摘要
幾丁質(chitin)的衍生物，不論是寡糖或是單糖的形式，都已經被證實具有廣泛應用的潛力。舉例來說，幾丁質和幾丁聚糖(chitiosan)的寡糖就具有很多的生理活性。包括各種抗癌活性、增強免疫力、抑制老鼠體內一些細菌的感染、抗真菌活性以及抗菌性。因此建立一個幾丁質酵素水解系統來水解幾丁質生成寡糖或單糖是相當重要的。
Aeromonas schubertii是一株於紅樹林土壤中篩出的本土菌株，可以利用粉狀的幾丁質來培養誘導生產幾丁質水解酵素。胞外分泌的蛋白質主要以硫酸胺沉澱法粗萃，經透析去鹽後，再以製備級IEF純化可得到數種幾丁質水解酵素。純化的酵素分別經過內含醣酐化幾丁質(glycol chitin)和未添加醣酐化幾丁質的SDS-PAGE來分析，發現具有SDS抗性。目前發現最明顯的幾丁質水解酵素其分子量為75 kDa。而酵素以膠狀紫色幾丁質(colloidal chitin azure)作為基質時，可以得到Michaelis constant為0.29 mM，而turnover number 為1 s-1。然而經過酒精處理的酵素，可以有效提升幾丁質水解的活性。其他經由相同IEF純化後收集管中的蛋白質分子量分別為30，38，110 kDa。經過IEF純化後收集的管中活性最高的pH值為4.8，據此可以推論這些幾丁質水解酵素是酸性的。本研究同時建立一種新的活性分析方法，以膠狀的紫色幾丁質作為基質與傳統的還原糖法對照之下，具有線性對應關係而且適用範圍更廣。另外本研究也使用二維膠體電泳分析其胞外分泌酵素的分佈情況，以及沉澱或溶劑處理後蛋白質的變化。
本研究結果發現，分子量75 kDa的蛋白質極有可能是38 kDa的雙聚物(dimer)。因此透過酒精，丙酮以及加熱的方式進行該蛋白質的SDS-PAGE的分析，明顯發現75 kDa的蛋白質位置消失。另外也將75 kDa和38 kDa的蛋白質分別以MALDI-TOF質譜儀測定其經過胰蛋白水解酵素切過之蛋白質片段分佈，證實Aeromonas schubertii的胞外幾丁質水解酵素75 kDa確實為38 kDa的雙聚物。並且發現以酒精或丙酮處理過的酵素溶液其活性明顯提高約兩倍，證實酒精和丙酮可有效將該蛋白質雙聚物分散成蛋白質單體。本研究另外也利用MALDI-TOF分析幾丁質水解產物以及找出粗萃幾丁質水解酵素固定化的最適化條件。

Abstract
Derivatives of chitin, either in the form of oligomer or monomer, have been paid attention due to their broad range of applications. Oligomers of chitin and chitosan, for example, have recently found to have many functional physiological activities, including the versatile antitumor activities, immuno-enhancing effects, protective effects against infection with some pathogens in mice, antifungal activity and antimicrobial activity. It is thus very important to develop processes for the enzymatic hydrolysis of macromolecular chitin to yield its oligomers or even monomer.
A locally isolated stain Aeromonas schubertii from soil of mangrove was cultured and induced by powdered chitin for the production of chitinases. Extracellular proteins were purified by ammonium sulfate precipitation, dialysis to remove salts, and then preparative isoelectric focusing (IEF) to yield several chitinases. The purified enzymes were analyzed by SDS-PAGE with or without glycol chitin and were found to be SDS-resistant. The chitinase present in the highest abundance was the one with an estimated molecular weight of 75 kDa. The Michaelis constant and turnover number were determined to be 0.29 mM and 1 s-1, respectively, using colloidal chitin azure as the substrate. However, the ethanol treatment of this enzyme could significantly increase its chitinolytic activity. Other chitinases obtained in the same IEF fraction were determined to have molecular weights of ca. 30, 38 and 110 kDa. Since the proteins with highest chitinase activity were collected from IEF fraction tube with pH value of 4.8, those chitinase were believed to be acidic. An activity assay method using colloidal chitin azure as the substrate was recommended since it possessed a broader range of linearity in comparison with conventional reducing sugar equivalent method. Two dimensional electrophoresis (2-DE) method was also used to analyze the extracellular proteins, precipitated by ammonium sulfate and proteins treated by organic solvent.
