Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Lyngby, Denmark.
Department of Chemistry, Technical University of Denmark, Lyngby, Denmark.
PLoS One. 2014 Jan 3;9(1):e83902. doi: 10.1371/journal.pone.0083902. eCollection 2014.
This paper reports rational engineering of Trypanosoma rangeli sialidase to develop an effective enzyme for a potentially important type of reactivity: production of sialylated prebiotic glycans. The Trypanosoma cruzi trans-sialidase and the homologous T. rangeli sialidase has previously been used to investigate the structural requirements for trans-sialidase activity. We observed that the T. cruzi trans-sialidase has a seven-amino-acid motif (197-203) at the border of the substrate binding cleft. The motif differs substantially in chemical properties and substitution probability from the homologous sialidase, and we hypothesised that this motif is important for trans-sialidase activity. The 197-203 motif is strongly positively charged with a marked change in hydrogen bond donor capacity as compared to the sialidase. To investigate the role of this motif, we expressed and characterised a T. rangeli sialidase mutant, Tr13. Conditions for efficient trans-sialylation were determined, and Tr13's acceptor specificity demonstrated promiscuity with respect to the acceptor molecule enabling sialylation of glycans containing terminal galactose and glucose and even monomers of glucose and fucose. Sialic acid is important in association with human milk oligosaccharides, and Tr13 was shown to sialylate a number of established and potential prebiotics. Initial evaluation of prebiotic potential using pure cultures demonstrated, albeit not selectively, growth of Bifidobacteria. Since the 197-203 motif stands out in the native trans-sialidase, is markedly different from the wild-type sialidase compared to previous mutants, and is shown here to confer efficient and broad trans-sialidase activity, we suggest that this motif can serve as a framework for future optimization of trans-sialylation towards prebiotic production.
本文报道了对 Trypanosoma rangeli 神经氨酸酶进行理性工程改造，以开发一种用于潜在重要反应性的有效酶：产生唾液酸化的前生物糖。此前，已经使用 Trypanosoma cruzi 转神经氨酸酶和同源的 T. rangeli 神经氨酸酶来研究转神经氨酸酶活性的结构要求。我们观察到，T. cruzi 转神经氨酸酶在底物结合裂缝的边界处具有七个氨基酸基序（197-203）。该基序在化学性质和取代概率上与同源神经氨酸酶有很大不同，我们假设该基序对转神经氨酸酶活性很重要。197-203 基序具有强烈的正电荷，与神经氨酸酶相比氢键供体能力有明显变化。为了研究该基序的作用，我们表达和表征了 T. rangeli 神经氨酸酶突变体 Tr13。确定了高效转神经氨酸酶的条件，并证明了 Tr13 的接受体特异性对接受体分子具有混杂性，能够使含有末端半乳糖和葡萄糖的聚糖甚至葡萄糖和岩藻糖的单体发生唾液酸化。唾液酸在与人乳寡糖结合时很重要，并且已经证明 Tr13 可以唾液酸化许多已建立和潜在的前生物。使用纯培养物对前生物潜力的初步评估表明，双歧杆菌的生长虽然不是选择性的，但具有选择性。由于 197-203 基序在天然转神经氨酸酶中突出，与野生型神经氨酸酶相比明显不同，并且与之前的突变体相比，它表现出能够赋予高效和广泛的转神经氨酸酶活性，因此我们建议该基序可以作为未来针对前生物生产优化转神经氨酸酶的框架。
