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Updated: May 25, 2025

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核酸脱氧核糖转移酶的分子进化,以增强向2'--2'-脱氧核酸转移酶的活性

Su-Been Yang¹, Yeon-Jin Yoo¹, Kanghyun Choi²

¹Department of Biological Sciences and Bioengineering, Inha University, Incheon 22212, Korea.

Journal of industrial microbiology & biotechnology

|February 25, 2025

概括

工程核酸脱氧基转移酶 (NDT) 突变体对合成非天然核酸类似物具有增强的活性. 这些改进的生物催化剂,fNDT-i1和fNDT-i4,对核酸相似物生产中的工业应用具有前景.

科学领域:

生物催化剂是一种生物催化剂.
酶工程是什么? 酶工程是什么?
合成生物学合成生物学

背景情况:

核酸脱氧基转移酶 (NDT) 对于合成核酸相似物至关重要.
野生类型的无核酸试验对非天然核酸类比的活性有限.
一个先前确定的突变,fNDT(L59Q),证明了对2'--2'-脱氧二 (2FDU) 的活性有所改善.

研究的目的:

为2FDU合成设计具有增强活性和稳定性的新型NDT突变物.
为了确定负责提高酶性能的关键突变.
评估工程NDT变体作为工业生物催化剂的潜力.

主要方法:

使用易出错的PCR进行分子进化.
选突变库以提高2FDU活动.
双重突变的构建和表征 (fNDT-i4).
酶动力学和热稳定性测试.

主要成果:

确定了两个高度活跃的突变,即fNDT-i1 (V52A) 和fNDT-i2 (L28I).
双重突变fNDT-i4在高温下表现出显著更高的活性 (40°C时为4.0倍,50°C时为20.6倍).
fNDT-i1和fNDT-i4的催化效率分别是野生类型的NDT的3.1倍和3.7倍.
特定突变 (L59Q,V52A,L28I) 与改善的热稳定性,基质亲和力和反应效率有关.

结论:

工程NDT突变物,特别是fNDT-i1和fNDT-i4,在合成非天然核酸相似物方面表现出卓越的性能.
这些增强的生物催化剂为工业规模生产核酸相似物提供了显著的潜力.
鉴定的突变为改善NDT功能的酶工程策略提供了洞察力.

关键词:

酶工程是什么?酶工程是什么?容易发生错误的PCR.核酸的类似物是核酸.核酸脱氧基转移酶的核酸脱氧基转移酶

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