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KMID : 1161420100130051153
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Journal of Medicinal Food
2010 Volume.13 No. 5 p.1153 ~ p.1160
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In Vitro Real-Time Interactions of Cranberry Constituents with Immobilized Fructosyltransferase
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Feldman Mark
Weiss Ervin I.
Ofek Itzhak
Shemesh Moshe
Steinberg Doron
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Abstract
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Cranberry has been proposed as an anti-biofilm agent that does not kill bacteria, but rather prevents the pathogen from survival in the host. This can be achieved by inhibiting the function of virulent factors essential for the pathogen to persist in a host environment. The oral bacterial enzyme fructosyltransferase (FTF) plays an important role in the pathogenesis of dental diseases. The real-time interaction of cranberry nondialyzable material (NDM) with immobilized FTF was investigated using the surface plasmon resonance (SPR) technique. To determine its binding efficiency, NDM at concentrations between 0?μg/mL and 200?μg/mL was applied onto the immobilized FTF. The effect of NDM or other polyphenols, myricetin, and epicatechin on FTF enzymatic activity was evaluated by applying the above compounds and sucrose onto immobilized FTF. Salivary amylase was applied with NDM onto immobilized FTF to explore the effect on NDM?FTF interaction. Our results show that NDM firmly attaches to immobilized FTF in a dose-dependent manner, whereas the presence of salivary amylase reduced this binding interaction. Using nonlinear regression we calculated that the affinity constant of NDM applied alone (106M?1) was fivefold higher than NDM in the presence of amylase (0.2?×?106M?1). At 200?μg/mL, NDM, introduced together with sucrose, inhibited the activity of immobilized FTF by 63% within minutes, in comparison with the control (sucrose alone). The effect of NDM was sustained even after it was washed off the immobilized FTF. Myricetin also strongly inhibited FTF activity, whereas epicatechin was less effective. The real-time SPR observation suggests that one of the anti-biofilm modes of action of NDM is an immediate and irreversible inhibitory effect on the activity of immobilized FTF, which is due to a strong binding affinity to the immobilized enzyme.
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KEYWORD
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cranberry nondialyzable material, enzyme, surface plasmon resonance
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