臺灣博碩士論文加值系統

白胺酸胜肽酶（leucine aminopeptidase; LAP）是一種針對蛋白質和胜肽之胺基端胺基酸殘基進行水解的外胜肽酶（exopeptidase），縱使LAPs廣存於生物體從細菌到人類，這類酵素之生物弁鄖丹滮揖憫馴廍F解。在這研究中，將分三個研究單元以探討Bacillus kaustophilus LAP (BkLAP)之可能應用及這酵素活性中心附近六個關鍵殘基之弁鄔囧丹�:

1. 融合BkLAP至 Bacillus sp. strain TS-23 脉 -澱粉酶（amylase）之澱粉結合區域以進行單一步純化。我們將Bacillus sp. strain TS-23 脉-澱粉酶的澱粉-吸附區域被導入BkLAP之C-端區域末端，以產生一個帶有生澱粉吸附活性（raw-starch-binding activity）之夢幻酵素(BkLAPsbd)。BkLAPsbd(分子量約65 kDa)能於Escherichia coli M15細胞中進行大量表現，且可以鎳-螯合層析管柱（nickel-chelate chromatography）純化至均質狀態。Native PAGE和色層分析之分析顯示純化之融合蛋白有一個六倍體結構（hexameric structure）。BkLAPsbd在70℃下之半衰期為12 min，相較於野生型酵素於相同受熱環境下約減少20%酵素活性。與野生型酵素相比，由於BkLAPsbd 之KM值增加約91%，導致催化效率約減少60%。澱粉-吸附分析顯示，此融合酵素（fusion enzyme）之Kd及Bmax值分別是2.3μM和0.35 μmol/g。粗粹之BkLAPsbd對生澱粉之吸附能力會受到澱粉濃度、pH值以及溫度影響。被吸附酵素液能以溶於20 mM Tris-HCl緩衝溶液（pH 8.0）之2%可溶性澱粉中溶析。粗粹液中有49% BkLAPsbd可經由一個吸附-溶析反覆循環純化且得到11.4倍純化效果。

　2. BkLAP固定化於Ca-褐藻膠/k-紅藻膠珠子之特性分析。我們也探討BkLAP固定化於Ca-褐藻膠/k-紅藻膠上之特性。對於未固定化和已固定化的BkLAP，其最適pH值分別為8.0和7.5，而最適溫度分別是55℃和60℃。BkLAP經固定化後，其熱穩定性增加；且其最大反應速率 (Vmax) 和Michalis-Menten常數 (KM) 也明顯地改變。固定化酵素可經由10次重複使用而沒有明顯活性減失。經由30天在4℃的培養後，發現固定化酵素的熱穩定性降為起始的90%。在50mM H2O2存在下，固定化BkLAP比未固定化酵素穩定，顯示出酵素的氧化穩定性可藉由固定化獲得改善。

3. BkLAP活性中心周圍殘基之選位突變。BkLAP上Thr346和Leu352殘基之重要性，可藉由選位突變來進行探討。這些位置被置換後之影響，係以在E. coli中進行表現的His6-BkLAP融合蛋白來評估。Thr346分別以Tyr、Arg、和Leu置換後，會造成LAP活性戲劇性減少。除L352V維持60%之野生型酵素活性外， L352E和L352R突變酵素之LAP活性完全喪失。從鋅含量分析以及蛋白質模擬推測BkLAP之Thr346和Leu352在維持鋅結合殘基配位環境上扮演重要角色。

序列比對顯示BkLAP之保留Ala348和Gly350殘基位在擬定的活性中心附近。我們更進一步藉由執行電腦模擬和選位突變來研究此兩個殘基的角色。根據此電腦模擬，Ala348分別藉由氫鍵與Asn345和Asn435 進行交互作用，而Gly350之羰基氧原子又分別與Ile353和Leu354之氫鍵牽連，而這些交互作用可能是在維持鋅配位殘基於正確位置。Ala348以Arg置換造成LAP活性戲劇性的減少。而A348E、A348V和Gly350突變酵素則完全喪失活性。野生型、Ala348和Gly350突變酵素之色胺酸發射螢光和偏圓二色吸收光譜（circular dichroism spectra）圖譜非常相似。由蛋白質模擬和選位突變推測Ala348和Gly350對BkLAP是必須的且係可能在維持催化活性中心之穩定環境。

