- 學位論文
探討酵母菌中 EXG1 轉醣合成昆布寡醣
Enzymatic transglycosylation of Saccharomyces cerevisiae EXG1 to synthesize laminarin-derived oligosaccharides
摘要
轉醣是醣基水解酶 (glycosyl hydrolase, GH) 的功能之一，它可以透過將醣基從受質供體 (substrate donor) 轉移到目標受體 (acceptor donor) 來形成新的醣苷鍵。用此方式可以合成複雜或新型的寡醣，也可能是滿足當今市場對功能性寡醣的高度需求的一種替代生產方法。先前研究發現酵母菌 Saccharomyces cerevisiae中的 EXG1蛋白 (ScEXG1) 具有轉醣而合成昆布寡醣的能力。但對於ScEXG1產生的寡醣結構組成或產量相關資訊仍有許多未知。因此，我們將進一步的了解在不同反應時間、受質濃度、酵素濃度條件下，ScEXG1 in vitro合成昆布寡醣的能力，以及其產物的結構表徵。在我們的研究中以Laminaribiose為受質，利用ScEXG1 新合成了三種不同的寡糖 (DP3、DP4-1、DP4-2)，其中最主要的寡醣 DP3鑑定為β-Glc-(1 → 6)-β-Glc-(1→ 3)-β-Glc。另外，當使用不同的酵素濃度時，原始受質和新合成的寡醣水解成葡萄糖的速率會有所不同。在固定的酵素濃度下，所有測試的Laminaribiose受質濃度 (4.38 – 32.85 mM) 都可以合成 DP3、DP4-1 和 DP4-2。但在 4.38 mM, 75 min 和 21.9 mM, 180 min下，分別合成了最低 (12.24 ± 0.18 µg/mL, p < 0.05) 和最高 (136.55 ± 5.21 µg/mL, p < 0.05) 的DP3。DP4-1 和 DP4-2 的含量均低於定量極限。為了提高ScEXG1合成能力仍需要進一步研究如何降低 ScEXG1 的水解活性，同時提高合成能力，以便能夠充分利用其酵素進行大規模寡醣合成。
並列摘要
Transglycosylation is a powerful tool that glycosyl hydrolases (GHs) possess and enables new glycosidic bonds formation by transferring a glycosyl moiety from the substrate donor to the desired acceptor. Complex or novel oligosaccharides can be synthesized with this method, which could be an alternative to fulfill the high market demand for functional oligosaccharides nowadays. Saccharomyces cerevisiae EXG1 (ScEXG1) has been reported previously to have the ability to synthesize the potential functional laminari-oligosaccharide through transglycosylation. However, the information on either the structure or the quantity of the oligosaccharide produced by ScEXG1 was very scarce and still have many unknowns. Therefore, we investigated the in vitro synthesis of ScEXG1 under different conditions (reaction times, substrate concentration, enzyme concentration) on producing laminarin-derived oligosaccharides, as well as the structural characterization of the product(s). In our work, we discovered that using laminaribiose as the substrate, ScEXG1 newly synthesized three different oligosaccharides (DP3, DP4-1, DP4-2), where the major oligosaccharide DP3 was characterized as β-Glc-(1 → 6)-β-Glc-(1→ 3)-β-Glc. Also, the transglycosylation reactions varied when different enzyme concentrations were used, and consequently, the hydrolysis rates of the original substrate and the newly synthesized oligosaccharide into glucose. Furthermore, under a fixed enzyme concentration, all the substrate concentrations given (4.38 – 32.85 mM) resulted in the synthesis of DP3, DP4-1 and DP4-2. The lowest (12.24 ± 0.18 µg/mL, p < 0.05) and highest (136.55 ± 5.21 µg/mL, p < 0.05) amounts of the synthesized DP3 were found under 4.38 mM for 75 min and 21.9 mM laminaribiose for 180 min, respectively. However, both DP4-1 and DP4-2 were under the limit of quantification. Future investigations on reducing the hydrolytic activity and increasing the synthesis ability of ScEXG1 are still needed to be able to fully exploit this enzyme for large-scale oligosaccharide synthesis.
參考文獻
延伸閱讀
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