Abstract：
Purification of amylases from digestive tracts of three freshwater fish species with Q-Sepharose Fast Flow and Sephacryl S-200 columns displayed two isoforms of amylases from Osteochilus hasselti (O1, O2) and three isoforms of those from both Hampala dispar (UB, H1, H2) and Puntioplites proctozystron (P1, P2, P3). The optimum pH values displayed at 7.0 and 8.0, while the optimum temperatures revealed at 40 and 50 °C. Almost isoenzyme activities were activated by NaCl and CaCl2, whereas EDTA and SDS strongly inhibited all enzymatic activities. Verification with an atomic absorption spectrophotometry exhibited the presence of Ca2+ ions in the range of 0.02-13.53 ppm per mg protein indicating that amylases are Ca2+ dependent. Molecular weight analysis revealed 12 to 147 kDa. The UB, O1, and H2 amylases with appropriate molecular masses of 64, 49, and 25 kDa validated with LC-MS/MS were selected. Three certain enzymes revealed high stability in a sample buffer after five cycles of freeze-thawing process upon storage at - 20 °C for 12 weeks. No protein degradation was observed on polyacrylamide gel, and the enzymes still displayed sharp and clear bands on zymograms. The result suggested that the purified fish amylases, which expressed high activities and stabilities, were potentially used as enzyme molecular weight markers for zymography.
摘要：
使用Q-SepharoseFastFlow和SephacrylS-200柱从三种淡水鱼种的消化道中纯化淀粉酶，显示了来自Osteochilushasselti（O1，O2）的淀粉酶的两种同工型和来自Hampaladispar的淀粉酶的三种同工型（UB，H1，H2）和Puntioplites原酶（P1，P2，P3）。最佳pH值显示在7.0和8.0，而最佳温度显示在40和50°C。NaCl和CaCl2几乎激活了同工酶活性，而EDTA和SDS强烈抑制了所有酶活性。用原子吸收分光光度法进行的验证表明，每mg蛋白质中Ca2离子的存在范围为0.02-13.53ppm，表明淀粉酶是Ca2依赖性的。分子量分析显示12至147kDa。UB,选择用LC-MS/MS验证的具有64、49和25kDa的适当分子量的O1和H2淀粉酶。三种特定的酶在-20°C下储存12周后在样品缓冲液中显示出高稳定性。在聚丙烯酰胺凝胶上没有观察到蛋白质降解，酶在酶谱上仍然显示清晰的条带。结果表明，纯化的鱼淀粉酶，表现出高度的活动性和稳定性，有可能用作酶谱的酶分子量标记。