It was found that the protein with molecular weight of 75 kDa was possibly the dimmer form of the protein with molecular weight of 38 kDa. The protein of 75 kDa treated by ethanol, acetone and heating was then analyzed by SDS-PAGE. The band of the protein with molecular weight of 75 kDa was visibly hyalinizing on the SDS-PAGE gel. Analyzing the peptide fragments of proteins of 75 kDa and 38 kDa that were digested by trypsin, with MALDI-TOF mass spectrograph also confirmed that the extracellular chitinase of 75 kDa from Aeromonas schubertii was a dimer of 38 kDa protein. It was proved that the ethanol and acetone could dissociate the protein-dimer into monomers through the observation of increasing activity of chitinase in two-fold by ethanol or acetone treatment. In addition to characterizing of chitinases, the hydrolytes of chitin were analyzed by using MALDI-TOF MS, and the conditions from the immobilization of crude chitinase were optimized.

目錄
中文摘要………………………………………………………………....I
英文摘要………………………………………………………..………III
目錄………………………………………………………...……...…....VI
圖目錄…………………………………………………………….....…XII
表目錄………………………………………………………….……..XVI
第一章 緒論…………………………………..…………………………1
1.1前言…………………….…………………………………………….1
1.2研究目的與緣由…….…………………………………..………...…2
1.3文獻回顧…………………………………………………………..…4
1.3.1幾丁質及其相關衍生物……………………………………………4
1.3.2幾丁質水解酵素的來源與相關研究……………..………………16
1.3.3來自Aeromonas屬的幾丁質水解酵素………………..…………19
1.3.4幾丁質水解酵素的純化分析……………………………….…….30
1.3.5幾丁質水解酵素雙聚物與分散的方式……….……….…………34
第二章 實驗藥品與設備………………………………………………39
2.1實驗藥品…………………………………………………………….39
2.1.1菌株培養與幾丁質水解酵素誘導………………………………..39
2.1.2活性測定方法……………………………………………………..40
2.1.2.1還原糖法………………………………………………………...40
2.1.2.2膠狀幾丁質法…………………………………………………...40
2.1.3蛋白質定量方法……………………………….………………....41
2.1.4沉澱蛋白質……………………………………………………….41
2.1.5透析的方法…………………………….…………………………41
2.1.6 2-D電泳與銀染…………………………………………………..42
2.1.6.1 IPG strip的rehydration………………………………………....42
2.1.6.2 IPG strip的平衡………………………………………………...43
2.1.6.3 SDS-PAGE(2D使用)…………………………………………...44
2.1.6.4銀染…………………………….………………………………..45
2.1.6.4.1銀氨錯離子法……………….………………………………...45
2.1.6.4.2硝酸銀法………………………………………………………46
2.1.7製備級 IEF 的操作……………..………………………………..46
2.1.8 SDS-PAGE操作(CBR染色)………..…………………………….47
