- 學位論文
溶菌酶還原變性之觀察、變性動力學之分析以及復性策略之探討
Observation and Kinetic Analysis on the Reductive Denaturation of Lysozyme and Investigation on the Strategy of Lysozyme Refolding
摘要
眾多因素會影響雞蛋白溶菌酶還原變性的程度，如: 蛋白質濃度、還原劑濃度與變性時間等等。 另外也有必要確認完全還原變性的溶菌酶具有哪些特徵。 在此篇研究中，利用自身螢光、水力半徑 (分子大小排斥層析)、螢光染劑IAF標記與活性回收率等方式來觀察溶菌酶變性的過程並且探討完全還原變性所需條件。 結果發現濃度 5 mg/mL 的溶菌酶必須溶在含有0.2 M二硫代蘇糖醇的變性溶液中經過30小時才能夠完全還原變性瓦解其立體結構。 而在還原變性過程中，觀察到了變性中間體的存在並且確認其結構屬於”殘存一個雙硫鍵的溶菌酶”。 經由這樣的發現，我們提出了”三階段還原變性”的動力學模式以下列步驟: 正確構型之溶菌酶至第一變性中間體，第一變性中間體至第二變性中間體，第二變性中間體至完全還原變性之溶菌酶此三步驟之反應速率常數各為: 0.61 mM-0.467h-1, 0.20 h-1以及0.0095 mM-0.333h-1。 而最後一步: 第二變性中間體至完全還原變性之溶菌酶則為反應速率決定步驟，另外經由螢光染劑的標記實驗結果得知: 第二變性中間體仍為殘存一個雙硫鍵的溶菌酶結構。 接下來我們以稀釋法進行復性發現當添加1.5 M的尿素至復性溶液中時，可以讓完全還原變性的溶菌酶逐漸復性達到80 % 的活性回收比且可以降低氧化態穀胱甘肽的使用量。 另外針對添加不同還原劑濃度或是不同變性時間所造成的不同還原變性程度之溶菌酶進行稀釋法復性時，若復性溶液中也添加了1.5 M 尿素，則可以觀察到不同變性程度的溶菌酶會有不同的復性速率與不同的活性回收比，因此復性效果優劣與變性的程度的高低密切相關。 一般而言，變性程度越高則復性速率越慢且活性回收比越低。 最後我們製備了巰基丙酸-戊二醛-幾丁聚醣微粒並用來促進溶菌酶的復性，配合透析復性法可以使溶菌酶的比活性增加到90 % 。
並列摘要
Many factors affect the extent of reductive denaturation of hen egg white lysozyme, e.g., the concentration of reducing agent such as dithiothreitol (DTT), the concentration of lysozyme and the time length of denaturation. It is thus crucial to identify the fully reductively denatured lysozyme. In this study, the reductive denaturation process of lysozyme was monitored with intrinsic fluorescence、hydrodynamic radius (size exclusion chromatography)、5-iodoacetamido fluroescein (IAF) labeling and activity recovery to characterize the fully reductively denatured lysozyme. It was found that lysozyme (5 mg/mL) was fully reductively denatured after being incubated in denaturing buffer which contained 0.2 M DTT for 30 hrs. Further, a reductively denatured intermediate of lysozyme was also identified as one-disulfide bond-left lysozyme. According to these findings, we proposed a “three-step reductive denaturation” kinetic model: N (native lysozyme) to I1 (intermediate 1) to I2 (intermediate 2) to U (fully unfolded lysozyme) to describe the existence of intermediates during reductive denaturation of lysozymes. The kinetic constants were estimated by fitting the shift in the wavelength of maximum emission intensity during denaturation with the three-step kinetic model. The rate constants for each steps were found to be 0.61 mM-0.467h-1, 0.20 h-1, and 0.0095 mM-0.333h-1, respectively, suggesting that I2 to U is the rate-limiting step. Additionally, the reductively denatured lysozyme intermediate I2 still has one uncleaved disulfide bond as revealed by IAF labeling. After that, by the method of dilution refolding, addition of 1.5 M urea in refolding buffer highly enhanced the refolding yield to 80 % and minimized the cost of GSSG. The denaturation of lysozyme in various extent could be generated by the addition of different concentrations of reducing agent (DTT) or different denaturing times under the influence of 1.5 M urea. The refolding of reductively denatured lysozyme was highly affected by the extent of denaturation. In general, a high DTT concentration and a long denaturing time resulted in slow refolding with low activity recovery. Finally, the beads of chitosan and its thiol derivative (MPA-GLA-chitosan) were prepared and used to facilitate the refolding of lysozyme. The use of MPA-GLA-chitosan beads as refolding additives increased the specific activity of lysozyme to 90 % after dialysis refolding.