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Amylase binding to starch granules under hydrolysing and non-hydrolysing conditions

By Sushil Dhital, Frederick J. Warren, Bin Zhang and Michael J. Gidley


Although considerable information is available about amylolysis rate, extent and pattern of granular starches, the underlying mechanisms of enzyme action and interactions are not fully understood, partly due to the lack of direct visualisation of enzyme binding and subsequent hydrolysis of starch granules. In the present study, α-amylase (AA) from porcine pancreas was labelled with either fluorescein isothiocyanate (FITC) or tetramethylrhodamine isothiocyanate (TRITC) fluorescent dye with maintenance of significant enzyme activity. The binding of FITC/TRITC-AA conjugate to the surface and interior of granules was studied under both non-hydrolysing (0 °C) and hydrolysing (37 °C) conditions with confocal microscopy. It was observed that enzyme binding to maize starch granules under both conditions was more homogenous compared with potato starch. Enzyme molecules appear to preferentially bind to the granules or part of granules that are more susceptible to enzymic degradation. The specificity is such that fresh enzyme added after a certain time of incubation binds at the same location as previously bound enzyme. By visualising the enzyme location during binding and hydrolysis, detailed information is provided regarding the heterogeneity of granular starch digestion

Topics: Alpha-amylase, Confocal microscopy, Enzyme binding, Starch granules, Surface structure, 2700 Medicine, 1605 Organic Chemistry, 2505 Materials Chemistry, 2507 Polymers and Plastics
Publisher: Pergamon Press
Year: 2014
DOI identifier: 10.1016/j.carbpol.2014.06.063
OAI identifier:

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