摘要
目的:选择不同的分离、纯化步骤并比对分析,筛选出纯化烟草中多酚氧化酶(PPO)的优化组合方案。方法:采用分段盐析、DEAE-SepharoseFastflow和SephadexG-150柱层析纯化PPO,通过测定和比较酶活性筛选最佳条件。结果:确定了最佳盐析浓度(40%)和柱层析条件,SDS-PAGE、FPLC以及动力学常数的检测结果表明,纯化出的蛋白质相对分子质量为42000,Km为1.2mmol/L,得到了纯化91倍的烟草多酚氧化酶Ⅱ。结论:优化方案减少了有机溶剂分级沉淀、阳离子交换层析等步骤,使纯化过程大大缩短。
Objective： To find out the optimal method purifying polyphenol oxidase（PPO） from tobacco. Method： PPO was purified through the different saturated concentration salt-out, DEAE-Sepharose Fastflow and Sephadex G-150. The optimal method was obtained through mensurating and comparing the activity of PPO. Results： The optimum ammonium sulfate concentration of salt-out is 40%, and it enhanced the time of purification to 8.4 fold. The optimal method purifying PPO through DEAE-Sepharose Fastflow and Sephadex G-150 was obtained. The relative molecular weight of purified pro- rein is 42 000 and Km is 1.2 mmol/L.therefore it was identified as the polyphenol oxidase II. Conclusion： For the manipulative steps, this experiment omitted the following steps： organic solvent fractional precipitation, cation-exchange chromatography and so on; and it also shorten the process greatly.
出处
《生物技术通讯》
CAS
2006年第2期207-209,共3页
Letters in Biotechnology
关键词
烟草
多酚氧化酶
分离
纯化
tobacco
polyphenol oxidase
separation
purification
