臺灣博碩士論文加值系統

摘要

本研究係自人類糞便中分離出能水解幾丁質的Clostridium perfringens，並試驗能使其產生幾丁質酶的最適培養基，再純化此酵素與探討其特性。此菌無需幾丁質或類似物的誘導，能在主要含蛋白腖及酵母萃之培養基中，於厭氧條件下在37℃培養至25小時後，產生活性最高之幾丁質酶。將粗酵素液先以硫酸銨沉澱，再經透析、濃縮及DEAE管柱及HIC管柱層析處理，可獲得兩種幾丁質酶（P-1及P-2）。此二幾丁質酶均能同時水解4-methylumbellifery-N-acetylchitobiose [4-MU-(NAG)2]與4-methylumbellifery-N-acetylchitotriose [4-MU-(NAG)3]。經Sephacryl S-300與SDS-PAGE檢定，P-1具有分子量139.7 kDa，由兩個次單元（87.3及52.5 kDa）構成；而P-2具有分子量196.8 kDa，亦由兩種次單元（97及49.9 kDa）構成。由於在溫度與pH穩定度、最適作用溫度及pH、和金屬離子致活上具有非常類似的特性，因此推測此二者應為同一種蛋白質。在同樣條件下，培養本菌至96小時後，收集粗酵素液，再以相同步驟處理粗酵素液，於DEAE管柱層析下，僅得一個具有水解幾丁質活性的波峰。再以HIC管柱層析後，獲得幾丁質酶(A-4)。幾丁質酶A-4經SDS-PAGE檢定後，可得一個單一蛋白帶，分子量為75 kDa，且仍同樣具有水解4-MU-(NAG)2與4-MU-(NAG)3之特性。以4-MU-(NAG)2為受質時，最適反應溫度為40℃，最適pH為6.0，Km值為0.14 mM，Vmax值為416.67 nmole/ min/ mg；以4-MU-(NAG)3為受質時，最適反應溫度為50℃，最適pH亦為6.0，Km值為0.34 mM，Vmax值為384.62 nmole/ min/ mg。以4-MU-(NAG)2或4-MU-(NAG)3為受質時，A-4僅受dithiothreitol與2-iodoacetamide1 (1 mM)致活；iodoacetic acid與phenyl methyl sulfonyl fluoride則會抑制其活性；金屬離子Fe2+、Fe3+或Hg+2也會抑制A-4的活性，而Ba2+、Ca2+、Cu2+、K+、Mg2+、Mn2+或Na+僅會活化該酵素水解4-MU-(NAG)2。幾丁質酶A-4水解不同來源幾丁質時，產物以N-乙醯幾丁二糖為主，且其中以水解β-form的魷魚鞘幾丁質為最有效率。由於粗酵素液中尚有其他酵素，能將幾丁質酶P-1與P-2修飾成A-4，而幾丁質酶A-4則為本菌最後穩定的幾丁質酶。

