Institute of Biological Engineering, Department of Chemical and Biological Engineering, Zhejiang University, Zheda Road 37#, Hangzhou 310027, Zhejiang, PR China.
Enzyme Microb Technol. 2011 May 6;48(6-7):438-44. doi: 10.1016/j.enzmictec.2011.03.008. Epub 2011 Mar 26.
With improved enzymatic activity and easy accessibility, the recombinant purine nucleoside phosphorylase (PNPase) could be a very promising alternative for nucleoside biosynthesis. In our work, the deoD gene encoding PNPase was successfully cloned from Escherichia coli MG1665 and overexpressed in E. coli BL 21(DE3). After optimization of expression conditions including temperature, induction timing and isopropyl-thio-β-D-galactoside (IPTG) concentration, over 70% of expressed total protein was His-tagged PNPase, in the soluble and functional form. Followed assays indicated that the recombinant enzyme exhibited similar substrate specificity and pH preference as the wild type PNPase. Furthermore, the immobilization technology was applied to develop the possible application of recombinant enzyme. Agar from four different polymer carriers was selected as a suitable matrix for whole recombinant cell entrapment. Subsequent enzyme assays, kinetic analysis and stability evaluation of free and immobilized recombinant cells were compared. The results indicated that although the immobilization process reduced the substrate affinity and catalytic efficiency of recombinant cells, it could significantly enhance the stability and reusability of these cells. Finally, the immobilized whole cell biocatalyst was applied to produce ribavirin, as a model nucleoside synthesis reaction. The obtained relative high productivity of ribavirin and quick reaction time suggested the great potential and feasibility of immobilized PNPase in efficient and valuable industrial utilizations.
具有改进的酶活性和易于接近性，重组嘌呤核苷磷酸化酶（PNPase）可能是核苷生物合成的非常有前途的替代物。在我们的工作中，成功地从大肠杆菌 MG1665 中克隆了编码 PNPase 的 deoD 基因，并在大肠杆菌 BL 21（DE3）中过表达。在优化表达条件（包括温度、诱导时机和异丙基硫代-β-D-半乳糖苷（IPTG）浓度）后，超过 70%的总表达蛋白以可溶和功能形式被 His 标记的 PNPase 标记。随后的测定表明，重组酶表现出与野生型 PNPase 相似的底物特异性和 pH 偏好。此外，还应用固定化技术来开发重组酶的可能应用。从四种不同的聚合物载体中选择琼脂作为整个重组细胞包埋的合适基质。比较了游离和固定化重组细胞的后续酶测定、动力学分析和稳定性评估。结果表明，尽管固定化过程降低了重组细胞的底物亲和力和催化效率，但它可以显著增强这些细胞的稳定性和可重复使用性。最后，将固定化的全细胞生物催化剂应用于生产利巴韦林，作为核苷合成反应的模型。获得的利巴韦林相对较高的生产率和快速的反应时间表明，固定化 PNPase 在高效和有价值的工业应用中具有巨大的潜力和可行性。
