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.
Centre for Nutrition and Food Sciences, Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St Lucia 4072, Qld., Australia.
Carbohydr Polym. 2014 Nov 26;113:97-107. doi: 10.1016/j.carbpol.2014.06.063. Epub 2014 Jul 1.
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.
尽管关于淀粉的水解速率、程度和颗粒形态有大量信息，但由于缺乏对酶结合和随后淀粉颗粒水解的直接可视化，酶作用和相互作用的基本机制仍未完全了解。在本研究中，用荧光素异硫氰酸酯（FITC）或四甲基罗丹明异硫氰酸酯（TRITC）荧光染料标记来自猪胰腺的α-淀粉酶（AA），同时保持显著的酶活性。使用共聚焦显微镜研究了 FITC/TRITC-AA 缀合物在非水解（0°C）和水解（37°C）条件下在颗粒表面和内部的结合情况。结果表明，与马铃薯淀粉相比，在这两种条件下，酶与玉米淀粉颗粒的结合更加均匀。酶分子似乎优先结合更易受酶降解的颗粒或颗粒的一部分。这种特异性是，在孵育一定时间后添加新鲜酶时，它会与先前结合的酶结合在相同的位置。通过在结合和水解过程中观察酶的位置，可以提供有关颗粒状淀粉消化异质性的详细信息。
