Institute of Bioengineering, Zhejiang University of Technology, Hangzhou 310014, People's Republic of China.
J Biotechnol. 2013 Mar 10;164(1):75-81. doi: 10.1016/j.jbiotec.2012.12.015. Epub 2013 Jan 11.
Recombinant β-glucuronidase (GUS) expressed in Pichia pastoris GS115 is an important glycoprotein, encoded by a gene with four potential N-glycosylation sites. To investigate the impact of N-linked carbohydrate moieties on the stability of recombinant GUS, it was deglycosylated by peptide-N-glycosidase F (PNGase-F) under native conditions. The enzymatic activities of the glycosylated and deglycosylated GUS were compared under various conditions such as temperature, pH, organic solvents, detergents and chaotropic agent. The results demonstrated that the glycosylated GUS retained greater fraction of maximum enzymatic activity against various types of denaturants compared with the deglycosylated. The conformational stabilities of both GUS were analyzed by monitoring the unfolding equilibrium by using the denaturant guanidinium chloride (dn-HCl). The glycosylated GUS displayed a significant increase in its conformational stability than the deglycosylated counterpart. These results affirmed the key role of N-glycosylation on the structural and functional stability of β-glucuronidase and could have potential applications in the functional enhancement of industrial enzymes.
毕赤酵母中表达的重组β-葡萄糖醛酸酶（GUS）是一种重要的糖蛋白，由一个基因编码，该基因有四个潜在的 N-糖基化位点。为了研究 N-连接的碳水化合物对重组 GUS 稳定性的影响，用肽-N-糖苷酶 F（PNGase-F）在天然条件下对其进行了去糖基化。比较了糖基化和去糖基化 GUS 在不同条件下（如温度、pH 值、有机溶剂、清洁剂和变构剂）的酶活性。结果表明，与去糖基化 GUS 相比，糖基化 GUS 在各种变性剂下保留了更多的最大酶活性。通过使用变性剂盐酸胍（dn-HCl）监测解折叠平衡来分析两种 GUS 的构象稳定性。糖基化 GUS 的构象稳定性显著增加，比去糖基化的 GUS 高。这些结果证实了 N-糖基化对β-葡萄糖醛酸酶结构和功能稳定性的关键作用，并且在增强工业酶的功能方面具有潜在的应用。