同樣也藉由執行電腦模擬以及選位突變來研究BkLAP保留殘基Asn345和Asn435之角色。根據此電腦模擬，這兩個殘基經由氫鍵與Ala348和Arg351交互作用，這些交互作用可能對於那些活性中心殘基位於適當位置上非常重要。野生型和突變酵素於重組E. coli M15大量表現後係以鎳金屬-敖合層析管柱純化至均質狀態。BkLAP Asn345以Gln或Leu置換，會造成酵素活性戲劇性減少；Asn435突變酵素則完全觀察到活性。野生型和所有突變酵素之偏圓二色吸收光譜（circular dichroism spectra）圖譜非常相似，然而內在色胺酸螢光譜之測量顯示在Asn345和Asn435被置換後，其芳香族胺基酸殘基之顯微環境有明顯改變。除N435R和N435L外，野生型BkLAP和其他突變酵素對於溫度-誘導變性有相似之敏感度。野生型和所有突變酵素之活性中心結構電腦模擬顯示，Asn345和Asn435突變酵素呈現部分或全部之氫鍵鍵結喪失。綜合這些結果，可以斷定BkLAP之Asn345和Asn435殘基在結構上扮演的角色仍為維持活性中心之穩定環境。

Leucine aminopeptidase (LAP) is an exopeptidase that catalyzes the hydrolysis of amino acid residues from the amino terminus of proteins and peptides. Although LAPs are widely distributed in organisms from bacteria to humans, the biological roles of these enzymes are not fully understood. In this study, three research units are conducted to explore the possible application of Bacillus kaustophilus LAP (BkLAP) and the functional role of six critical residues around the active center of the enzyme:

1. Construction and one-step purification of Bacillus kaustophilus leucine aminopeptidase fused to the starch-binding domain of Bacillus sp. strain TS-23 a-amylase. The starch-binding domain of Bacillus sp. strain TS-23汹脉-amylase was introduced into the C-terminal end of BkLAP to generate a chimeric enzyme (BkLAPsbd) with raw-starch-binding activity. BkLAPsbd, with an apparent molecular mass of approximately 65 kDa, was overexpressed in Escherichia coli M15 cells and purified to homogeneity by nickel–chelate chromatography. Native PAGE and chromatographic analyses revealed that the purified fusion protein has a hexameric structure. The half-life for BkLAPsbd was 12 min at 70℃, while less than 20% of wild-type enzyme activity retained at the same heating condition. Compared with the wild-type enzyme, the 60% decrease in the catalytic efficiency of BkLAPsbd was due to a 91% increase in KM value. Starch-binding assays showed that the Kd and Bmax values for the fusion enzyme were 2.3 μM and 0.35 μmol/g, respectively. The adsorption of the crude BkLAPsbd onto raw starch was affected by starch concentration, pH, and temperature. The adsorbed enzyme could be eluted from the adsorbent by 2% soluble starch in 20 mM Tris–HCl buffer (pH 8.0). About 49% of BkLAPsbd in the crude extract was recovered through one adsorption-elution cycle with a purification of 11.4-fold.
2. Characterization of BkLAP immobilized in Ca-alginate/k-carrageenan beads. Properties of BkLAP immobilized in alginate/k-carrageenan hydrogel were examined. For the free and immobilized BkLAP, optimum pH was found to be 8.0 and 7.5,　respectively. The optimum temperature of the free and immobilized enzymes was also　observed to be 55 and 60°C, respectively. Thermal stability of BkLAP was increased as a result of immobilization. Maximum reaction rate (Vmax) and Michalis-Menten constant (KM) of BkLAP were changed significantly upon immobilization. The immobilized enzyme could be reused up to 10 cycles without any further loss of　activity. After incubating at 4°C for 30 days, the stability values for the immobilized　enzyme system were found to be 90%. In the presence of 50 mM H2O2, the immobilized BkLAP was more stable respective to the free enzyme, indicating that the oxidative stability of the enzyme can be improved by immobilization.

3. Site-directed mutagenesis of the conserved residues situated at the putative active site of BkLAP. The importance of Thr-346 and Leu-352 residues in BkLAP was explored by site-directed mutagenesis. The impact of substitutions at these positions was evaluated with His6-BkLAP fusion proteins expressed in E. coli. Substitution of Thr346 with Tyr, Arg, and Leu, respectively, resulted in a dramatic reduction in LAP activity. A complete loss of activity was observed in L352E and L352R variants with the exception of L352V, which retained approximately 60% of the wild-type activity. Zinc content analysis and protein modeling suggested that Thr346 and Leu352 of BkLAP play a role in maintaining the coordination environment for the zinc-binding residues.

