOs became more accurate after SLP participation. There were significant correlations between students who had negative perceptions of GMOs and negative opinions or inaccurate knowledge about them. Students recognized the effect of peer teaching on their knowledge and perceptions, identifying the repeated peer teaching as an important factor in knowledge gain. Our results suggest students developed an informed opinion about the use of GMOs through first learning the science of genetic engineering and then teaching this information to younger students.

RBOH-Dependent ROS Synthesis and ROS Scavenging by Plant Specialized Metabolites To Modulate Plant Development and Stress Responses.

Reactive oxygen species (ROS) regulate plant growth and development. ROS are kept at low levels in cells to prevent oxidative damage, allowing them to be effective signaling molecules upon increased synthesis. In plants and animals, NADPH oxidase/respiratory burst oxidase homolog (RBOH) proteins provide localized ROS bursts to regulate growth, developmental processes, and stress responses. This review details ROS production via RBOH enzymes in the context of plant development and stress responses and defines the locations and tissues in which members of this family function in the model plant Arabidopsis thaliana. To ensure that these ROS signals do not reach damaging levels, plants use an array of antioxidant strategies. In addition to antioxidant machineries similar to those found in animals, plants also have a variety of specialized metabolites that scavenge ROS. These plant specialized metabolites exhibit immense structural diversity and have highly localized accumulation. This makes them important players in plant developmental processes and stress responses that use ROS-dependent signaling mechanisms. This review summarizes the unique properties of plant specialized metabolites, including carotenoids, ascorbate, tocochromanols (vitamin E), and flavonoids, in modulating ROS homeostasis. Flavonols, a subclass of flavonoids with potent antioxidant activity, are induced during stress and development, suggesting that they have a role in maintaining ROS homeostasis. Recent results using genetic approaches have shown how flavonols regulate development and stress responses through their action as antioxidants.

User-Tailored Metal-Organic Frameworks as Supports for Carbonic Anhydrase.

Carbonic anhydrase (CA) was previously proposed as a green alternative for biomineralization of carbon dioxide (CO2). However, enzyme's fragile nature when in synthetic environment significantly limits such industrial application. Herein, we hypothesized that CA immobilization onto flexible and hydrated "bridges" that ensure proton-transfer at their interfaces leads to improved activity and kinetic behavior and potentially increases enzyme's feasibility for industrial implementation. Our hypothesis was formulated considering that water plays a key role in the CO2 hydration process and acts as both the reactant as well as the rate-limiting step of the CO2 capture and transformation process. To demonstrate our hypothesis, two types of user-synthesized organic metallic frameworks [metal-organic frameworks (MOFs), one hydrophilic and one hydrophobic] were considered as model supports and their surface characteristics (i.e., charge, shape, curvature, size, etc.) and influence on the immobilized enzyme's behavior were evaluated. Morphology, crystallinity and particle size, and surface area of the model supports were determined by scanning electron microscopy, dynamic light scattering, and nitrogen adsorption/desorption measurements, respectively. Enzyme activity, kinetics, and stability at the supports interfaces were determined using spectroscopical analyses. Analysis showed that enzyme functionality is dependent on the support used in the immobilization process, with the enzyme immobilized onto the hydrophilic support retaining 72% activity of the free CA, when compared with that immobilized onto the hydrophobic one that only retained about 28% activity. Both CA-MOF conjugates showed good storage stability relative to the free enzyme in solution, with CA immobilized at the hydrophilic support also revealing increased thermal stability and retention of almost all original enzyme activity even after heating treatment at 70 °C. In contrast, free CA lost almost half of its original activity when subject to the same conditions. This present work suggests that MOFs tunable hydration conditions allow high enzyme activity and stability retention. Such results are expected to impact CO2 storage and transformation strategies based on CA and potentially increase user-integration of enzyme-based green technologies in mitigating global warming.

Abscisic Acid-Induced Reactive Oxygen Species Are Modulated by Flavonols to Control Stomata Aperture.

Abscisic acid (ABA) increases reactive oxygen species (ROS) in guard cells to close Arabidopsis (Arabidopsis thaliana) stomata. In tomato (Solanum lycopersicum), we find that ABA-increased ROS is followed by stomatal closure and that both responses are blocked by inhibitors of ROS-producing respiratory burst oxidase enzymes. ABA-induced ROS sensor fluorescence accumulates in the nucleus, chloroplasts, and endomembranes. The accumulation of flavonol antioxidants in guard cells, but not surrounding pavement cells, was visualized by confocal microscopy using a flavonol-specific fluorescent dye. Decreased flavonols in guard cells in the anthocyanin reduced (are) mutant and elevated levels in the anthocyanin without (aw) mutant were quantified by confocal microscopy and in leaf extracts by mass spectrometry. Consistent with flavonols acting as antioxidants, higher levels of ROS were detected in guard cells of the tomato are mutant and lower levels were detected in aw both at homeostasis and after treatment with ABA. These results demonstrate the inverse relationship between flavonols and ROS. Guard cells of are show greater ABA-induced closure than the wild type, reduced light-dependent guard cell opening, and reduced water loss, with aw having opposite responses. Ethylene treatment of wild-type tomato plants increased flavonol accumulation in guard cells; however, no flavonol increases were observed in Neverripe (Nr), an ethylene receptor mutant. Consistent with lower levels of ROS due to elevated flavonols, ethylene treatments decreased ABA-induced stomatal closure in the wild type, but not Nr, with ethylene responses attenuated in the are mutant. Together, these results are consistent with flavonols dampening the ABA-dependent ROS burst that drives stomatal closure and facilitating stomatal opening to modulate leaf gas exchange.