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  • Thus this work aims to evaluate the effect of

    2021-10-27

    Thus, this work aims to evaluate the effect of tannic FIPI sale on β-galactosidase activity, by observing the variations in the molecular interaction kinetic parameters ( and ). Besides, these data were correlated with the modifications that the tannin caused to the enzyme’s secondary structure (CD), tertiary structure (fluorescence spectroscopy), and thermodynamic interaction parameters (ITC).
    Materials and methods
    Results and discussion
    Conclusion The adverse effect of tannic acid on β-galactosidase catalytic activity was evaluated by kinetic analysis, and these data were correlated to the changes caused by the tannin on the enzyme structure and molecular interactions via spectroscopy and calorimetry techniques. A decrease in β-galactosidase activity due to the presence of tannic acid was verified. Both kinetic parameters ( and ) decreased, which could be an indication that the tannic acid reduces and modifies the enzyme activity. Fluorescence spectroscopy indicated the tannin had a great affinity for the protein and the interaction occurred between the compounds by a static mechanism. Modifications to the protein secondary structure (α-helix, β-turn, and random coil) in the presence of different concentrations of the tannin, impacted the overall protein structure, properties, and catalytic activity. The ITC results demonstrated that tannic acid modifies the β-galactosidase–ONPG molecular interaction mechanism, reducing the protein–substrate binding affinity, and the molecular interaction spontaneity. The present study may help to better understand the tannin effects on β-galactosidase structure and how the compound reduces the enzyme catalytic activity. The alterations in the conformation and activity of the enzyme should be taken into consideration when dairy products are consumed with tannin-rich food.
    Acknowledgments Funding: This work was supported by the Graduation Program of Food Engineering (PPGEAL-UFPR), CAPES – Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, a Brazilian government agency, CNPq (Proc. No. 310905/2015-0), the National Institute of Science and Technology on Biological Nitrogen Fixation (INCT-UFPR), and Granolab Granotec.
    Introduction Strong industrial interests are attributed to β-galactosidases due to their hydrolase and transferase activities. Because of their hydrolytic activity, β-galactosidases are broadly employed in the agro-food industry essentially for reducing lactose concentration in milk and its products with the aim of overcoming lactose intolerance with which almost 75% of the Asian population is affected (Adalberto et al., 2010). β-Galactosidase (βGS) is also used to prevent lactose crystallization subjected to low storage and transportation temperatures and to improve the relative sweetness of the product. This enzyme is mostly found in microorganisms, plants and animal tissues (Haider and Husain, 2007a). Commercialized βGS mainly derives from Escherichia coli, Aspergillus niger, Aspergillus oryzae, yeasts, some species from the Lactobacillus genus and others (Husain, 2010). βGS from microbial sources exhibit great industrial relevance mainly due to their ease of handling, enhanced catalytic activity and increased production yield (Cardoso et al., 2017). However, only a few microbial sources of βGS are generally recognized as safe (GRAS) and eligible for usage in the pharmaceutical and food industries (Saqib et al., 2017). Extracellular β-galactosidases from the fungi Aspergillus niger and Aspergillus oryzae have been classified as GRAS by the Food and Drug Administration. Immobilization of enzymes is a powerful tool to improve enzyme properties like activity, stability, specificity, resistance against inhibitors and chemical agents etc. (Rodrigues et al., 2013, Guzik et al., 2014, Silva et al., 2017). The employment of enzyme on support matrix promises biocatalyst recovery and recyclability thus allowing continuous operation and making them economically viable for large-scale processes (Sheldon & Pelt, 2013). Moreover, this technology has been recognized to shield enzymes against denaturing microenvironments (Husain, 2016). However, enzymes often encounter activity reduction upon immobilization associated with conformational changes in the secondary structure (Secundo, 2013). There is a great demand to explore new technology for fabricating enzyme carriers such that the enzyme activity is not compromised (Rodrigues et al., 2013).