Sequence alignment showed that the conserved Ala348 and Gly350 residues of BkLAP are located in the vicinity of the putative active site. We further investigated the roles of these two residues by performing computer modeling and site-directed mutagenesis. Based on the modeling, Ala348 interacts with Asn345 and Asn435 respectively by a hydrogen bond, and the carbonyl oxygen of Gly350 is also involved in the hydrogen bonding with Ile353 and Leu354 respectively, where these interactions might maintain the zinc-coordinated residues at their correct positions. Replacement of Ala348 with Arg resulted in a dramatic reduction in LAP activity. A complete loss of the activity was also observed in A348E, A348V, and the Gly350 variants. Tryptophan emission fluorescence and circular dichroism spectra were nearly identical for wild-type and all mutant enzymes. Protein modeling and site-directed mutagenesis suggest that residues Ala348 and Gly350 are essential for BkLAP and might involve in the maintenance of a stable active-site environment for the catalytic reaction.

Role of the conserved Asn345 and Asn435 residues of BkLAP was also investigated by performing computer modeling and site-directed mutagenesis. Based on the modeling, these two residues interact with Ala348 and Arg351 through hydrogen bonds, where these interactions might be critical for the active-site residues situated at their proper positions. The wild-type and mutant enzymes were over-expressed in the recombinant E. coli M15 and purified by nickel-chelate chromatography to near homogeneity. Replacement of BkLAP Asn345 with Gln or Leu resulted in a dramatic reduction in enzymatic activity. A complete loss of the LAP activity was observed in Asn435 variants. Circular dichroism spectra were nearly identical for wild-type and all mutant enzymes, while measurement of intrinsic tryptophan fluorescence revealed the significant alterations of the microenvironment of aromatic amino acid residues in Asn345 and Asn435 replacements. Except N435R and N435L, wild-type BkLAP and other mutant enzymes showed a similar sensitivity towards temperature-induced denaturation. Computer modeling of the active-site structures of wild-type and mutant enzymes shows a partial or complete loss of the hydrogen bonding in Asn345 and Asn435 variants. Taken together, it can be concluded that residues Asn345 and Asn435 of BkLAP might play a structural role to maintain a stable active-site environment for the catalytic reaction.

目錄

目錄 I

圖目錄 XII

表目錄 VIII

中文摘要 1
英文摘要 4
全文前言 8

第一章 構築Bacillus kaustophilus白胺酸胜肽酶與Bacillus sp. strain TS-23汹脉-澱粉酶澱粉結合區域之融合蛋白以單一步驟純化重組酵素 13
1.1 前言 14
1.2 材料與方法 21
1.2.1 材料、菌種和質體 21
1.2.2 表現質體之構築 21
1.2.3 BkLAP 和BkLAPsbd之金屬螯合管柱色層分析（Metal-chelate column chromatography） 23
1.2.4 蛋白質方法 24
1.2.5 酵素分析、動力學特性和熱穩定性 24
1.2.6 澱粉吸附分析 25
1.2.7 BkLAPsbd之吸附-沖提純化（Adsorption-elution purification of BkLAPsbd） 26
1.3 結果 27
1.3.1 BkLAP和BkLAPsbd之生產與純化 27
1.3.2 BkLAPsbd的特性 28
1.3.3 純化BkLAPsbd對生澱粉之吸附 29
1.3.4 藉由生澱粉之吸附-沖提 ( adsorption-elution ) 來進行BkLAPsbd之分離純化 30
1.4 討論 37