2.1.9糖酐化幾丁質製備………………………………………………..48
2.1.10膠內幾丁質水解酵素分析……………………………………....49
2.1.11凝膠過濾法……………………………………………………....49
2.1.12質譜儀分析雙聚物的蛋白質片段……………………………....50
2.1.13水解產物之MALDI-TOF分析………………………………….50
2.1.14幾丁質水解酵素固定化………………………………………...51
2.1 實驗儀器…………………………………………………………..51
第三章 實驗步驟………………………………………………………54
3.1菌種保存與幾丁質誘導培養………………………………………54
3.1.1菌種保存………………………………………………………….54
3.1.2幾丁質誘導培養…………………………………………………..55
3.2活性分析方法……………………………………………………….56
3.2.1還原糖法…………………………………………………………..56
3.2.2膠狀幾丁質法(colloidal chitin azure)(Chen et al.，2002)………..57
3.2.3膠狀幾丁質法與還原糖法的活性對照實驗……………………..58
3.3蛋白質定量方法…………………………………………………….59
3.4不同培養條件下幾丁質水解酵素的誘導實驗…………………….59
3.5 Aeromonas schubertii胞外分泌蛋白質的分佈…………………….60
3.5.1菌液培養及處理……………………………….………………….60
3.5.2二維膠體電泳(田兆真，2002)…………….……………………..61
3.6幾丁質水解酵素的抗酒精與丙酮沉澱測試……………………….66
3.7純化幾丁質水解酵素……………………….………………………67
3.7.1硫酸胺沉澱………………………………………………………..67
3.7.2製備級IEF純化…………………………………………………..70
3.7.3以SDS-PAGE鑑定經由製備級 IEF 所純化的樣品…………...70
3.7.4凝膠過濾(Gel filtration)步驟……………………………………..74
3.8 膠中幾丁質水解酵素活性測定…………………………………...75
3.8.1製作糖酐化幾丁質膠片(Chen et al.，2002)……………………..76
3.8.2糖酐化幾丁質(glycol chitin)合成方法(Yamada and Imoto，1981)……………………………………………………………….76
3.9酵素動力學實驗…………………………………………………….77
3.10幾丁質水解酵素雙聚物的分析…………………………………...78
3.10.1酒精、丙酮、E液和沉澱的影響……………………………….78
3.10.2製備級IEF純化後酵素加熱的影響……………………..……..79
3.10.3酒精丙酮和E液加熱前後的影響……………………….……..79
3.10.4用質譜儀分析雙聚物的蛋白質片段…………………….……...80
3.11水解產物之MALDI-TOF分析…………………………….……..81
3.12酵素固定化………………………………………………….……..81
3.12.1固定化載體的製備……………………………………….……...81
3.12.2粗萃幾丁質水解酵素的固定化………………………….……...83
第四章 結果與討論……………………………………………………85
4.1酵素活性的測定…………………………………………………….85
4.2幾丁質水解酵素誘導與分泌……………………………………….91
4.3 Aeromonas schubertii胞外分泌蛋白質的分佈……….…………....94
4.4幾丁質水解酵素經酒精與丙酮沉澱測試………………………...102
4.4.1酵素經丙酮處理後的二維膠體電泳圖………………………....106
4.5胞外幾丁質水解酵素的純化……………………………………...107
4.5.1製備級IEF純化活性與pH值分佈…………………………….108
4.5.2製備級IEF後經SDS-PAGE分析………………………………109
4.5.4製備級IEF純化後第4管經膠體過濾後的活性………………..112
4.5.5膠體過濾後的SDS-PAGE分析圖……………………………....114
4.5.6 Aeromonas schubertii胞外幾丁質水解酵素的純化…………....117
4.6 幾丁質水解酵素的SDS抗性性質……………………………….118
4.6.1製備級IEF後經膠內幾丁質水解酵素分析…………………….118
4.6.2膠體過濾後的膠內幾丁質水解酵素分析圖…………………....121
4.7酵素動力學………………………………………………………...121
4.8雙聚物的假設與證實……………………………………………..125
