臺灣博碩士論文加值系統

幾丁質水解酵素廣泛存在於自然界各物種之間，此類酵素可以水解自然界中含量第二豐富的多醣聚合物—幾丁質，屬於醣解酵素的成員之ㄧ。幾丁質水解酵素在特定生物體內如節肢動物及魚類，分別扮演執行組織上的新陳代謝與消化含有幾丁質食物的必要酵素；然而，在人類體內也存在多種的幾丁質水解酵素及類似幾丁質酶的蛋白質，此類酵素及蛋白質的生理功能大多仍然無法有明確的定義。
本研究利用兩種不同的液體層析分離法試圖鑑別人類血漿中之幾丁質水解酵素及相關蛋白質。第一種層析分離方式是以Blue Sepharose 6親和性液體層析管柱與DEAE Sepharose液體層析管柱進行人類血漿中的蛋白質的部份分離，搭配西方墨點法鑑別出一個分子量約為150 kDa且可與Bacillus菌屬chitinase A1的chitin binding domain片段所產生的抗體結合。第二種層析分離方式則是利用幾丁質親和性液體層析管柱與DEAE Sepharose液體層析管柱對人類血漿中的蛋白質進行部份分離，再以SDS-聚丙烯胺電泳及活性染色等方法鑑別出一個分子量約為30 kDa且具有水解glycol chitin能力的幾丁質水解酵素。

Chitinolytic enzymes exist in various species in nature. As a glycosyl hydrolases, they can catalyze the hydrolysis of chitin, the second most abundant polysaccharide on earth. Chitinolytic enzymes are necessary in the development of metabolism for arthropoda and fish. In human, there are many kinds of chitinolytic enzymes and chitinase-like proteins. Their physiological functions are yet appreciated.
In this study, chitinase and related proteins from human plasma were identified using liquid chromatography. In one method, Blue Sepharose 6 affinity liquid chromatography and DEAE Sepharose ion exchange liquid chromatography were used to separate proteins from human plasma. A protein with molecular weight of 150 kDa, could be identified by western blot using ChBD antiserum. In other way, chitin affinity liquid chromatography and DEAE Sepharose ion exchange liquid chromatography were used to separate proteins from human plasma. A protein with molecular weight of 30 kDa was identified with ability to catalyze the hydrolysis of glycol chitin by in-situ activity staining on SDS-PAGE.