第二章 Bacillus kaustophilus白胺酸胜肽酶固定化於Ca-褐藻膠/k-紅藻膠（Ca-alginate/k-carrageenan）珠子之特性分析
40
2.1 前言 41
2.2 實驗 46
2.2.1 材料、菌種及生長條件 46
2.2.2 BkLAP之純化 46
2.2.2.1 利用IMAC技術純化His6-tagged BkLAP 46
2.2.2.2 重組酵素之大量表現及純化 47
2.2.3 固定化酵素 48
2.2.3.1 膠體製備 48
2.2.3.2 製作對照組之固定化球珠 48
2.2.3.3 製作BkLAP 固定化球珠 48
2.2.3.4 配置受質 49
2.2.3.5 Biocatalyst beads之反應前處理 49
2.2.4 不同膠體濃度之BkALP biocatalyst beads之活性反應 50
2.2.5 不同膠體濃度之BkALP biocatalyst beads之再利用性 51
2.2.6 固定化效率 51
2.2.7 掃描式電子顯微鏡 51
2.2.8 合成RITC-BkLAP衍生物 52
2.2.9 pH和溫度對固定化BkLAP活性 52
2.2.10 未固定和固定化BkLAP之動力學常數 53
2.2.11 固定化BkLAP之保存穩定性 53
2.2.12 固定化BkLAP之重複使用性 53
2.2.13 固定化對氧化穩定性之影響 53
2.3 結果 55
2.3.1 BkLAP之固定化 55
2.3.2 掃描式電子顯微鏡 55
2.3.3 RITC-BkLAP衍生物 56
2.3.4 pH和溫度對固定化BkLAP影響 59
2.3.5 動力參數 62
2.3.6 重覆使用性 62
2.3.7 保存穩定性 64
2.3.8 氧化穩定性 65
2.4 討論 66

第三章 Bacillus kaustophilus白胺酸胜肽酶活性中心之結構模擬及以選位突變定義其周圍殘基角色 69
3.1 前言 70
3.1.1 M17 LAP之結構 72
3.1.2 LAP之催化活性中心 75
3.1.3 螢光光譜及圓二色光譜 78
3.1.3.1 螢光光譜原理 78
3.1.3.2 蛋白質之螢光分析 79
3.1.3.3 圓二色光譜原理 80
3.1.3.4 蛋白質之圓二色光譜分析 81
3.1.4 BkLAP特性與先前研究 83
3.1.5 本章研究重點 85
3.1.5.1 Thr346和Leu352對B. kaustophilus白胺酸胜肽酶有獨特弁鄐妣垠n性 86
3.1.5.2 在B. kaustophilus白胺酸胜肽酶的活性中心上針對保留之Ala348和Gly350殘基做選位突變 86
3.1.5.3 藉由選位突變評估來自B. kaustophilus的白胺酸胜肽酶之保留胺基酸殘基Asn345和Asn435的角色 87
3.2 材料及方法 90
3.2.1 藥品 90
3.2.2 所使用菌株、質體及培養基 90
3.2.3 序列比對 91
3.2.4 質體DNA抽取 92
3.2.5 DNA洋菜膠體電泳分析 93
3.2.6 DNA 純度鑑定及定量分析 93
3.2.7 選位突變 94
3.2.7.1 突變DNA之製備 94
3.2.7.1.1 設計定點突變引子 94
3.2.7.1.2 聚合酶鏈鎖反應 96
3.2.7.1.3 產物之降解 97
3.2.7.1.4 檢測勝任細胞的存活 97
3.2.7.1.5 XL1-Blue supercompetent 細胞之轉型 97
3.2.7.1.6 DNA定序 98
3.2.8 轉型至E.coli M15 98
3.2.8.1 勝任細胞之製備 98
3.2.9 利用IMAC技術純化His6-tagged BkLAP 99
3.2.10 野生與突變酵素之大量表現 99
3.2.11 超音波震盪 100
3.2.12 利用Ni2+-NTA resin純化酵素 100
3.2.13 蛋白質分析方法 101
3.2.13.1 蛋白質電泳分析與活性染色 101
3.2.13.2 製膠流程 102
3.2.13.3 蛋白質電泳之菌體處理 102
3.2.13.4 進行PAGE分析 103
3.2.13.5 PAGE染色 103
3.2.13.6 蛋白質濃度的測定 104
3.2.13.7 呈色法測定酵素活性 105
3.2.14 野生型與突變酵素之酵素動力學 106
3.2.14.1 測最適反應基質濃度 106
3.2.14.2 作標準曲線 106
3.2.14.3 酵素之呈色試驗 107
3.2.14.4 計算反應初速度（V0） 107
3.2.14.5 Vmax 及 KM 測定 107
3.2.14.6 Lineweaver-Burk 雙倒數作圖法 108
3.2.14.7 推算KM及Vmax 108
3.2.14.8 計算kcat及比活性 109
3.2.14.9 催化效率（kcat/KM） 109
3.2.15 光譜結構特性分析 109
3.2.15.1 BkLAP之螢光光譜分析 110
3.2.15.2 原子吸收光譜 111
3.2.15.3 BkLAP之圓二色光譜測量 111
3.3 結果 113
3.3.1 BkLAP之殘基Thr346和Leu352對酵素之弁鄖丹� 113
3.3.1.1 LAPs羧基端區域之序列比對與建立BkLAP結構模型 113
3.3.1.2 Thr346和Leu352之置換 115
3.3.1.2.1 野生型與突變酵素之表現與純化 115
3.3.1.2.2 螢光光譜 115
3.3.1.2.3 原子吸收光譜 119
3.3.1.2.4 酵素動力學與熱穩定性 120
3.3.2 BkLAP推定活性中心附近保留殘基丙胺348和甘胺酸350之選位突變 122
3.3.2.1 BkLAP活性中心周圍殘基Ala348和Gly350之同源性電腦模擬 122
3.3.2.2 野生型和突變酵素之表現與純化 124
3.3.2.3 野生型和突變酵素之特性 127
3.3.2.4 野生型和突變酵素之鋅含量 128
3.3.2.5 BkLAP和突變酵素之結構分析 128
3.3.2.6 野生型與突變酵素之活性中心構形 131
3.3.3 藉由選位突變評估BkLAP之保留胺基酸殘基Asn345和Asn435角色 132
3.3.3.1 BkLAP活性中心周圍殘基Asn345和Asn435之同源性電腦模擬 132
3.3.3.2 野生型和突變酵素之純化與特色 135
3.3.3.3 N345和N435突變酵素之結構分析 136
3.3.3.4 突變對活性位置之影響 137
3.4 討論 143
3.4.1 Thr346和Leu352對BkLAP之重要性 143
3.4.2 BkLAP活性中心周遭保留性Ala348 和Gly350殘基之選位突變 143
3.4.3 置換殘基Asn345和Asn435對BkLAP活性之影響 144

