title: Amylase binding to starch granules under hydrolysing and non-hydrolysing conditions.
abstract: 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.
authors.affiliation: ARC Centre of Excellence in Plant Cell Walls, Centre for Nutrition and Food Sciences, Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St Lucia 4072, Qld., Australia; Centre for Nutrition and Food Sciences, Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St Lucia 4072, Qld., Australia.
authors.forename: Sushil
authors.initials: S
authors.lastname: Dhital
authors.affiliation: Centre for Nutrition and Food Sciences, Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St Lucia 4072, Qld., Australia.
authors.forename: Frederick J
authors.initials: FJ
authors.lastname: Warren
authors.affiliation: ARC Centre of Excellence in Plant Cell Walls, Centre for Nutrition and Food Sciences, Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St Lucia 4072, Qld., Australia; Centre for Nutrition and Food Sciences, Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St Lucia 4072, Qld., Australia.
authors.forename: Bin
authors.initials: B
authors.lastname: Zhang
authors.affiliation: ARC Centre of Excellence in Plant Cell Walls, Centre for Nutrition and Food Sciences, Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St Lucia 4072, Qld., Australia; Centre for Nutrition and Food Sciences, Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St Lucia 4072, Qld., Australia. Electronic address: m.gidley@uq.edu.au.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Gidley
mesh_terms: D000818:Animals; D001665:Binding Sites; D006868:Hydrolysis; D010179:Pancreas / Q000201:enzymology; D011198:Solanum tuberosum; D013213:Starch / Q000737:chemistry / Q000378:metabolism*; D034421:Sus scrofa / Q000378:metabolism; D013696:Temperature; D003313:Zea mays; D000516:alpha-Amylases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D013213:Starch; D000516:alpha-Amylases
keywords: Alpha-amylase; Confocal microscopy; Enzyme binding; Starch granules; Surface structure