臺灣博碩士論文加值系統

本研究主要利用 urea 和 guanidine hydrochloride (GdnHCl) 對 C端帶有6×His tag的N-carbamoyl-D-amino acid amidohydrolase (DCaseH) 進行變性反應，以了解其結構變化及特性。為了探討其變性程度，分別以不同濃度的變性試劑在不同時間條件下反應，除利用HPLC分析其殘餘活性外，並以螢光光譜儀及動態雷射光散射 (DLS) 分析其結構變化。

殘餘活性分析結果顯示，DCaseH經過了4 M urea 變性處理20分鐘後，殘餘活性開始明顯地下降，而當urea 濃度為4.5 M時殘餘活性已降至原本的 50% ，在urea 濃度達6 M時已幾乎完全失活 。由螢光光譜分析結果，當DCaseH 經 4.3 M urea 變性處理 20分鐘後開始出現紅位移現象，且在以6 M urea 反應10分鐘後完全偏移。 DLS結果則發現其經過 urea變性處理30分鐘後，粒徑隨 urea濃度增加而有逐漸變大趨勢，但並無膠體狀的超大聚集體(colloid-emulsion) 產生，研判當DCaseH 利用 urea 做變性處理時較不激烈且為簡單的展開反應。在變性試劑GdnHCl研究的方面，當 DCaseH 經 GdnHCl 0.7 M 變性處理 5分鐘後，殘餘活性開始下降，在GdnHCl 達0.8 M 時衰減為 50%，而在GdnHCl 為 2 M時完全失活，表示GdnHCl對DcaseH的變性反應較urea來得強烈。 而當酵素以GdnHCl 1 M處理 10分鐘後，螢光光譜開始呈現紅位移現象，表酵素結構開始展開，此時DLS 結果顯示粒徑開始變化為超大粒徑(範圍為600 ~ 1000 nm)，而當GdnHCl 濃度達 2 M時其粒徑則變回10~11 nm間。以結果顯示經GdnHCl變性反應的酵素，其結構一經展開後會立即變成膠體聚集的型態，最後在達一定程度的變性後再提高GdnHCl濃度，則聚集體會被再次溶解成為完全分散的小分子。

The purpose of this study is to investigate the denaturation of C-terminally His-tagged N-carbamoyl-D-amino acid amidohydrolase (DCaseH) with urea and guanidine hydrochloride (GdnHCl). The denaturation degrees were determined with residual activity, fluorescence, and dynamic laser scattering (DLS) analyses under different denaturant concentrations for various time. The activity assays showed that the residual activities of DCaseH started to significantly decline when the urea concentration was beyond 4 M for 20 min, reached a 50% value when the enzyme was treated with 4.5 M, and almost completely vanished right after the 6-M treatment. For the fluorescence analysis, a red-shift spectrum was first observed when DCaseH was treated with 4.3 M urea for 20 min, followed by a complete denaturation after a 10-min 6-M treatment. The DLS results showed that particle sizes gradually increased with urea concentrations for the 30 min treatment without the observation of any colloid-emulsion formation, suggesting that the inactivation of DCaseH with urea was mainly caused by a simple unfolding mechanism. As for the GdnHCl study, a dramatic declination of the residual activities was first observed at 0.7 M for a 5-min treatment, a 50% residue at 0.8 M, and a nearly complete inactivation at 2 M, indicating that GdnHCl is a much hasher denaturant for DCaseH than urea. The fluorescence analysis indicated that DCaseH started to unfold and consequently display a red-shift spectrum when it was simply treated with 1 M GdnHCl for 10 min. The DLS studies showed the particle sizes of DCaseH first increased to a high value (600 ~ 1000 nm) and then returned to a native size (10~11 nm) throughout the process of increasing GdnHCl concentrations, with an observation of formation of emulsion droplets which were subsequently dissolved, suggesting that both unfolding and aggregation took place during the denaturation of DCaseH with GdnHCl.