第四章 後續之研究- 以分析式超高速離心機來測量BkLAP之四級結構 146
4.1 前言 147
4.2 材料與方法 149
4.2.1 蛋白質之製備 149
4.2.2 分析式超高速離心機實驗 149
4.2.2.1 樣品的準備 149
4.2.2.2 分析級超高速離心機沉降速率實驗 150
4.2.2.3 實驗數據分析 150
4.3 結果與討論 152

參考文獻 154
附錄一 QuikChange 定點突變方法之流程圖 171
附錄二 BkLAP之核甘酸及胺基酸序列 173
附錄三 Acrylamide resolving gel（分離膠；下層膠）製膠配方 174
附錄四 Stacking gel（上層膠；焦集膠）製膠配方 175
附錄五 176




圖目錄

全文前言
圖一 蛋白酶之分類。 9

第一章 構築Bacillus kaustophilus白胺酸胜肽酶與Bacillus sp. strain TS-23汹脉-澱粉酶澱粉結合區域之融合蛋白以單一步驟純化重組酵素

圖二 澱粉分解酵素之澱粉結合區域。 15
圖三 包含SBD之澱粉分解酵素演化樹。 17
圖四 B. circulans環糊精醣基轉移酶 (A) 與CBM20家族之SBD結構圖(B)。 20
圖五 B. kaustophilus LAP融合Bacillus sp. strain TS-23 脉-澱粉酶澱粉吸附區域之構築。 23
圖六 純化BkLAP and BkLAPsbd 之SDS-PAGE 分析。 28
圖七 BkLAP（實心三角型）和BkLAPsbd（實心圓型）之熱穩定性。 29
圖八 純化BkLAPsbd對生澱粉之吸附。 30
圖九 BkLAPsbd對生澱粉之吸附。 32
圖十 pH值和溫度對BkLAPsbd吸附至生澱粉之影響。 34
圖十一 以SDS-PAGE分析粗酵素液、生澱粉吸附後上清液，以及純化BkLAPsbd。 36

第二章 Bacillus kaustophilus白胺酸胜肽酶固定化於Ca-褐藻膠/k-紅藻膠（Ca-alginate/k-carrageenan）珠子之特性分析