Abstract

This research was conducted to isolate chitinolytic Clostridium perfringens from human feces, to develop a medium that rendered the bacterium to produce higher chitinase activity, and to purify and characterize the chitinase(s) produced by the bacterium. A medium mainly containing peptone and yeast extract was employed to cultivate the bacterium for production of higher chitinase activity after cultivation (37℃) for 25 h at anaerobic condition. When the proteins in crude enzyme solution of C. perfringens were precipitated by ammonium sulfate and then followed by dialysis, condensation and gel filtration, two chitinases (P-1 and P-2) were fractionated. Both chitinases could hydrolyse 4-methylumbellifery-N-acetylchitobiose [4-MU-(NAG)2] and 4-methylumbellifery-N-acetylchitotriose [4-MU-(NAG)3]. After the chromatography using Sephacryl S-300 and SDS-PAGE, chitinase P-1 revealed the molecular weight of 139.7 kDa composed of two subunits (87.9 and 52.4 kDa); P-2 showed the molecular weight of 196.8 kDa composed of two subunits (97 and 49.9 kDa). Due to the similar characteristics on the stabilities of reaction temperatures and pHs and the activation of metal ions, both enzymes were supposed to be the same protein. When the bacterium was incubated at the same condition for 96 h and the crude enzyme solution was dealt with the same treatments, there was only a chitinolytic peak revealed on DEAE or HIC chromatography, which was coded as chitinase A-4. The molecular weight of A-4 was 75 kDa and could hydrolyse both 4-MU-(NAG)2 and 4-MU-(NAG)3. Using 4-MU-(NAG)2 as a substrate, the optimum reaction temperature was 40℃ and the values of Km and Vmax were 0.14 mM and 416.67 nmole/ min/ mg, respectively; while using 4-MU-(NAG)3 as the substate, the optimum reaction temperature was 50℃, and Km and Vmax values were 0.34 mM and 384.62 nmole/ min/ mg, respectively. When using both substrates above-mentioned, the optimum reaction pH was the same (6.0) and the enzyme activity was enhanced by dithiothreitol or 2-iodoacetamide (1 mM), but inhibited by iodoacetic acid, phenyl methyl sulfonyl fluoride, Fe2+, Fe3+ or Hg2+. However, Ba2+, Ca2+, Cu2+, K+, Mg2+, Mg2+, Mn2+ or Na+ could only activate chitinase A-4 to hydrolyse 4-MU-(NAG)2. N-acetychitobiose [(NAG)2] was the main chitinolytic product when A-4 was employed to hydrolyse chitins from different sources. Among them, the highest quality of (NAG)2 was obtained from squid pen chitin after hydrolysation. Due to the existence of other enzymes in the crude enzyme solution, chitinase P-1 and P-2 could be modified to form A-4. Consequently, A-4 was the final stable chitinase of C. perfringens.