目錄

中文摘要………………………………………………………………………... I

英文摘要………………………………………………………………………... II


目錄………………………………………………………………………........... IV
表目錄………………………………………………………………………....... VII
圖目錄………………………………………………………………………....... VIII

第一章 緒論……………………………………………………………………. 1

第二章 文獻回顧………………………………………………………………. 3
2-1 D型胺基酸D-p-HPG……………………………………………………… 3
2-1-1 D型胺基酸…………………………………………………………... 3
2-2 DCase ……………………………………………………………………… 4
2-2-1 DCase的原生種來源及特性…………………………………......... 4
2-2-2 DCase的晶體結構和單體間作用力………………………………… 8
2-2-3 DCase的活性區域與胺基酸水解反應機制………………………… 11
2-3 變性試劑對酵素結構的影響……………………………………………... 12
2-3-1變性試劑對酵素催化活性的影響………………………………….. 22
2-3-2變性試劑對酵素結構變化的影響………………………………….. 24
2-3-3還原試劑DTT對酵素變性過程穩定性之影響……………………. 25
2-4 固定化金屬層析法(IMAC)……………………………………………….. 26
2-5 蛋白質結構分析方法……………………………………………………... 32
2-5-1螢光光譜儀(fluorescence spectroscopy)……………………………... 32
2-5-1-1原理………………………………………………………….. 32
2-5-1-2螢光光譜於蛋白質及胜肽結構分析上的應用…………….. 33
2-5-2動態雷射光散射(dynamic light scattering, DLS)…………………… 33
2-5-2-1原理………………………………………………………….. 34
2-5-2-2原理公式及應用…………………………………………….. 36

第三章 實驗目的................................................................................................. 40

第四章 實驗……………………………………………………………………. 41
4-1 實驗流程…………………………………………………………………... 41
4-2 實驗材料…………………………………………………………………... 41
4-2-1菌株....................................................................................................... 41
4-2-2蛋白質純化層析擔體、純化管柱及透析膜………………………… 41
4-2-3其他....................................................................................................... 41
4-3 實驗藥品…………………………………………………………………... 41
4-4 實驗設備…………………………………………………………………... 43
4-5 實驗步驟…………………………………………………………………... 44
4-5-1酵素之生產………………………………………………………….. 44
4-5-1-1 DCaseH原生種於E. coli BL21(DE3)之生產....................... 44
4-5-1-2 DCaseH原生種之純化…...………………………………… 45
4-5-2 DCaseH酵素之變性反應後與活性分析........................................... 45
4-5-2-1 DCaseH酵素之活性單位 (U)……………………………… 45
4-5-2-2 D-p-HPG及N-carbamoyl-D-p-HPG之HPLC分析條件...... 46
4-5-2-3純化DCaseH酵素之變性反應及活性分析.......................... 46
4-5-2-4配製不同濃度urea及D-p-HPG溶液以HPLC分析所得之
檢量線製作…………………………………………………..
47
4-5-2-5蛋白質之濃度分析…………………………………………. 47
4-5-3 DCaseH酵素之變性反應與螢光光譜儀分析……………………… 47
4-5-4 DCaseH酵素之變性反應與DLS分析…………………………….. 48

第五章 結果與討論 50
5-1 DCaseH酵素在不同變性試劑濃度及變性時間反應後之殘餘活性分析.. 50
5-2 DCaseH酵素受變性試劑反應後之結構分析……………………………. 52
5-2-1螢光光譜分析………………………………………………………. 52
5-2-2動態雷射光光譜(DLS)分析原生種DCaseH酵素………………… 59

第六章 結論…………. …………. …………. …………. …………. ………... 73

參考文獻…………. …………. …………. …………. …………. …………. ... 74

附錄…………. …………. …………. …………. …………. …………. ……... 79

表目錄

Table 2-1 生產DCase酵素之各個原生菌種之特性...................................... 9
Table 5-1 DCaseH純化酵素在不同urea濃度及各變性反應時間後之殘餘活性分析.......................................................................................... 53
Table 5-2 DCaseH純化酵素在不同GdnHCl濃度及各變性反應時間後之殘餘活性分析.................................................................................. 54
Table 5-3 DCaseH純化酵素在不同urea濃度下之粒徑大小及分佈分析... 68
Table 5-4 DCaseH純化酵素在不同GdnHCl濃度下之粒徑大小及分佈分析....................................................................................................... 69













