Department of Inorganic and Analytical Chemistry, University of Debrecen, P.O. Box 400, H-4002, Debrecen, Hungary.
Carbohydr Polym. 2018 Mar 1;183:263-266. doi: 10.1016/j.carbpol.2017.12.037. Epub 2017 Dec 18.
Despite being widely used, there is no standard protocol for α-amylase activity measurement with starch azure substrate. Boiling pre-treatment of starch azure suspension increased the reaction rate of hydrolysis catalysed by human salivary α-amylase (HSA) or porcine pancreatic α-amylase (PPA) and the sensitivity of spectrophotometric activity measurement has been improved. Kinetic constants, K, and v, obtained from parallel isothermal titration calorimetric (ITC) measurements on natural and starch azure revealed, that the blue starch derivative does not differ significantly from its natural counterpart from kinetic point of view. Interestingly, substrate inhibition was observed in starch azure hydrolysis characterised by dissociation constants 49 mg/mL and 16.4 mg/mL for HSA and PPA, respectively. In this work a new protocol has been suggested for α-amylase activity measurement using boiled insoluble starch azure as substrate at 5 mg/mL concentration.
尽管α-淀粉酶活性的淀粉蓝底物检测法被广泛应用，但目前仍没有标准化的检测方法。淀粉蓝悬浮液的煮沸预处理提高了人唾液α-淀粉酶（HSA）或猪胰腺α-淀粉酶（PPA）催化水解的反应速率，提高了分光光度法检测活性的灵敏度。通过对天然淀粉和淀粉蓝进行平行等温滴定量热法（ITC）测量得到的动力学常数 K 和 v 表明，从动力学角度来看，蓝色淀粉衍生物与其天然对应物没有显著差异。有趣的是，淀粉蓝水解过程中观察到了底物抑制作用，其对 HSA 和 PPA 的解离常数分别为 49mg/mL 和 16.4mg/mL。在这项工作中，我们提出了一种新的方法，使用 5mg/mL 浓度的煮沸不溶性淀粉蓝作为底物来检测α-淀粉酶的活性。
