臺灣博碩士論文加值系統

白胺酸胜肽胺基切割酶是一種胜肽外切酶，可自胜肽鏈的N端將白胺酸殘基移除。本研究將Bacillus stearothermophilus NCIB 8924之白胺酸胜肽胺基切割酶II基因選殖到表現載體pQE32上，並且利用T5啟動子的調節來進行基因表現。此攜帶6個His標記之酵素以鎳離子螯合的層析分離法進行均質純化後，可得到具有425 U/mg比活性及回收率達76%的蛋白質。此純化蛋白質之次單位利用SDS-PAGE測得的分子量為44.5 kDa；其最適操作溫度、pH值分別是60℃與8.0。Co2+對此重組型酵素具有增強效應；而Cu2+、Hg2+和DTT、EDTA等金屬螯合劑則對其具有強烈的抑制作用。經EDTA處理後的失活酵素可由Co2+刺激而恢復活性，由此可知其為Co2+依賴型的金屬酵素。綜觀以上生化特性，我們發現此重組型白胺酸胜肽胺基切割酶II (rLAPII)與其原生型具有極明顯的相似性。
氧化穩定性的分析顯示，rLAPII對於H2O2所造成的氧化傷害會隨著溫度上升而增加感受性。經300 mM H2O2處理的酵素，其二級結構有明顯的改變。為了探討Met殘基在酵素活性受氧化作用抑制的過程中所扮演的角色，我們將rLAPII的5個Met殘基以定點突變法置換成Leu，純化後得到Mr值為44.5 kDa的突變酵素。Met82Leu、Met88Leu、Met254Leu及Met382Leu等突變酵素的比活性與野生型相似；而則Met136Leu降低。動力學參數顯示，Met136Leu的kcat值減少了47%，因此催化效率(kcat/Km)也降低了50%。所有Met突變酵素都比野生型酵素對氧化劑敏感。由此可知，B. stearothermophilus LAPII的Met殘基在氧化反應中具有保護酵素的功能。
金屬胜肽切割酶依其催化作用之類別共分為33個家族，白胺酸胜肽胺基切割酶II為M29家族的成員之一，其與金屬結合的胺基酸殘基至今仍不清楚。為了想進一步探討此酵素之Co2+結合部位，經比對了不同之胜肽胺基切割酶的胺基酸序列後，找出一些可能與酵素活性有關的高保留區胺基酸殘基。在rLAPII中，我們挑出Asp-61、Asp-180、Asp-308、Asp-339、Asp-380、Asp-391、Asp-396、His-191、His-227、His-345和His-378等殘基進行定點突變。其中，7個Asp殘基被置換成Ala，而4個His殘基則被置換成Leu。酵素活性分析方面，Asp380Ala、His345Leu和His378Leu三者均無活性表現；而Asp61Ala與鈷離子的親合性明顯降低。動力學參數顯示，其他突變型酵素的Km值皆比野生型酵素增加30﹪以上。在kcat值的比較中，Asp391Ala、Asp396Ala、His191Leu和His227Leu四者均增加；而Asp339Ala、Asp61Ala和Asp308Ala則降低了40﹪至60﹪。Asp61Ala之催化效率(kcat/Km)降低達83﹪之多，Asp308Ala和Asp339Ala也降低了70﹪，Asp-180、Asp-391、Asp-396、His-191和His-227的催化效率並無明顯差異。以EDTA處理酵素後，我們發現野生型、Asp180Ala、Asp308Ala、Asp339Ala、Asp391Ala、Asp396Ala、His191Leu和His227Leu等在添加CoCl2後可恢復酵素活性。經EDTA處理的Asp61Ala變異酵素之活性恢復情形很不明顯；但是EDTA處理的Asp61Glu酵素活性恢復則明顯可見。此外，apo-Asp380Ala、apo-His345Leu、apo-His345Arg、apo-His378Leu及apo-His378Arg在添加CoCl2後仍無法恢復酵素活性。根據以上結果推測，Asp-61的羧基支鏈可能在鈷離子結合上扮演重要角色，而Asp-380、His-345和His-378則可能為酵素結構之關鍵胺基酸。

Leucine aminopeptidases (LAPs) are exopeptidases that remove the N-terminal L-leucine from peptide substrates. For expression of Bacillus stearothermophilus NCIB 8924 leucine aminopeptidase II (LAP II) in Escherichia coli regulated by a T5 promoter, the gene was amplified by polymerase chain reaction and cloned into expression vector pQE-32 to generate pQE-LAPII. The His6-tagged enzyme was overexpressed in IPTG-induced E. coli M15 (pQE-LAPII) as a soluble protein and was purified to homogeneity by nickel-chelate chromatography to a specific activity of 425 U/mg protein with a final yield of 76%. The subunit molecular mass of the purified protein was estimated to be 44.5 kDa by SDS-PAGE. The temperature and pH optima for the purified protein were 60oC and 8.0, respectively. Under optimal condition, the purified enzyme showed a marked preference for Leu-ρ-nitroanilide, followed by Arg- and Lys-derivatives. The His6-tagged enzyme was stimulated by Co+2 ions, but was strongly inhibited by Cu+2 and Hg+2 and by the chelating agents, DTT and EDTA. The EDTA-treated enzyme could be reactivated with Co+2 ions, indicating it is a cobalt-dependent exopeptidase. Taking the biochemical characteristics together, we found that the recombinant LAPII (rLAPII) exhibits significant similarities from those properties described for the native enzyme.
