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Pectin depolymerase is widely utilized in various industrial sectors. However, the traditional methods for determining its enzymatic activity have limitations, such as cumbersome operations and a significant impact of enzyme solution dilution ratios on activity. The 3-methyl-2-benzothiazolinone hydrazone (MBTH) method can be employed to address these issues, but pectin precipitation and strong background commonly arise in this method. We have successfully overcome these challenges by employing a low-temperature and high-alkaline environment, and further optimized the reagent compositions and detection wavelength to improve the method. Consequently, enzyme hydrolysis follows a zero-order reaction within 60 min, which is helpful for the endpoint measurement of pectinase activity. The developed calibration curve for pectinase concentration and hydrolysis rate demonstrates linearity (R2 = 0.9945) within the range of 2.5-15.8 mU/mL of pectinase. This method exhibits high sensitivity, accuracy, and stability, making it suitable for routine determination of pectin depolymerase activity in research and applications.
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BACKGROUND: Phyllanthus emblica Linn. (PE) is rich in polyphenols, which can be categorized into free and bound phenolics (PEFP and PEBP). This study evaluated the inhibitory effect of PEFB and PEBP on α-amylase for the first time. The mechanism of the inhibition effect of PEFP and PEBP on α-amylase was investigated by enzyme inhibition kinetics, multispectral analysis, thermodynamics, and molecular docking. RESULTS: Free and bound phenolics inhibited α-amylase activity effectively in a mixed type of inhibition. Fluorescence quenching and thermodynamic analyses showed that the binding of PEFP and PEBP to α-amylase occurred through a static quenching process (Kq = 6.94 × 10¹² and 5.74 × 10¹² L mol-1 s-1), which was accompanied by a redshift (λem from 343 to 347 nm), leading to a change in the microenvironment. This process was found to be a spontaneous exothermic reaction (ΔG < 0). Circular dichroism (CD) analysis confirms that the secondary structure of α-amylase was altered, in particular a decrease in α-helixes and an increase in random coils. Molecular docking studies showed that PEFP and PEBP interacted with α-amylase through hydrogen bonding and hydrophobic interactions. CONCLUSION: The present study provides valuable insights into the mechanism of action of PEFP and PEBP on α-amylase, which will provide a theoretical basis for their possible use as novel natural α-amylase inhibitors. © 2024 Society of Chemical Industry.
RESUMO
Phyllanthus emblica Linn. (PE) fresh fruits contain high concentrations of polyphenolics, of which free and bound phenolics are rich in biological activities. In this study, the inhibitory activity and mechanism of PEFP and PEBP on α-glucosidase (α-GLU) were investigated using spectroscopic techniques, kinetic analysis, and molecular docking. The results showed that 13 PEFP and 12 PEBP were identified by UPLC-MS/MS analysis, and Bis-HHDP-hexose and castalagin (vesgalagin) were found for the first time in PE fresh fruits. Kinetic analysis of enzyme inhibition showed that a mixture of free and bound phenolics inhibited α-GLU, and the effect of the conformational relationship of PEFP and PEBP with α-GLU on hypoglycemia was further explored by fluorescence quenching, circular dichroism (CD) spectroscopy, and molecular docking analysis. The findings demonstrated the inhibitory activity and mechanism of free and bound phenolics on α-GLU and provided a theoretical basis for PE polyphenolics as α-GLU inhibitors for hypoglycemia.