TABLE OF CONTENTS
ACKNOWLEDGEMENTS……..…………….…....………………………..i
ABSTRACT…….…..............…………………………………………….…ii
CHINESE ABSTRACT……...…….........………...…………….…….……iii
TABLE OF CONTENTS….…………............………..………...………….iv
LIST OF TABLES……….......……………………………..…………...…...x
LIST OF FIGURES…............……………………………...…..…………...xi
NOTATIONS….…..……………………………………..……...…....……xiv
CHAPTERS
CHAPTER 1 INTRODUCTION………………......…………….………….1
CHAPTER 2 LITERATURE REVIEWS ….…..…..........…………..………2
2.1. Chitin and chitosan…………..……….………………….………2
2.1.1. Chitosan…………………..……………………………..…3
2.1.2. N-acetyl chito-oligosaccharides and chito-oligosaccharides.3
2.2. Chitinolytic enzymes…...………….……………..……...……....4
2.2.1. Chitinase……….…………….……………………………..4
2.2.2. N-acetyl-β-hexosamindase……….….....…………………..5
2.2.3. Chitobiase..............................................................................5
2.2.4. Chitin deacetylase.................................................................5
2.2.5. Chitosanase...........................................................................6
2.3. The distribution of chitinolytic enzymes in nature…...................6
2.3.1. The chitinolytic enzymes in microoganisms.........................6
2.3.1.1. The chitinolytic enzymes in bacteria.......................6
2.3.1.2. The chitinolytic enzymes in fungi............................7
2.3.1.3. The chitinolytic enzymes in malaria parasites….....7
2.3.2. The chitinolytic enzymes in plants........................................8
2.3.3. The chitinolytic enzymes in animals...................................10
2.3.3.1. Oviduct-specific glycoprotein................................11
2.3.3.2. TSA 1902...............................................................12
2.3.3.3. YKL-39..................................................................12
2.3.3.4. Eosinophil chemptactic cytokine...........................13
2.3.3.5. Chitotriosidase.......................................................13
2.3.3.6. Acidic mammalian chitinase..................................14
2.4. The analysis of chitinase activity................................................16
2.4.1. Reducing sugar assay..........................................................16
2.4.2. Radioactivity assay..............................................................16
2.4.3. Turbidity assay....................................................................16
2.4.4. N-acetyl-glucosamine assay................................................17
2.4.5. Viscosity assay....................................................................17
2.4.6. Substrate-labeled assay.......................................................17
2.4.7. Fluorescence assay..............................................................18
2.4.8. Others..................................................................................18
CHAPTER 3 MATERIALS AND METHODS........................................…19
3.1. Materials……………………….....……….…………..…….…19
3.1.1. Chemicals………………………..…..…………………...19
3.1.2. Gels…………..…………………….……………….…....20
3.1.3. Antibodies…….……………….……….………….……..20
3.1.4. Instruments…………………...………………….……….20
3.2. Methods….……………………….……...……….……..……..22
3.2.1. The assay of proteins from human plasma………...….….22
3.2.2. The activity assay of chitinolytic enzymes from human plasma……………………………………………………22
3.2.2.1. The assay with potassium ferricyanide reagent…………………………………………...22
3.2.2.2. The assay with DNS reagent……………….……23
3.2.3. Sodium dodecyl sulfate polyacrylamide gel electrophoresis……………………….…………………...24
3.2.3.1. Preparation of separating gel……….………....…24
3.2.3.2. Preparation of stacking gel……………………....25
3.2.3.3. SDS-PAGE……………………………………....25
3.2.4. The staining of SDS-PAGE……………….……………...25
3.2.4.1. Coomassie brilliant blue R250 staining……….…25
3.2.4.2. Silver staining………………………………..…..25
3.2.5. The analysis of chitinolytic enzyme in SDS-PAGE with glycol chitosan…………………………………………...26
3.2.6. In situ activity staining of chitinolytic enzyme on SDS-PAGE……………………….………………….…...27
3.2.6.1. Coomassie brilliant blue G250 staining….......….27
3.2.6.2. Fluorescent brightener 28 staining……...……….27
3.2.7. Western blot.……………………………..….....…………28
3.2.8. Chromatography………………………….…………....…29
3.2.8.1. Blue Sepharose 6 affinity chromatography……...29
3.2.8.2. DEAE ion exchange chromatography…………...29
3.2.8.3. Chitin-affinity chromatography…………….........30
CHAPTER 4 RESULTS…………..………………………...............…….31
4.1. The sensitivity comparison of reducing sugar assays………....31
4.2. The analysis of higher molecular weight (HMW) chitinase-like protein from human plasma……………………………………32
4.2.1. Blue Sepharose 6 affinity liquid chromatography……….32
4.2.2. DEAE Sepharose ion exchange liquid chromatography………………………………….……….32