圖十二 甘露糖醛酸與古洛糖醛酸之二級結構(A)以及鈣離子依靠型epimerization程序以形成膠體(B)。 45

圖十三 固定化BkLAP 對人工受質之Leu-p-NA之水解。右為對照組，左為Ca-褐藻膠/k-紅藻膠BkLAP球珠。 50
圖十四 未固定與固定BkLAP之Ca-褐藻膠/k-紅藻膠固定化球珠顆粒表面SEM顯微照相圖（A）Ca-褐藻膠/k-紅藻膠固定化球珠（B）Ca-褐藻膠/k-紅藻膠/BkLAP固定化球珠。57
圖十五 （A）以SDS-PAGE 分析 RITC conjugated BkLAP。（B）UV-vis 吸收圖譜。 58
圖十六 pH值對游離態與固定化酵素之活性(A)與穩定性(B)影響。 60
圖十七 溫度對游離態與固定化酵素之活性(A)與穩定性(B)影響。 61
圖十八 使用次數對固定化酵素活性影響。 63
圖十九 游離態和固定化BkLAP之保存穩定性。 64
圖二十 游離態和固定化BkLAP之氧化穩定性。 65

第三章 Bacillus kaustophilus白胺酸胜肽酶活性中心之結構模擬及以選位突變定義其周圍殘基角色

圖二十一 由序列相似性所建立演化樹狀圖。 71
圖二十二 牛眼睛LAP之單體結構。 74
圖二十三 牛眼睛LAP之三倍及六倍體結構。 75
圖二十四 BlLAP活性中心與鋅配位或催化有關殘基。 76
圖二十五 推論之BlLAP催化機制。 77
圖二十六 Jablonski 圖。 79
圖二十七 左旋及右旋圓偏極光組成之偏極光。 81
圖二十八 蛋白質二級結構之遠紫外光圓二色光譜。 82
圖二十九 BlLAP之活性中心結構。 88
圖三十 BlLAP和EcPepA的LAPs活性中心周圍殘基之序列比對。 89
圖三十一 （A與B）以E. coli PepA之結構做為模板，構築B. kaustophilus LAP之同源性模型和活性中心結構。（C與D）圖示為野生型酵素Thr346和Leu352殘基之氫鍵鍵結和疏水性反應。 114
圖三十二 在突變前後B. kaustophilus LAP Thr346與Leu352殘基週遭位置之DNA序列。 116
圖三十三 已純化野生型和突變酵素之SDS-PAGE（A）和native-PAGE（B）。 117
圖三十四 已純化野生型和突變B. kaustophilu LAP酵素之螢光發射光譜圖。 118
圖三十五 溫度對已純化野生型 BkLAP（實心圓圈）和L352V突變酵素（空心圓圈）之活性（A）及穩定性影響（B）。 121
圖三十六 （A）以EcPepA之結晶結構做為模板構築BkLAP之活性中心結構。（B）圖示為野生型酵素之Ala-348和Gly-350殘基之氫鍵鍵結。 123
圖三十七 突變前後B. kaustophilus LAP Ala348與Gly350殘基週遭位置之DNA序列。 125
圖三十八 以SDS-PAGE分析粗粹液(A)與已純化野生型及突變蛋白質(B)。 126
圖三十九 BkLAP與其突變酵素的色胺酸螢光發射光譜。 129
圖四十 BkLAP與其突變酵素之CD光譜。Far-UV光譜是在22℃下紀錄。 130
圖四十一 以222 nm CD訊號監測BkLAP級其突變酵素溶於20 mM Tris-HCl buffer (pH 8.0)之熱變性。 130
圖四十二 BkLAP與其突變酵素的活性中心之重疊。以程式PyMOL繪製活性中心結構。 131
圖四十三 一些M17酵素之C-端區域序列比對以及野生型BkLAP假定活性中心附近之局部結構。 133
圖四十四 突變前後B. kaustophilus LAP Asn345與Asn435殘基週遭位置之DNA序列。 138
圖四十五 已純化野生型與Asn345和Asn435突變酵素之SDS-PAGE分析。 139
圖四十六 野生BkLAP及Asn345和Asn435突變酵素之圓二色光譜（A）與色胺酸螢光圖譜（B）。 140
圖四十七 野生BkLAP及Asn345和Asn435突變酵素之熱變性。於222 nm下觀測溶於10 mM Tris-HCl buffer (pH 8.0)之蛋白質樣品CD訊號。 141
圖四十八 模擬突變酵素在位置345和435附近的結構。 142

後續之研究- 以分析式超高速離心機來測量BkLAP之四級結構

圖四十九 牛眼睛LAP之四級結構。A. 由上往下看。B.側面圖。 148
圖五十 以分析式離心機進行BkLAP之連續性沉降分析。 151

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