4.8.1雙聚物的假設……………………………………………………125
4.8.2雙聚物假設的證實實驗………………………….……………...127
4.8.3酵素經酒精丙酮處理後及其沉澱物的SDS-PAGE分析……...130
4.8.4加熱對於幾丁質水解酵素雙聚物的影響……………………....133
4.8.5綜合各種的變因對幾丁質水解酵素雙聚物的影響…………....137
4.8.6 MALDI-TOF MS分析幾丁質水解酵素的結果………………..138
4.8.7雙聚物的機構推論………………………………………………141
4.9水解產物之MALDI-TOF分析…………………………………....142
4.10酵素固定化的最適化條件……………………………………….147
第五章 結論………………………………………………………......150
第六章 參考文獻……………………………………………………..153
附錄……………………………………………………………………162
圖目錄
圖1.1幾丁質(上)與幾丁聚醣(下)的構造。…………………………….6
圖1.2由蝦、蟹殼製造幾丁質的方法。…………………..……………8
圖4.1還原糖法測出的吸光值與N-乙醯葡萄糖胺(GlcNAc)的檢量線。……………………………………………………………....88
圖4.2偵測幾丁質水解酵素的活性。(A)是使用還原糖法，(B)是膠狀紫色幾丁質法。…………………..……………………………..89
圖4.3還原糖法與膠狀紫色幾丁質法的對應關係。其中()是GlcNAc濃度對A550的關係，()是活性對A550的關係。 ……..……...90
圖4.4 Aeromonas schubertii在不同培養環境所測得的幾丁質水解酵素的活性(經由△A550轉換而來)，其中實心符號表示不同兩次幾丁質培養實驗的結果，空心符號則是表示用LB medium培養的結果。……………………………………………………………...92
圖4.5 Aeromonas schubertii經由 LB 誘導產生的蛋白質2-D電圖。
(培養第三天，樣品處理為透析去糖，蛋白質含量10.3μg，染色方法為銀氨錯離子法。)…………………………………...94
圖4.6 Aeromonas schubertii經由 chitin medium 誘導產生的蛋白質2-D電泳圖。(培養第三天，樣品處理為透析去糖未經沉澱，蛋白質含量10.3μg。染色方法為銀氨錯離子法。)…………...95
圖4.7 Aeromonas schubertii經由 LB 誘導產生的蛋白質2-D電圖。(培養第四天，樣品處理為三氯醋酸沉澱，蛋白質含量100μg，染色方法為硝酸銀法。)……………………………………....97
圖4.8 Aeromonas schubertii經由 chitin medium 誘導產生的蛋白質2-D電泳圖。(培養第四天，樣品處理為三氯醋酸沉澱，蛋白質含量100μg，染色方法為硝酸銀法。)……………………...98
圖4.9 Aeromonas schubertii經由 LB 誘導產生的蛋白質2-D電圖。(培養第四天，樣品處理為三氯醋酸沉澱，蛋白質含量50μg，染色方法為硝酸銀法。)………………………………………100
圖4.10 Aeromonas schubertii經由 chitin medium 誘導產生的蛋白質2-D電泳圖。(培養第四天，樣品處理為三氯醋酸沉澱，蛋白質含量50μg。染色方法為硝酸銀法。)………….……...101
圖4.11不同濃度酒精處理下的幾丁質水解酵素溶液的比活性與上清液蛋白質含量，分別以(●)與(○)為代表。……….………103
圖4.12不同濃度丙酮處理下的幾丁質水解酵素溶液的比活性與上清液蛋白質含量。符號說明同圖4.11。………………….……....104
圖4.13幾丁質水解酵素經丙酮處理後的二維膠體電泳圖…….…...105
圖4.14由Aeromonas schubertii得到粗萃幾丁質水解酵素經製備IEF
純化後的結果。其中()表示活性，()為pH值都是由製備級IEF純化後各管所得。……………………………..………….108
圖4.15經製備級IEF純化後蒐集的第1到6管的SDS-PAGE分析圖。………………………………………………….………...110
圖4.15經製備級IEF純化後蒐集的第7到18管的SDS-PAGE分析
圖。…………………………………………………………….111
圖4.17經製備級IEF純化後的第四管所進行的凝膠過濾情形。其中()
表示每管的活性，而()表示每管的蛋白質含量。………..113
圖4.18 經過製備級純化的後的第四管，再經過凝膠過濾後第5、6、7和8管所進行的SDS-PAGE分析圖。…………………...115
圖4.19 經過製備級純化的後第四管，再經過凝膠過濾後第13和14管所進行的SDS-PAGE分析圖。…………………………..116
