Herein, a new and straightforward approach for the fabrication of a stable and multifunctional white light emitting (WLE) hydrogel is reported. For the first time, the utility of such hydrogels for protein packaging with enhanced activity and stability is presented. Initially, a WLE composite with color chromaticity of (0.33, 0.27) is fabricated by engineering the surface of an orange light emitting Mn2+‐doped ZnS quantum dot (QD) using a blue‐emitting choline‐tosylate ionic liquid (IL). The color chromaticity can be tuned by altering the concentration of the IL and excitation wavelengths. The WLE hydrogel is constructed through the conjugation of the WLE QD‐IL composite with an alginate biopolymer. Remarkably, the WLE QD‐IL composite and WLE hydrogel show preservation of their structural and luminescence properties for an extended time and thus indicate a potential for storage applications. When cytochrome c (Cyt C) is caged within the WLE hydrogel matrix, the peroxidase activity increases by more than 1.7‐times compared with native Cyt C and a Cyt C‐loaded QD hydrogel at room temperature. Also, Cyt C‐immobilized WLE hydrogel shows a 3.5‐fold increase in activity (compared with native Cyt C) at a higher temperature (120 °C) and in the presence of a denaturation agent.
Enhancing the structural stability and catalytic activity of Cytochorme c (Cyt C) against harsh process conditions would boost its use in biocatalysis. Herein, a new protein engineering strategy with improved efficacy is demonstrated through judicious task-specific functionalization of Cyt C with quantum dots (QDs) and ionic liquids (ILs). Mn 2+ doped ZnS QD and ILs ([Cho][Ac]; [Cho][Dhp]) were concomitantly used to decorate Cyt C, which was characterized using various analytical tools. The peroxidase activity at room temperature of engineered Cyt C (Cyt C-QD-IL) increased markedly (1.2 to 3.5-fold) as compared to that for bare Cyt C, Cyt C with QD, and Cyt C with ILs. Further, Cyt C-QD-IL showed better catalytic activity under various stresses such as high temperature (110 °C), presence of a chemical denaturant (6 M GuHCl), high oxidative stress (30 min H 2 O 2 ), and presence of proteases. Molecular docking results indicate that QD interacted with the active site of Cyt C and IL interacted with side chain amino acids via electrostatic and H-bonding interactions. Such favorable allosteric interactions might be behind the improved activity of Cyt C-QD-IL. The observed catalytic activity is in harmony with the structural stability of the protein as confirmed by UV−vis, ATR-IR, and CD analysis. Thus, the unveiled strategy represents an innovative dimension of protein packaging foreseeing the development of more robust biocatalysts that can be used at high temperatures.
Green and nano-structured catalytic media are vital for bio-catalysis to attenuate the denaturation tendency of biocatalysts under severe reaction conditions. Hydrotropes with multi-faceted physiochemical properties represent promising systems for sustainable...
Herein, we present a simple approach to fabricate protein nanoconstructs by complexing cytochrome C (Cyt C) with silk nanofibrils (SNF) and choline dihydrogen phosphate ionic liquid (IL). Molecular docking revealed...
Green and nanostructured catalytic media are vital in biocatalysis to attenuate the denaturation tendency of biocatalysts under severe reaction conditions. Hydrotropes with multifaceted physiochemical properties can be envisaged as promising systems for sustainable protein packaging. Herein, the suitability of adenosine-5'-triphosphate (ATP) and cholinium salicylate ([Cho][Sal]) ionic liquid (IL) to form nanostructures and to nanoconfine Cytochrome c (Cyt c) were disclosed envisioning enhancement of stability and activity under multiple stresses. Experimental and computational validations were undertaken to explain nanostructuring phenomenon of ATP and IL, structural organizations of nanoconfined Cyt c, and site-specific interactions that stabilize protein structure. Both, ATP and IL form nanostructures in aqueous media and caged Cyt c via multiple non-specific soft interactions. Remarkably, the engineered molecular nano-cages of ATP (5-10 mM), IL (300 mg/mL), and ATP+IL around Cyt c resulted in 9-72 folds higher peroxidase activity than native Cyt c with exceptionally high thermal tolerance (110 degrees C). The polar interactions mediated by hydrotropes with the cardiolipin binding site of Cyt c well corroborated with the increased peroxidase activity. Further, higher activity trends were observed in the presence of urea, GuHCl, and trypsin without any protein degradation. Specific binding of hydrotropes at highly mobile regions of Cyt c (Omega 40-54 residues) and enhanced H-bonding with Lys and Arg offered excellent stability under extreme conditions. Additionally, ATP effectively counteracted reactive oxygen species (ROS) induced denaturation of Cyt c, which was enhanced by [Sal] counterpart of IL. Overall, this study explored the robustness of nanostructured hydrotropes having a higher potential for protein packaging with improved stability and activity in extreme conditions. Thus, the present work brings out a novel strategy for real-time industrial biocatalysis to protect mitochondrial cells from ROS-instigated apoptosis.
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