ABSTRACT
In this study, sweet potato ß-amylase (SPA) was modified by methoxy polyethylene glycol maleimide (molecular weight 5000, Mal-mPEG5000) to obtain the Mal-mPEG5000-SPA modified ß-amylase and the interaction mechanism between SPA and Mal-mPEG5000 was investigated. the changes in the functional groups of different amide bands and modifications in the secondary structure of enzyme protein were analyzed using infrared spectroscopy and circular dichroism spectroscopy. The addition of Mal-mPEG5000 transformed the random curl in the SPA secondary structure into a helix structure, forming a folded structure. The Mal-mPEG5000 improved the thermal stability of SPA and protected the structure of the protein from breaking by the surrounding. The thermodynamic analysis further implied that the intermolecular forces between SPA and Mal-mPEG5000 were hydrophobic interactions and hydrogen bonds due to the positive values of ΔHθ and ΔSθ. Furthermore, the calorie titration data showed that the binding stoichiometry for the complexation of Mal-mPEG5000 to SPA was 1.26, and the binding constant was 1.256 × 107 mol/L. The binding reaction resulted from negative enthalpy, indicating that the interaction of SPA and Mal-mPEG5000 was induced by the van der Waals force and hydrogen bonding. The UV results showed the formation of non-luminescent material during the interaction, the Fluorescence results confirmed that the mechanism between SPA and Mal-mPEG5000 was static quenching. According to the fluorescence quenching measurement, the binding constant (KA) values were 4.65 × 104 L·mol-1 (298K), 5.56 × 104 L·mol-1 (308K), and 6.91 × 104 L·mol-1 (318K), respectively.
Subject(s)
Ipomoea batatas , beta-Amylase , Circular Dichroism , Thermodynamics , Polyethylene Glycols , Maleimides , Protein Binding , Spectrometry, Fluorescence/methods , Binding Sites , Molecular Docking SimulationABSTRACT
A method based on a reversed-phase HPLC method was established, optimized, and validated for the separation and quantitation of 10 polyphenols extracted from the peel of apple species. A bidentate reversed-phase C18 column was used as stationary phase, and an acidified water buffer and methanol were used as mobile phase. The polyphenols were well separated and detected using UV at 280 and 320 nm. Validation parameters, such as linearity, LOD, LOQ, accuracy, and precision, were acceptable for all 10 polyphenols. The proposed method has enough linearity with correlation coefficient >0.99 within the investigated range for all tested polyphenols. The LOD was 0.24 µg/mL for ellagic acid and <0.2 µg/mL for all other polyphenols. The LOQ was 9.39 × 10(-2) µg/mL for chlorogenic acid, and ellagic acid, 2.82 × 10(-2) µg/mL for caffeic acid and >0.1 µg/mL for all other polyphenols. Recovery was within the acceptable range from 98.38 to 100.39% for all polyphenols standards. Satisfactory precision was achieved for both intra- and interday assay, with RSD <2%. The method was successfully applied for simultaneous analysis of polyphenols from apple peel.