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標題: | 利用唾液酸醛縮脢的定向演化增進L-唾液酸合成效率 Directed Evolution of N-acetylneuraminic Acid Aldolase for Synthesis of L-sialic Acid |
作者: | Li-Jhou Yang 楊禮州 |
指導教授: | 翁啟惠(Chi-Huey Wong) |
關鍵字: | 唾液酸,醛縮酶, sialic acid,aldolase, |
出版年 : | 2013 |
學位: | 碩士 |
摘要: | 能夠與天然界醣分子作用的胜肽 (peptide),藉由高親和力相結合,間接地阻止蛋白質與細胞表面的醣分子反應,可以當作藥物開發的方法之一。當有專一性的L-peptide 被篩選出,其相對應的鏡像異構物D-peptide即可利用化學合成製造,作用於細胞表面的D-sugar,阻止蛋白質與醣分子反應以作為藥物,因此合成L-sugar (為了篩選L-peptide)在此方法中便成了先決條件;這些非天然界的D-peptide之所以可能發展為新的藥物,最重要的原因就是因為其在生物體中相較於L-peptide,對蛋白水解酶抵抗性較好,在人體及動物體內不易被水解酶影響。
在本論文研究中,著手於利用酵素去合成非天然界的醣分子;首先利用L-ManNAc先合成L-sialic acid,之後再以N5B2醛縮酶為模板,利用隨機突變進行酵素的演化,並以L-sialic acid為基質進行篩選以找出對L-sialic acid立體選擇性較佳的酵素。在第一階段的實驗,合成L-sialic acid的過程中,發現利用乾填法的純化方式可以有效的將非鏡像異構物純化,包括L-glucose 上保護基後alpha/ beta的anomer,以及L-sialic acid不必策略性地上保護基團便可將C-4 hydroxy非鏡像異構物成功純化。在第二階段,實驗企圖進一步提升酵素 (N5B2醛縮酶)對L-sialic acid的專一性以合成L-sialic acid;演化的酵素經由蛋白質純化後,進行動力學參數測量。實驗的結果發現並未篩選出較好的酵素,其理由以及解決的方法會進一步的進行討論。 Peptides that can bind to natural D-sugar with high affinity and specificity can act as functional inhibitors to block glycan-protein interaction, and may have the potentials to be developed as therapeutics. When high affinity L-peptides that bind to unnatural L-sugar was identified, corresponding unnatural D-peptides can then be artificially synthesized by chemical methods to bind to natural D-sugars to interfere protein-carbohydrate interactions and used as potential inhibitors. Hence, preparation of L-sugar is a prerequisite in this approach. The advantages of D-peptides compared to natural L-peptides include being resistant to protease and increased serum half-lives. In this study, enzymatic synthesis was attempted to produce unnatural L-sugar. L-sialic acid was chemically prepared from L-ManNAc and then N5B2 sialic acid aldolase was improved to increase its stereoselectivity toward L-sialic acid by directed evolution. For the synthesis of L-sialic acid, it was found that diastereomer with partial protection groups can be successfully purified by using a dry loading method to facilitate diastereomer separation during chromatography. Furthermore, evolution of N5B2 sialic acid aldolase resulted in no successful improvements in reactivity towards L-sialic acid. The reasons and possible resolutions will be discussed. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/62848 |
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顯示於系所單位: | 化學系 |
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