Isolation, purification and characterization of 5'-phosphodiesterase from <i>Aspergillus fumigatus</i>

5′-Phosphodiesterase (5′-PDE) catalyzes the hydrolysis of ribonucleic acid to obtain a mixture of ribonucleotides, such as 5′-guanosine monophosphate and 5′-adenosine monophosphate. In this study, a 5'-PDE was newly isolated and purified from Aspergillus fumigatus. Following purification, this enzyme exhibited a specific activity of 1036.76 U/mg protein, a molecular weight of 9.5 kDa, and an optimal temperature and pH for enzyme activity of 60°C and 5.0, respectively. However, its activity was partially inhibited by Fe3+, Cu2+, and Zn2+, but slightly improved by the presence of K+ and Na+. Additionally, chemical-modification experiments were also applied to investigate the structural information of 5'-PDE, in which the residues containing carboxyl and imidazole groups were essential for enzyme activity based on their localization in the 5′-PDE active site. Furthermore, purified 5′-PDE could specifically catalyze the synthesis of ribonucleotides with a Vmax 0.71 mmol/mg·min and a KM of 13.60 mg/mL.5′-磷酸二酯酶（5′-Phosphodiesterase，5′-PDE）可催化核糖核酸（ribonucleic acid）水解，生成以5′-鸟苷一磷酸、5′-腺苷一磷酸为代表的核糖核苷酸混合物。本研究从烟曲霉（Aspergillus fumigatus）中全新分离纯化得到一株5′-PDE。经纯化后，该酶的比酶活达1036.76 U/mg蛋白，分子量为9.5 kDa，催化反应的最适温度与pH分别为60℃与5.0。然而，Fe³+、Cu²+与Zn²+会对其酶活产生部分抑制作用，而K+与Na+则可小幅提升其酶活性。此外，本研究还通过化学修饰实验探究了5′-PDE的结构信息，结果表明，定位于该酶活性位点内的羧基与咪唑基残基对其酶活至关重要。进一步实验显示，纯化得到的5′-PDE可特异性催化核糖核苷酸合成，其最大反应速率（Vmax）为0.71 mmol/mg·min，米氏常数（KM）为13.60 mg/mL。