5'- nucleotidase (5'-NT) is a key enzyme in nucleoside/nucleotide metabolic pathway, it plays an important role in the biosynthesis of cordycepin in caterpillar fungus. In this study, a 5'-NT gene was identified and mined from genomic DNA of caterpillar fungus, which was 1968 bp in length and encoded 656 amino acid residues. The recombinant 5'-NT was first time heterologously expressed in Pichia pastoris GS115, subsequently purified and functionally characterized. The optimal reaction temperature for 5'-NT was 35 °C, and it retained 52.8% of its residual activity after incubation at 50 °C for 1 h. The optimal reaction pH was 6.0 and it exhibited high activity over a neutral pH range. Furthermore, 5'-NT exhibited excellent Km (1.107 mM), Vmax (0.113 μmol/mg·min) and kcat (4.521 S-1) values compared with other typical 5'-nucleotidase. Moreover, substrate specificity analyses indicated that 5'-NT exhibited different phosphatase activity towards the substrates containing different basic groups. The work presented here could be useful to 5'-NT applications and provide more scientific basis and new ideas for the biosynthesis of artificial control cordycepin.

中文翻译：

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通过有效的基因组挖掘，从毛虫真菌中表达，纯化和鉴定5'-核苷酸酶。

5'-核苷酸酶（5'-NT）是核苷/核苷酸代谢途径中的关键酶，在虫草菌丝的生物合成中起着重要作用。在这项研究中，从毛虫真菌的基因组DNA中鉴定出5'-NT基因，该基因长1968 bp，编码656个氨基酸残基。重组5'-NT首次在巴斯德毕赤酵母GS115中异源表达，随后纯化并进行功能表征。5'-NT的最佳反应温度为35°C，在50°C孵育1小时后，其保留剩余活性的52.8％。最佳反应pH为6.0，在中性pH范围内表现出高活性。此外，与其他5'-NT相比，其5'-NT具有极好的Km（1.107 mM），Vmax（0.113μmol/ mg·min）和kcat（4.521 S-1）值。-核苷酸酶。此外，底物特异性分析表明5'-NT对含有不同碱性基团的底物表现出不同的磷酸酶活性。此处介绍的工作可能对5'-NT应用很有用，并为人工控制虫草素的生物合成提供了更多的科学依据和新的思路。