4.2.3. The SDS-PAGE analysis and western blot of chitinolytic enzyme from human plasma……………………………..33
4.2.4. The SDS-PAGE analysis and western blot of HMW chitinase-like protein treated with β-mercaptoethanol…...34
4.3. The analysis of lower molecular weight (LMW) chitinolytic enzyme from human plasma………………………………..…34
4.3.1. The chitin-affinity chromatography……………………...34
4.3.2. DEAE Sepharose ion exchange liquid chromatography…………………………….…………….35
4.3.3. The in situ activity staining on SDS-PAGE of fractioned proteins in each chromatography…………..……….........35
4.3.4. The western blot of LMW chitinolytic enzyme…….........36
CHAPTER 5 DISCUSSION………….………..........................................37
REFERENCES…..........................………………...…….…………..……..40
LIST OF TABLES
Table 1.1 The characterization comparison of chitinases from various species……………………………………………………………………...50
Table 1.2 The characterization comparison of chitinase-like proteins from mammalians.…..……..………………………………………...…………...51
Table 1.3 The introduction of substrates in various assays of chitinase activity...……..……………..………………………………………............52
LIST OF FIGURES
Figure 1.1 The conformation structures of α-chitin (a) and β- chitin (b)………………………………………………….……………………….53
Figure 1.2 The structures of chitin (a), chitosan (b) and cellulose (c)…………………………………………………….…………………..…54
Figure 1.3 Schematic representation of the structural differences in various chitinase classes…….……………………………………………..………..55
Figure 1.4 The (αβ)8 barrel structure from bacterial chitinase……..…….56
Figure 1.5 The expression of chitinase in lung tissue of mice with asthma……………………………………………………………………....57
Figure 1.6 Proposed effect of acidic mammalian chitinase (AMCase) on chemokine expression and inflammation in asthma………………........…..58
Figure 1.7 Schematic representation of the primary structure in mammalian chitinase-like proteins…………………………………...…….59
Figure 1.8 The amino sequences alignment of AMCase, TSA1902, chitotriosidase, YKL-39, ECF-L, OGP and B. circulans chitinase A1…….60
Figure 1.9 The 3-dimensional structures of 3 kind ChBD types……........65
Figure 2.1 The flowchart of experiments......…….…………….………...66
Figure 3.1 The standard curve of reducing sugar analysis…………….…67
Figure 3.2 Blue Sepharose 6 affinity chromatography profile…………...68
Figure 3.3 DEAE Sepharose ion exchange chromatography profile…….69
Figure 3.4 The 12% SDS-PAGE analysis of each fraction in Blue Sepharose 6 affinity chromatography and DEAE Sepharose ion exchange chromatography……………………………………………………..……...70
Figure 3.5 The analysis of HMW chitinase-like protein……...…...……..71
Figure 3.6 The modificaton of β-mercaptoethanol to HMW chitinase-like protein…………………………………………………................................72
Figure 3.7 Chitin-affinity chromatography profile…...………………….73
Figure 3.8 The 12% SDS-PAGE analysis of each fraction from chitin affinity chromatography……....….…………………………………….…..74
Figure 3.9 DEAE Sepharose ion exchange chromatography profile of fraction 1 in chitin affinity column………………………………………...75
Figure 3.10 DEAE Sepharose ion exchange chromatography profile of fraction 2 in chitin affinity column………………..……………...……..….76
Figure 3.11 The 12% SDS-PAGE analysis of pooled protein from DEAE column chromatography…………………………………………………....77
Figure 3.12 The SDS-PAGE active stain of LMW chitinolytic enzyme……………………………………………………..……….……....78
Figure 3.13 The SDS-PAGE active stain comparison of LMW chitinolytic enzyme……………………………………………………………………...79
Figure 3.14 The western bloting comparision of HMW chitinase-like protein and LMW chitinolytic enzyme………….........................................80

NOTATIONS
4MU 4-methylumelliferyl-β-D-N,N’,N’’ triacetyl
AMCase acidic mammalian chitinase
APS ammonium persulifate
BSA bovine serum albumin
cDNA complementary deoxyribonucleic acid
ChBD chitin binding domanin
CM carboxymethyl chitin
DA degree of deacetylation
DAB diaminobenzide
DEAE dimethylamino ethyl
DNS 3,5-dinitrosalicylic acid
ECF-L eosinophil chemotactic cytokine
ENA epithelial-derived neutrophil-activating protein
HMW high molecular weight
IL interleukin
LMW low molecular weight
No number
MCP macrophage inflammatory chemokine
NAG N-acetyl D-glucosamine
OGP oviduct-specific glycoprotein
PAGE polyacrylamide gel electrophoresis
pI isoeletric point
RBV Remazol Brilliant Violet
SDS sodium dodecyl sulfate
TCA trichloroacetic acid
Th2 T helper type 2
UV ultraviolet

Units：
kDa kilodalton
M molar
ml milliliter
mM milimolar concentration
mm milimeter
nm nanometer
rpm revolutions per minute
U unit
×g in terms of earth gravity
μg microgram
μl microliter
℃ degree Centigrade

Superscript：
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