Development of a reusable synthetic enzyme with broad substrate specificity

In this study, hemoglobin (Hbg)-based nanoenzymes, which have broad substrate specificity in vitro, were synthesized as an alternative to the limited substrate specificity of natural enzymes. An artificial heme complex was synthesized to mimic the active site of Hbg and was compared with natural Hbg. The synthesized nano Hbg and zeolitic imidazole methacryloyl histidine framwork (ZMHF) based structures were characterized in detail by Fourier Transform Infrared spectroscopy (FT-IR), Scanning Electron Microscopy (SEM), and Zeta Sizer. The SEM images have presented the morphology of the nano Hbg structures in detail. Significant changes in morphology were observed due to the metacryloylated functional monomers added to the zeolitic structure. Zeolytic imidazole cages were added to optimize the activity and stability of the mimic nano Hbg enzymes over extended pH and temperature ranges. For each synthesized enzyme, its affinity for substrate, reaction rate, catalytic activity, and specific activity were determined by detailed kinetic analysis. Within the scope of application studies, novel procedures for detecting catalytic products of Myeloperoxidase and Heme Oxygenase-1 enzymes were developed, and satisfactory results were obtained. A zymography-like technique established a simple, low-cost, and visually detectable procedure. The findings indicate that mimic nano Hbg and ZMHF-based enzymes offer significant potential for biocatalysis and biosensor applications. The findings show that mimic nano Hbg and ZMHF-based enzymes offer significant potential for biocatalysis and biosensor applications. The detailed characterisation data and successful application results make this study an important contribution to the field. Keywords: Hemoglobin, Nanoenzyme, Zeolitic imidazole frameworks, Activity.