通过添加非自然氨基酸来改善天然酶活性
Isaac N Ugwumba1, Kiyoshi Ozawa, Zhi-Qiang Xu
1Commonwealth Scientific and Industrial Research Organization, Black Mountain, Canberra, Australia.
Journal of the American Chemical Society
|December 18, 2010
概括
使用非自然氨基酸的工程细菌三酶显示,杀虫剂水解率增加了8-11倍. 这表明设计者氨基酸可以扩展酶功能和进化超出自然界限.
科学领域:
- 生物化学 生物化学
- 酵素工程是什么? 酶工程是什么
- 合成生物学 合成生物学
背景情况:
- 细菌三酶在化农药氧方面非常有效.
- 他们的自然进化极限对于氧溶解活性被认为是接近.
- 外围活性部位残留物在非化学催化步骤中发挥作用.
研究的目的:
- 调查非自然氨基酸是否可以改善三酶的催化周转率.
- 探讨外围活性部位残留在基质结合和产品释放中的作用.
- 在细菌三酶中改进农药水解活性.
主要方法:
- 在位置309用非天然的氨基酸取代了氨酸:L-(7-hydroxycoumarin-4-yl) ethylglycine (Hco) 和L-(7-methylcoumarin-4-yl) ethylglycine.
- 包括天然氨基酸白,氨酸和氨酸进行比较.
- 进行了动力学分析,以评估催化效率并确定速度限制步骤.
主要成果:
- 非自然的Hco氨基酸显著增加了限制产品释放速度的步骤.
- 当Hco的7-基组被去质子化时,它将负电荷的产物以静电方式排斥出去.
- 通过单个理性设计的突变,在催化活性中实现了8-11倍的改善.
- 这种改进超过了使用天然氨基酸突变的努力.
结论:
- 非天然的氨基酸为增强现有的酶功能提供了强大的工具.
- 设计者氨基酸为酶进化提供了新的序列和功能空间.
- 这种方法证明了工程酶超出其自然能力的巨大潜力.
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