Single amino acid bionanozyme for environmental remediation

Enzymes are complex catalytic structures with significant biological and technological importance. However, their use in environmental remediation is hampered by high production costs, low stability, and difficult recovery/reuse. This necessitates the development of minimalistic biomolecular nanomaterials, or bionanozymes, that can mimic enzymatic function while overcoming these limitations. Laccase enzymes, which catalyze the oxidation of environmentally toxic phenolic compounds, are particularly attractive targets for mimicry. While laccases effectively transform toxic phenols into less harmful substances using molecular oxygen as the electron acceptor, their inherent drawbacks limit their practical applications. This research explores the possibility of creating a laccase-mimicking bionanozyme using the self-assembly of a single amino acid, aiming for a cost-effective and robust alternative to natural enzymes. The design is inspired by the laccase catalytic site, focusing on creating a simplified structure that retains high catalytic efficiency and stability. This minimalist approach offers a potential breakthrough in enzyme engineering and nanozyme development, paving the way for applications in diverse fields including environmental remediation and analytical chemistry.

Extensive research has focused on exploring enzyme-mimicking nanomaterials, termed nanozymes, to overcome the limitations of natural enzymes. Various nanomaterials, including metal oxides, nanoparticles, and carbon-based materials, have been shown to exhibit enzyme-mimetic activities. However, many of these nanozymes suffer from drawbacks such as harsh synthesis conditions, complicated fabrication, and limited stability. Peptide-based nanomaterials, due to their protein-like nature, biodegradability, and ease of synthesis, have emerged as a promising alternative. The minimalist approach to protein design, focusing on creating simplified sequences that retain function, has led to the development of simple biomolecular nanozymes. However, creating a laccase-mimicking bionanozyme remained a challenge due to the complexity of the laccase active site and catalytic mechanism. This study directly addresses this challenge, taking a significantly more minimalistic approach than previously explored.

The research employed a novel approach, using phenylalanine (F) and copper(II) ions (Cu²⁺) to create a laccase-mimicking bionanozyme. Phenylalanine, chosen for its self-assembling properties and metal-chelating functionalities, spontaneously coordinates with Cu²⁺ ions to form a two-dimensional (2D) layered structure. These 2D crystals, analyzed through single-crystal X-ray crystallography, reveal an octahedral copper coordination complex with tetragonal distortion. The crystals exhibit a layered structure held together by van der Waals forces, allowing for mechanical exfoliation into ultrathin nanosheets using ultrasonication. Various techniques were utilized to characterize the structure and properties of the F-Cu bionanozyme, including optical microscopy, high-resolution scanning electron microscopy (HRSEM), atomic force microscopy (AFM), powder X-ray diffraction (PXRD), UV-Vis spectroscopy, FTIR spectroscopy, energy dispersive X-ray (EDX) spectroscopy, and thermal gravimetric analysis (TGA). The catalytic activity of the F-Cu nanosheets was evaluated using the oxidation reaction of 2,4-dichlorophenol (2,4-DP) with 4-aminoantipyrine (4-AP), a benchmark reaction for laccase activity. Kinetic parameters (Vmax, Km, kcat, kcat/Km) were determined using Michaelis-Menten kinetics. Control experiments using various combinations of F, Cu²⁺, and other amino acids were performed to confirm the role of F-Cu nanosheets in the catalysis. The mechanism was further investigated using electron paramagnetic resonance (EPR) spectroscopy and density functional theory (DFT) calculations. The stability, recyclability, and substrate universality of the F-Cu bionanozyme were also assessed under various conditions (pH, ionic strength, temperature, storage time) and with different substrates. Finally, the ability of the F-Cu bionanozyme to detect catecholamine neurotransmitters, such as epinephrine, was explored through colorimetric analysis.

The study successfully synthesized a highly crystalline 2D layered F-Cu bionanozyme. Single-crystal X-ray crystallography revealed the detailed structure of the F-Cu complex, showing an octahedral copper coordination with tetragonal distortion. The F-Cu bionanozyme displayed exceptional laccase-mimicking activity, significantly outperforming natural laccase in terms of efficiency (four orders of magnitude higher) and sensitivity (36 times higher). The Michaelis-Menten analysis revealed a significantly higher catalytic efficiency (kcat/Km) for F-Cu compared to laccase. Control experiments confirmed the crucial role of both F and Cu²⁺ in the catalytic activity, and a comparison with other amino acid-copper complexes showed phenylalanine's superior performance. The F-Cu bionanozyme exhibited remarkable stability under extreme conditions (pH, ionic strength, temperature, storage time), maintaining high catalytic activity even after prolonged storage and high-temperature incubation. Importantly, the bionanozyme demonstrated excellent recyclability, retaining its activity after fifteen cycles. The F-Cu bionanozyme showed broad substrate specificity, effectively oxidizing various toxic phenolic compounds. DFT calculations provided insights into the reaction mechanism, suggesting a crucial role of hydrogen atom transfer to the carboxylate group of phenylalanine. Notably, the F-Cu bionanozyme enabled a highly sensitive and cost-effective colorimetric detection of catecholamine neurotransmitters, such as epinephrine, showing a significantly lower detection limit than natural laccase. Overall, the F-Cu bionanozyme proved highly superior to laccase in terms of cost-effectiveness (5400 times more cost-effective than laccase), efficiency, sensitivity, stability, recyclability and substrate range.

The findings address the research question of developing a highly efficient and robust laccase-mimicking bionanozyme by demonstrating the successful creation and characterization of a single amino acid-based catalyst. The exceptional performance of the F-Cu bionanozyme in comparison to natural laccase highlights the potential of minimalist design in overcoming the limitations of natural enzymes. The superior stability, recyclability, and broad substrate specificity make this bionanozyme highly attractive for practical applications, particularly in environmental remediation and analytical chemistry. The use of a single amino acid significantly reduces the complexity and cost associated with enzyme production, opening new avenues for industrial-scale applications. The insights from DFT calculations aid in understanding the reaction mechanism and further optimize the catalyst design. The sensitive detection of catecholamine neurotransmitters adds another dimension to the potential applications of this bionanozyme. The work contributes significantly to the field of nanozymes and biocatalysis, providing a promising model for future development of cost-effective and highly effective biomimetic catalysts.

This study successfully developed a highly efficient, stable, and reusable single amino acid bionanozyme (F-Cu) that effectively mimics laccase activity. This minimalist approach offers a significant advance over natural laccases and other nanozymes. The F-Cu bionanozyme presents substantial potential for environmental remediation, analytical chemistry, and biotechnology applications. Future research could focus on exploring other amino acids and metal ions to design even more efficient and versatile bionanozymes. Investigating the potential of this bionanozyme in real-world environmental remediation scenarios will also be valuable. Furthermore, exploring the broader implications of this minimalist design for understanding the evolution of enzymes could provide insightful contributions to origins-of-life research.

While the F-Cu bionanozyme demonstrates exceptional performance, some limitations exist. The current study primarily focuses on in vitro experiments, and further research is needed to assess its efficacy in real-world environmental settings. The long-term stability and potential toxicity of the F-Cu bionanozyme in environmental contexts require further investigation. Moreover, the optimization of the reaction conditions for different substrates might be necessary to achieve maximal catalytic efficiency in diverse applications. The current DFT calculations offer a valuable insight into the mechanism; however, more detailed and possibly more complex modelling could provide deeper understanding of the complex processes taking place.