圖目錄

Figure 2-1 Steps involved in industrial amoxycllin production…………........ 5
Figure 2-2 Chemo-enzymatic or enzymatic production of D-amino acid........ 6
Figure 2-3 Schematic ribbon diagram of the DCase homotetramer structure viewed along the Xm axis from Agrobacterium sp. strain KNK712………………………………………………………….. 13
Figure 2-4 (a) Front view of the protein fold of DCase, (b)Topological diagram of the protein fold of DCase…………………………….. 14
Figure 2-5 (a)Ribbon representation of the homotetrameric structure of DCase from Agrobacterium radiobacter CCRC 14924, (b) The subunit of DCase, (c) Topology of subunit A…………………….. 15
Figure 2-6 Structural anaylsis of three catalytically important residues His129、His144 and His215 as identified by mutagenesis……………. 17
Figure 2-7 Sequence alignment of DCase……………………………………. 18
Figure 2-8 (a) Superimposed structures between C172S-DCase and C172S-HPG enzyme-substrate complex, (b) Schematic diagram of HPG bound to C172S………………………………………….. 19
Figure 2-9 The model of the DCase-N-carbamoyl-D-phenylalanine complex…………………………………………………………... 20
Figure 2-10 Proposed mechanism of N-carbamyl-D-amino acid hydrolysis….. 21
Figure 2-11 Unfolding of a monomeric protein……………………………….. 27
Figure 2-12 (a) Molecular formulas of urea and GdnHCl, (b) Urea induces the unfolding of a protein composed of α-helices and β-sheets, (c) The protein disulfide bonds would be broken following the reaction with urea and mercaptoethanol………………………….. 28
Figure 2-13 Schematic depiction of protein…………………………………… 30
Figure 2-14 Schematic representation of the urea-induced structural and functional changes in FabG………………………………………. 31
Figure 2-15 DTT is highly efficient at reducing disulfides, since a single molecule can reduce the intermediate mixed disulfide by forming a ring tructure…………………………………………………….. 31
Figure 2-16 (a) TALON of CO2+ metal chelated affinity resin, (b) structure of imidazole and histidine………………………………………… 32
Figure 2-17 光照射發光分子之部份能階圖………………………………… 35
Figure 2-18 (a) The propagated wave from light scattered by particles, (b) Curve of current (I) as a function of time (t)……………………... 38
Figure 2-19 The light from the source was reflected perpendicularly to the sample…………………………………………………………….. 39
Figure 2-20 Illustration of Stoke’s law for the fictional force exerted on spherical objects with very small Reynolds numbers (e.g., very small particles) in a continuous viscous fluid…………………….. 39
Figure 4-1 實驗流程圖..................................................................................... 42
Figure 4-2 (a)以HPLC分析20 mM之D-p-HPG(滯留時間為1.774 min)之圖圖譜, (b)以HPLC分析1 M urea(滯留時間為1.776 min)之圖譜 49
Figure 5-1 DCaseH純化酵素和不同濃度變性試劑反應後殘餘活性對各變性反應時間作圖。(a) Urea、(b) GdnHCl……………………. 55
Figure 5-2 DCaseH純化酵素和變性試劑反應不同時間後殘餘活性對各變性試劑濃度作圖。(a) Urea、(b) GdnHCl……………………. 56
Figure 5-3 DCaseH純化酵素和變性試劑反應不同時間後的結構展開分率(fU)分析。(a) Urea、(b) GdnHCl………………………………. 57
Figure 5-4 當DCaseH純化酵素和低於(含) 4.0 M urea進行變性反應後，螢光光譜強度隨變性試劑濃度升高而遞減，並無任何位移發生………………………………………………………………… 60
Figure 5-5 當DCaseH純化酵素和高於(含) 4.2 M urea進行變性反應後，螢光光譜開始呈現紅位移狀態(red shift)………………………. 61
Figure 5-6 當DCaseH純化酵素和低於(含) 0.9 M GdnHCl進行變性反應後，螢光光譜強度隨變性試劑濃度升高而遞減，並無任何位移發生……………………………………………………………. 63
Figure 5-7 當DCaseH純化酵素和高於(含) 1 M GdnHCl進行變性反應後，螢光光譜開始呈現紅位移狀態(red shift)………………….. 64
Figure 5-8 不同變性反應時間後之CSM值和變性試劑濃度之關係 65
Figure 5-9 以DLS分析DCaseH純化酵素於不同濃度urea變性反應後30 min之粒徑變化.............................................................................. 70
Figure 5-10 熱處理溫度對不同突變酵素之熱失活表觀(apparent)速率常數的影響…………………………………………………………… 71
Figure 5-11 以DLS分析DCaseH純化酵素於不同濃度GdnHCl變性反應後30 min之粒徑變化…………………………………………… 72

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