分离地龙DNA裂解蛋白过程中组织破碎法、pH及温度的选择(英文)

Choice of the crushing method,pH buffers and temperature during the process of extracting earthworm deoxyribonuclease

目的观察分离过程中三种处理方法制备的地龙组织匀浆及pH、温度对地龙DNA裂解蛋白(EWDNase)的影响。方法采用糖溶解法、机械法和超声破碎法制备地龙组织匀浆;不同温度、pH条件下进行EWDNase的粗提取。测定地龙组织匀浆及不同温度、pH条件下制备的粗提取液中EWDNase活性和蛋白含量,计算其比活性。结果三种方法获得地龙组织匀浆中EWDNase比活性大小无显著性差异(P>0.05);EWDNase耐热(75℃,30min仍有活性),pH3.4-5.4具有较高比活性。结论机械法因其操作简便、经济实用、误差小、重复性高可以用来处理地龙组织制备组织匀浆。提取EWDNase过程中宜采用酸性缓冲体系和较高温度(65-75℃)的热变性条件。

Objective To choose the optimal crushing tissue method during extracting earthworm deoxyribonuclease（ EWDNase）, and to search the suitable pH buffers and temperature at which the EWDNase is stable. Methods To achieve the tissue homogenates, earthworms were dealt by three crushing methods such as dissolved by sugar, minced with scissors-machine, and minced with ultrasonicmachine, respectively. At different temperatures and pH buffers, the coarse extracts of EWDNase were achieved. Then, the activities of EWDNase in the coarse extracts were determined as well as its protein content. The comparative activities of EWDNase were calculated. Results The EWDNase comparative activities of tissue homogenates achieved by three crushing methods showed no significant diffe- rence（ P 〉 0.05 ）. The coarse extracts of EWDNase were heating-stable such as 75 ℃ ,30 min. The EWDNase still had strong comparative activities at pH buffers in a range from 3.4 to 5.4. Cortclusion The crushing method of scissor machine is time-saving, easy to operate and hold in a large-scale industrial production for extracting the EWDNase. And the pH buffers in low pH and temperature from 65 ℃ to 75℃ are suitable.