Jin Zhang,Shengzhen Hou,Yanru Chen,Jing Zhou,Hao Chen,Yebang Tan
The strategy of dual cross-linking was investigated by enhancing the performance of dynamic hydrogels. To this end, phenylalanine modified ε-polylysine was synthesized and employed as the polymer backbone of hydrogels. The phenylalanine moieties and amine groups of the polymer could be cross-linked with cucurbit[8]uril (CB[8]) and the dialdehyde cross-linker, respectively. Single CB[8] linkage with fast dynamics led to an increase in the viscosity of the polymer solution, and single imine linkage with slow dynamics led to the formation of weak and brittle hydrogels. However, the two linkages were combined together to form a dual-cross-linked hydrogel and the performance of the hydrogel could be well enhanced. Compared with the single imine cross-linked hydrogel, the dual-cross-linked hydrogel demonstrated a higher mechanical strength, better extensibility and faster self-healing rate. It is anticipated that this line of research could provide a useful method to enhance the performance of dynamic hydrogels.
Many fluorometric methods have been developed for detecting tyrosinase activity, yet many of them require the sophisticated synthesis of relatively complex fluorescent molecules. Here, we report the utilization of a label-free supramolecular tandem assay for measuring tyrosinase activity based on an easily available reporter pair. Cucurbit[8]uril (CB[8]) can bind with thioflavin-T (ThT) and increase its fluorescence intensity , while the tripeptides YLA and Dopa-LA can decrease the fluorescence intensity of CB[8]-ThT complex. YLA and Dopa-LA can be oxidized by tyrosinase, and the products have lower binding affinity with CB[8], leading to an increase of fluorescence in the intensity of CB[8]-ThT-YLA or Dopa-LA. We were able to continuously monitor tyrosinase activity with CB[8]-ThT-YLA or Dopa-LA.
Yun-Bin Han,Liu-Qing Chen,Zhuo Li,Yu-Meng Tan,Yan Feng,Guang-Yu Yang
Endoglycoceramidases (EGCases) specifically hydrolyze the glycosidic linkage between the oligosaccharide and the ceramide moieties of various glycosphingolipids, and they have received substantial attention in the emerging field of glycosphingolipidology. However, the mechanism regulating the strict substrate specificity of these GH5 glycosidases has not been identified. In this study, we report a novel EGCase I from Rhodococcus equi 103S (103S_EGCase I) with remarkably broad substrate specificity. Based on phylogenetic analyses, the enzyme may represent a new subfamily of GH5 glycosidases. The X-ray crystal structures of 103S_EGCase I alone and in complex with its substrates monosialodihexosylganglioside (GM3) and monosialotetrahexosylganglioside (GM1) enabled us to identify several structural features that may account for its broad specificity. Compared with EGCase II from Rhodococcus sp. M-777 (M777_EGCase II), which possesses strict substrate specificity, 103S_EGCase I possesses a longer α7-helix and a shorter loop 4, which forms a larger substrate-binding pocket that could accommodate more extended oligosaccharides. In addition, loop 2 and loop 8 of the enzyme adopt a more open conformation, which also enlarges the oligosaccharide-binding cavity. Based on this knowledge, a rationally designed experiment was performed to examine the substrate specificity of EGCase II. The truncation of loop 4 in M777_EGCase II increased its activity toward GM1 (163%). Remarkably, the S63G mutant of M777_EGCase II showed a broader substrate spectra and significantly increased activity toward bulky substrates (up to >1370-fold for fucosyl-GM1). Collectively, the results presented here reveal the exquisite substrate recognition mechanism of EGCases and provide an opportunity for further engineering of these enzymes.
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