圖4.20 經製備級IEF純化後的第1到9管蛋白質經膠內幾丁質水解酵素活性的電泳圖。其中AS表示經硫酸胺沉澱後經過透析的粗萃酵素，經過稀釋10倍。…………………………….119
圖4.21 經製備級IEF純化後的第9到18管蛋白質經膠內幾丁質水解酵素活性的電泳圖。其中0表示經硫酸胺沉澱後經過透析的粗萃酵素，未經稀釋。……………………………………..120
圖4.22取經過製備級IEF純化後的第4管經過凝膠過濾的第4到8管的膠內幾丁質水解酵素活性分析。……………………….122
圖4.23取經過製備級IEF純化後的第4管經過凝膠過濾的第9到14管的膠內幾丁質水解酵素活性分析。……………………….123
圖4.24 雙聚物存在的推論流程。…………………………………...126
圖4.25 經酒精丙酮處理過的製備級IEF純化第六管與經稀釋粗萃幾丁質水解酵素溶液經SDS-PAGE分析的結果。圖上0表示稀釋後的透析溶液，6表示經IEF純化後第6管Eth表示經30%酒精處理，Ace表示經30%丙酮處理。……………………128
圖4.26經酒精丙酮處理過的製備級IEF純化第六管與經稀釋粗萃幾丁質水解酵素溶液經過In-gel chitinase 分析的結果。圖上0表示稀釋後的透析溶液，6表示經IEF純化後第6管Eth表示經30%酒精處理，Ace表示經30%丙酮處理。…………....129
圖4.27粗萃的幾丁質水解酵素經製備級IEF純化後第四管，分別經過不同處理後的SDS-PAGE分析圖，其中1表示原來的第4管經過E液處理，2表示經過30%酒精處理後的沉澱物，3表示經過30%丙酮的沉澱物，4表示經過30 %酒精處理的上清液，5表示經過30 % acetone處理的上清液。…………………………..……………………………….131
圖4.28粗萃的幾丁質水解酵素經製備級IEF純化後第四管，分別經過不同處理後的In-gel chitinase分析圖，其中1表示原來的第4管經過E液處理，2表示經過30%酒精處理後的沉澱物，3表示經過30%丙酮的沉澱物，4表示經過30 %酒精處理的上清液，5表示經過30 % acetone處理的上清液。…………………………………………………..………..132
圖4.29粗萃的幾丁質水解酵素溶液經製備級IEF純化後，各管未經加熱處理的SDS-PAGE分析圖。…………………………...134
圖4.30粗萃的幾丁質水解酵素溶液經製備級IEF純化後，各管經加熱處理的SDS-PAGE分析圖。……………………………....135
圖4.31幾丁質水解酵素經過不同處理後的蛋白質SDS-PAGE分析圖，其中
1是經製備級IEF純化後第4管經過E液處理，未加熱。
2是經製備級IEF純化後第4管加上E液且加熱處理。
3是經製備級IEF純化後第4管加上30 %酒精且加熱處理。
4是經製備級IEF純化後第4管經過3 % acetone且加熱處理。
…………………………………………...……………………136
圖4.32 MALDI-TOF質譜儀測定分子量75 kDa與38 kDa的幾丁質水解酵素經過胰蛋白水解酵素切過之蛋白質片段分佈圖。….139
圖4.33 matrix-blank的MALDI-TOF分析圖譜。…………………..143
圖4.34 colloid-chitin的MALDI-TOF分析圖譜。………………….144
圖4.35 Lysozyme水解幾丁質，其產物的MALDI-TOF圖譜。…...145
圖4.36 使用粗萃幾丁質水解酵素水解幾丁質，其產物的MALDI-TOF圖譜。…………………………………………………….…....146
圖4.37在不同濃度粗萃幾丁水解酵素下測量顆粒固定後的活性，固定化酵素的量，以及比活性的關係。……………………….148
圖4.38測量粗萃幾丁質水解酵素固定化最佳時間。………………149
表目錄
表1.1幾丁質及其衍生物的實際應用(Shahidi et al.，1999；Jeon et al.，　
2000；蘇遠志，2001；Patil et al.，2000；Braham et al.，2003)…………………………………………………………..….14
表1.2 Aeromonas屬相關的幾丁質研究。………….…………………27
表3.1菌株保存培養基………………………………………………....54
表3.2幾丁質誘導培養基………………………………………………56
表3.3 SDS-PAGE配膠組成(12.5% acrylamide)。…………………….63
表3.4配膠(以下單位為ml)…………………………………………....72
表3.5甲基丙烯醯胺共聚顆粒製備配方。……………….…………...82
表 4.1 Aeromonas schubertii胞外幾丁質水解酵素的純化。………..117
表4.2 純化後幾丁質水解酵素的動力學參數……………………….124
表4.3 影響雙聚物分散的因素及效果……………………………….138

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