基于Phi29 DNA聚合酶的新酶挖掘及表征

Mining and characterization of new enzymes based on Phi29 DNA polymerase

摘要:

近年来，噬菌体Φ29(Phi29) DNA聚合酶因其具有恒温高保真扩增的能力，成为热点关注对象。为了进一步推进这类等温聚合酶的工业化应用，本研究将已表征的Phi29 DNA聚合酶序列针对微生物宏基因组进行新酶挖掘与表征。在植物宿主群体的宏基因组中，鉴定出一种新酶—— Php29 DNA聚合酶，该酶具有更高的链置换活性(与噬菌体Φ29的相似度达到59.5%)。实验验证结果表明，该酶也具备3ʹ→5ʹ核酸外切酶活性，其扩增产物可用于进一步的催化反应。Php29 DNA聚合酶的挖掘和验证为未来等温扩增酶的工业化应用提供了有益参考。

Abstract:

In recent years, the bacteriophage Φ29 (Phi29) DNA polymerase has garnered increasing attention due to its high-fidelity amplification capacity at constant temperatures. To advance the industrial application of this type of isothermal polymerases, this study mined and characterized new enzymes from the microbial metagenome based on the known Phi29 DNA polymerase sequence. The results revealed that a new enzyme, Php29 DNA polymerase, was identified in the microbial metagenome with plants as the hosts. This enzyme exhibited higher strand displacement activity, with a 59.5% similarity to bacteriophage Φ29. Experimental validation demonstrated that the enzyme had 3ʹ→5ʹ exonuclease activity, and its amplification products can serve as substrates for further catalytic reactions. The discovery and validation of Php29 DNA polymerase gives insights into the future industrial application of isothermal polymerases.