Oxidative stability assay showed that the rLAPII was sensitive to oxidative damage by hydrogen peroxide at the elevated temperature. The H2O2-treated enzyme experienced obvious changes in the secondary structure when the oxidant concentration increased to 300 mM. To investigate the role of methionine residues on the oxidative inactivation, each of the five methionine residues in the rLAPII was replaced with leucine by site-directed mutagenesis. The mutant enzymes with an apparent Mr of approximately 44.5 kDa. The specific activities for Met82Leu, Met88Leu, Met254Leu, and Met382Leu were similar to that of the wild-type enzyme, whereas a reduced activity was observed in Met136Leu. The 50% decrease in the catalytic efficiency (kcat/Km) for Met136Leu was caused by 47% decrease in kcat value. As compared with the wild-type enzyme, all mutant proteins were more sensitive to the oxidant, implying that the methionine residues of B. stearothermophilus LAP II are important for the protection of the enzyme from oxidative inactivation.
Metallopeptidases form the most diverse of the catalytic types of peptidases, about 33 families being recognized. LAP is a member of family M29 of metallopeptidases, and the metal-binding residues remain unknown. In order to identify the Co2+-binding domain, we performed the alignment of amino acid sequence of LAPs from different microorganisms to determine the conserved amino acid residues, which might be essential for enzyme activity. The conserved amino acid residues of rLAPII include Asp-61, Asp-180, Asp-308, Asp-339, Asp-380, Asp-391, Asp-396, His-191, His-227, His-345 and His-378 were changed by site-directed mutagenesis. Replacement of 7 Asp residues with Ala and 4 His residues with Leu. There is no enzymatic activity were detected in Asp380Ala, His345Leu and His378Leu. Co2+ affinity of Asp61Ala was decreased obviously. The Km values for the other mutants were increased more than 30% by comparison with wild-type enzyme. The kcat values for Asp391Ala, Asp396Ala, His191Leu and His227Leu were increased, while kcat decreased 40 to 60%for Asp339Ala, Asp61Ala and Asp308Ala. The 83% decreased in the catalytic efficiency (kcat/Km) for Asp61Ala and the 70% decreased was exhibited by Asp308Ala and Asp339Ala. Alterations in Asp-180, Asp-391, Asp-396, His-191 and His-227 did not cause a significant difference on the catalytic efficiency. The EDTA-treated enzyme experienced obvious reactivation in the wild-type, Asp180Ala, Asp308Ala, Asp339Ala, Asp391Ala, Asp396Ala, His191Leu and His227Leu by CoCl2 addition. The significant reactivation of EDTA-treated Asp61Glu was observed, while the reactivation of EDTA-treated Asp61Ala was feebly. Moreover, apo-Asp380Ala, apo-His345Leu, apo-His345Arg, apo-His378Leu and apo-His378Arg could not restore the LAP activity by CoCl2 addition. Based on these results, it is suggested that the carboxyl group of Asp-61 may be play an important role in cobalt-binding, while Asp-380, His-345 and His-378 were the putative essential residues for enzyme structure.

