活性污泥中一氧化氮还原酶的提取和测定优化

Optimizations of extraction and activity determination methods of nitric oxide reductase in activated sludge

通过正交实验,研究了超声破碎法和低温高压破碎法提取一氧化氮还原酶(nitric oxide reductase,nor)的活性,分析了超声强度和次数,破碎压力和次数,裂解液的添加量等因素对于胞内可溶性蛋白和核酸(DNA)的释放与提取nor活性的影响.根据活性污泥中酶的催化特性以及其他实验条件,完善了nor催化活性的测定方法.结果表明超声法提取nor的参数应设定为,超声次数为100次,超声强度为500W,裂解液的添加量为0.1mL.低温高压破碎法提取nor的参数应设定为破碎次数为4次,破碎压力为50MPa,裂解液的添加量为0.1mL.低温高压破碎法下的胞内可溶性蛋白和DNA的释放量与nor的最大催化活性要高于超声破碎法.此外,在nor催化活性的测定方法中,nor活性测定终点时间应为15min.

Orthogonal experiments were carried out to study the effects of different fragmentation methods(e.g.fragmentation by ultrasonic,fragmentation by low temperature and high-pressure)on the catalytic activity of nitric oxide reductase(nor).The release of intracellular soluble protein,nucleic acid(DNA)and the catalytic activity of nor were analyzed under different ultrasonic intensity and frequency,breaking pressure and frequency,and the amount of lysate addition.According to the catalytic characteristics of enzymes in activated sludge under different experimental conditions,the method for measuring the catalytic activity of nor was improved.According to the results,the parameters of nor extraction by ultrasonic fragmentation were suggested to set at 100 times(ultrasonic times),500 W(ultrasonic intensity),and 0.1 mL(amount of lysate addition).The parameters of nor extraction by low temperature and high-pressure fragmentation were suggested to set at 4 times(breaking times),50 Mpa(breaking pressure),and 0.1 m L(amount of lysate addition).The release of intracellular soluble protein,DNA and the maximum catalytic activity of nor by low temperature and high-pressure fragmentation were higher than that by ultrasonic fragmentation.Last but not least,the end time for activity determination of nor was suggested at 15 min.