目錄
頁次
中文摘要………………………………………………………………… I
英文摘要………………………………………………...……………….. III
ㄧ、前言………………………………………………………………….1
二、文獻整理………………………………………………………………3
1. 產氣莢膜桿菌（Clostridium perfringens）……………………………..3
(1) 分類地位…………………………………………………………….3
(2) 自然棲息地………………………………………………………….3
(3) 型態………………………………………………………………….3
(4) 培養特性…………………………………………………………….3
(5) 生化特性…………………………………………………………….6
(6) 致病及症狀…………………………..………………………….6
2. 幾丁質和N-乙醯幾丁寡醣……………………………………………7
(1) 分布與特性………………………………………………………….7
(2) 製備……………………………………………………………… ..10
3. 幾丁質酶……………………………………………………………...11
(1) 幾丁質酶的天然分佈……………………………………………...11
(2) 幾丁質酶的分類…………………………………………………...13
(3) 幾丁質酶的水解模式……………………………………………...14
(4) 幾丁質酶的轉醣特性……………………………………………...16
(5) 幾丁質酶的結構…………………………………………………...17
(6) 幾丁質酶與溶菌酶的比較………………………………………...19
(7) 幾丁質酶活性的測定…………………………………………….21
a. 傳統方法………………………………………………………….21
b. 快速檢測法……………………………………………………….23
(8) 幾丁質酶的純化…………………………………………………...24
4. 動物體內消化幾丁質的情形………………………………………...25
(1) 動物體中具幾丁質酶活性的分佈情形…………………………...25
(2) 幾丁質對動物消化道中微生物生長的影響……………………...27
(3) 幾丁質及N-乙醯幾丁寡醣的免疫活性………………………28
三、實驗流程………………………………………………………31
四、材料與方法……………………………………………………………32
1. 材料…………………………………………………………………...32
(1) 有機溶劑…………………………………………………………...32
(2) N-乙醯幾丁寡醣標準品……………………………………………32
(3) 酵素基質…………………………………………………………...32
(4) 電泳試劑…………………………………………………………...33
(5) 層析膠體…………………………………………………………...33
(6) 酸、鹼及鹽類……………………………………………………….34
(7) 培養基……………………………………………………………...34
(8) 其他………………………………………………………………...34
2. 設備…………………………………………………………………...35
(1) 厭氧菌培養………………………………………………………...35
(2) HPLC設備及管柱規格…………………………………………….35
(3) FPLC設備…………………………………………………………..35
(4) 蛋白質電泳………………………………………………………...36
(5) 其他………………………………………………………………...36
3. 方法…………………………………………………………………...36
(1) 培養基的製備……………………………………………………...36
a. Tryptose-sulfite-D-cycloserine agar………………………………...36
b. 添加不同量膠體幾丁質於brain heart infusion broth…………….37
c. 添加不同量膠體幾丁質於peptone yeast medium……………...38
d. 添加不同量膠體幾丁質於salt medium……………..…………...38
(2) 膠體幾丁質的製備………………………………………………...39
(3) 糞便取樣與菌株分離……………………………………………...40
(4) 菌株保存…………………………………………………………...40
(5) 菌株活化…………………………………………………………...40
(6) 幾丁質酶活性分析………………………………………………...40
a. 反應試劑的製備…………………………………………………...40
b. 酵素的反應步驟………………………………………………….41
(7) 粗酵素液的製備…………………………………………………...41
(8) 利用硫酸銨分劃粗酵素液中的蛋白質…………………………...41
(9) 透析………………………………………………………………...41
(10) 超過濾膜濃縮…………………………………………………...42
(11) 蛋白質定量……………………………………………………...42
(12) 弱陰離子交換樹酯 (DEAE fast flow)的層析步驟……………..42
a. 層析(Chromatography)…………………………………………….42
b. 樹酯再生(Regeneration)…………………………………………...42
(13)疏水性基交換樹脂 (Phenyl sepharose 6 fast flow)的層析步驟………………………………………………………..……43
a. 層析………………………………………………………………...43
b. 樹酯再生…………………………………………………………...43
(14) 電泳分析………………………………………………………….43
a. Native-PAGE膠體製備…………………………………………….44
b. 5× sampling buffer………………………………………………..44
c. Native-PAGE electrophoresis buffer………………………………..44
d. 幾丁質酶膠體活性染色…………………………………………...44
e. SDS-PAGE膠體製備……………………………………………….45
f. 5× SDS sampling buffer…………………………………………..45
g. SDS-PAGE electrophoresis buffer……………………….................45
h. SDS-PAGE staining solution…………………………………..46
i. SDS-PAGE destaining solution……………………………………...46
(15) 幾丁質酶性質的試驗…………………………………………….46
a. 幾丁質酶分子量的測定…………………………………………...45
b. 溫度影響酵素活性的測定………………………………………...46
(i). 最適作用溫度…………………………………………………...46
(ii). 不同溫度下之穩定性…………………………………………..46
c. pH影響酵素活性的測定…………………………………………...47
(i). 最適作用pH值………………………………………………….47
(ii). 不同pH值下的穩定性…………………………………………47
d. 金屬離子對酵素活性的試驗……………………………………...47
e. 化學試劑對酵素活性的試驗……………………………………...48
(16) 水解產物的分析………………………………………………….48
a. 流動相液體的製備………………………………………………...48
b. N-乙醯幾丁寡醣標準品的製備…………………………................48
c. 酵素水解不同來源幾丁質與水解後產物的製備………………...49
d. HPLC的分析條件…………………………………………………49
(17) 酵素動力學的測定……………………………………………….49
(18) 溶菌酶活性的測定……………………………………………….49
(19) 醣蛋白之鑑別………………………………………………...50
(20) 胺基酸比對…………………………………………………….....50
4. 統計分析…………………………………………………………...50
五、結果…………………………………………………………………..51
六、討論…………………………………………………………………....56
七、結論…………………………………………………………………..98
八、參考文獻……………………………………………………………..99
附錄………………………………………………………………………112



