目 錄
目錄………………………………………………………………....1
中文摘要………………………………………………………….....5
英文摘要…………………………………………………………….8
前言………………………………………………………………...11
一、 蛋白分解酶(Proteases)………………………………………….11
二、 Bacillus stearothermophilus……………………………………..15
三、 Leucine aminopeptidases (LAPs)………………………………..16
四、 過氧化氫對酵素穩定性之影響………………………………...17
五、 拉曼光譜學(Raman spectroscopy)在生化上之應用…………...18
六、 金屬胜肽切割酶(Metallopeptidases)…………………………...20
七、 酵素催化機制之研究…………………………………………...23
八、 論文研究方向…………………………………………………...24
材料與方法……………………………………………………….....27
一、 藥品……………………………………………………………....27
二、 菌株、質體及培養條件…………………………………………27
三、 E. coli質體DNA的抽取……………………………………......27
四、 B. stearothermophilus染色體DNA之抽取………………….....28
五、 DNA的洋菜膠體電泳分析………………………………….......29
六、 DNA片段之回收與純化…………………………………….......30
七、 聚合酶鏈鎖反應………………………………………………….31
八、 DNA黏接反應………………………………………………33
九、 質體的轉形作用……………………………………………..33
十、 酵素純化……………………………………………………..34
十一、 蛋白質的電泳分析…………………………………………35
十二、 呈色法測定酵素活性………………………………………36
十三、 rLAPII特性分析……………………………………………37
十四、 定點突變法………………………………………………….38
十五、 酵素之氧化穩定性及對過氧化氫之抗性………………….39
十六、 氧化酵素次級結構之拉曼光譜儀分析…………………….40
十七、 鈷離子親合性……………………………………………….40
十八、 EDTA處理之酵素再活化反應…………………………….40
十九、 酵素動力學參數之測定……………………………………41
結果……………………………………………………………....43
一、 rLAPII的大量表現及純化…………………………………..43
1. B. stearothermophilus LAP II基因之選殖
2. IPTG誘導之最適濃度與時間
3. 酵素之純化
二、 rLAPII的特性分析………………………………………….44
1. pH值及溫度對酵素活性之影響
2. 酵素對受質的選擇性
3. 金屬離子與化學試劑對酵素活性之影響
三、 酵素中甲硫胺酸(methionine)殘基經定點突變後對過氧化
氫之抗性……………………………………………………..45
1. 過氧化氫對rLAPII之影響
2. 突變基因之表現與酵素純化
3. 酵素之氧化穩定性
4. 酵素比活性與動力學特性
四、 探討天門冬酸(aspartic acid)殘基在酵素催化作用中扮演
的角色……………………………………………………….47
1. 突變基因之表現與酵素純化
2. 酵素比活性與動力學特性
3. Asp變異酵素之鈷離子親合性
4. EDTA處理後Asp變異酵素之再活化作用
五、 探討組織胺酸(histidine)殘基在酵素催化作用中扮演
的角色……………………………………………………….48
1. 突變基因之表現與酵素純化
2. 酵素比活性與動力學特性
3. His變異酵素之鈷離子親合性
4. EDTA處理後His變異酵素之再活化作用
六、 天門冬酸與組織胺酸殘基第二次突變之分析……………..49
1. 變異酵素之鈷離子親合性
2. EDTA處理後變異酵素之再活化作用
討論………………………………………………………………...51
一、 rLAPII之純化與生化特性……………………………………..51
二、酵素中Met殘基經定點突變後對過氧化氫之抗性……………53
三、天門冬酸(aspartic acid)殘基在酵素催化作用中所扮
演的角色………………………………………………………….54
四、組織胺酸(histidine)殘基在酵素催化作用中所扮演
的角色…………………………………………………………….55
參考文獻………………………………………………………………….....57
圖表目錄………………………………………………………………….....65
附錄………………………………………………………………………….99
個人資料…………………………………………………………...108

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