用现有蛋白质支架设计和演变新的催化活性
Hee-Sung Park1, Sung-Hun Nam, Jin Kak Lee
1Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1, Kusung-Dong, Yusung-Gu, Daejon 305-701, Korea.
概括
蛋白质工程成功地将β-乳糖酶活性引入了一个新的酶支架. 这种进化的酶有效地水解了cefotaxime,增强了细菌的耐药性.
科学领域:
- 蛋白质工程是指蛋白质工程.
- 酶设计 酶设计
- 生物催化剂是一种生物催化剂.
背景情况:
- 设计具有新功能的酶是蛋白质工程的一个关键目标.
- 修改现有的蛋白质支架为创造新的生物催化剂提供了一个有希望的策略.
研究的目的:
- 开发一种方法,通过修改活性位点循环来改变酶催化活性.
- 将β-乳糖酶活性引入氧酶II支架中.
主要方法:
- 同时插入,删除和替换活动位点循环.
- 通过点突变微调酶活性.
- 设计了glioxalase II的alphabeta/betaalpha金属化酶支架.
主要成果:
- 一个新的酶,evMBL8 (进化金属β-乳糖酶8),被创造出具有β-乳糖酶活性.
- 改造的酶失去了其原始的glyoxalase II活性.
- EvMBL8有效地催化了cefotaxime的水解,其 (kcat/Km) app为1.8 x 10 ((2) (摩尔/升) ((-1) 秒) ((-1).
结论:
- 修改活性位点循环的策略对于创建具有新功能的酶是有效的.
- 改造的evMBL8表现出显著的β-乳糖酶活性,使其在cefotaxime.xime上对大肠杆菌生长产生了相当大的耐药性.
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