圖目錄
圖1.自人體糞便中篩出的C. perfringens在含蛋黃的TSA agar上之菌落型態。…………………………………………………………....64
圖2. C. perfringens在添加不同量的膠體幾丁質的BHI medium培養，產生幾丁質酶[以4-MU-(NAG)2為受質]的情形。…………...……….65
圖3. C. perfringens在添加不同量的膠體幾丁質的PY medium培養，產生幾丁質酶[以4-MU-(NAG)2]為受質的情形。…………………....66
圖4. C. perfringens以添加1% (w/ v) yeast extract的salts medium培養，產生幾丁質酶[以4-MU-NAG、4-MU-(NAG)2或4-MU-(NAG)3為受質]的情形。…………………………………….…………67
圖5. C. perfringens以添加0.1% (w/ v)膠體幾丁質的salts medium培養，產生幾丁質酶[以4-MU-NAG、4-MU-(NAG)2或2-MU-(NAG)3為受質]的情形。…………………………………………………….68
圖6. C. perfringens以添加0.3% (w/ v)膠體幾丁質的salts medium培養，產生幾丁質酶[以4-MU-NAG、4-MU-(NAG)2或2-MU-(NAG)3為受質]的情形。………………………………………………….69
圖7. C. perfringens以添加0.5% (w/ v)膠體幾丁質的salts medium培養，產生幾丁質酶[以4-MU-NAG、4-MU-(NAG)2或2-MU-(NAG)3為受質]的情形。………………………………………………….70
圖8. C. perfringens以PY medium培養，產生幾丁質酶[以4-MU-NAG、4-MU-(NAG)2或4-MU-(NAG)3為受質]的情形。……………….71
圖9. C. perfringens幾丁質酶[4-MU-(NAG)2或4-MU-(NAG)3為受質]的DEAE管柱層析圖。………………………………………………72
圖10. C. perfringens幾丁質酶P-1[4-MU-(NAG)2或4-MU-(NAG)3為受質]的HIC管柱層析圖。…………………………………………73
圖11. C. perfringens幾丁質酶P-2[4-MU-(NAG)2或4-MU-(NAG)3為受質]的HIC管柱層析圖。…………………………………………74
圖12. C. perfringens幾丁質酶P-1與P-2在Native-PAGE分析之活性染色。…………………………………………………………...76
圖13. C. perfringens幾丁質酶P-1與P-2以SDS-PAGE分析之電泳圖。……………………………………………………..………..77
圖14. C. perfringens幾丁質酶P-1與P-2分子量測定檢量曲線。…………………………………………………………….78
圖15. C. perfringens幾丁質酶A-4 [以4-MU-(NAG)2或4-MU-(NAG)3為受質]的DEAE管柱層析圖。………………………………81
圖16. C. perfringens幾丁質酶A-4 [以4-MU-(NAG)2或4-MU-(NAG)3為受質]的HIC管柱層析圖。…………………………………82
圖17. C. perfringens幾丁質酶A-4在Native-PAGE分析之活性染色。84
圖18. C. perfringens幾丁質酶A-4以SDS-PAGE分析之電泳圖。……………………………………………..…………...85
圖19. 溫度對C. perfringens幾丁質酶A-4活性之影響。…………….86
圖20. C. perfringens幾丁質酶A-4自不同溫度下之安定性。…………87
圖21. pH對C. perfringens幾丁質酶A-4活性之影響。………………88
圖22. C. perfringens幾丁質酶A-4自不同pH下之安定性。……………89
圖23. C. perfringens幾丁質酶A-4之酵素動力學分析圖。……………93
圖24. C. perfringens幾丁質酶A-4胜肽片段胺基酸序列之比對。…….96
附錄圖1. C. perfringens幾丁質酶A-4水解蝦殼幾丁質24及48小時後，以HPLC分析其水解產物。……………………………...112
附錄圖2. C. perfringens幾丁質酶A-4水解蟹殼幾丁質24及48小時後，以HPLC分析其水解產物。……………………………..113
附錄圖3. C. perfringens幾丁質酶A-4水解膠體幾丁質24及48小時後，以HPLC分析其水解產物。……………………………..114
附錄圖4. C. perfringens幾丁質酶A-4水解魷魚鞘幾丁質24及48小時後，以HPLC分析其水解產物。……………………………..115
























表目錄
表1. C. perfringens幾丁質酶P-1與P-2純化摘要。……………………...75
表2. C. perfringens幾丁質酶P-1與P-2性質。…………………………...79
表3. C. perfringens幾丁質酶P-1與P-2之比較。………………………...80
表4. C. perfringens幾丁質酶A-4純化摘要。………………………….74
表5. 不同濃度之金屬離子對C. perfringens chitinase A-4分別以4-MU-(NAG)2與4-MU-(NAG)3為受質時，活性之影響。……….90
表6. 化學試劑對C. perfringens幾丁質酶A-4活性之影響。…………...91
表7. C. perfringens幾丁質酶A-4對4-MU共軛受質水解特性。………..92
表8. C. perfringens幾丁質酶A-4對不同來源幾丁質經24與48小時水解後之產物分析。……………………………………..…………...94
表9. C. perfringens幾丁質酶A-4以Micrococcus lysodeiticus BCRC 13079為受質時，溶菌酶活性之測定。…………………………….95
表10. C. perfringens幾丁質酶P-1、P-2與A-4之比較。…………….97

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