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研究生:施怡秀
研究生(外文):Yi-xiu Shih
論文名稱:日本麴菌液態醱酵生產幾丁聚醣
論文名稱(外文):Production of Chitosan by Submerged Culture of Asperjillus japonicus
指導教授:許垤棊許垤棊引用關係
指導教授(外文):Dey-Chyi Sheu
學位類別:碩士
校院名稱:大同大學
系所名稱:生物工程學系(所)
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:英文
論文頁數:90
中文關鍵詞:日本麴菌幾丁聚醣β-呋喃果糖苷酶
外文關鍵詞:Asperjillus japonicuschitosanβ-fructofuranosidase
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利用日本麴菌的液態醱酵生產幾丁聚醣。當使用葡萄糖作為碳源,添加5 ppm鋅離子不僅能夠促進細胞生長,細胞乾重達每公升8.2公克,亦能增加幾丁聚醣的分子量達到65 kDa以及去乙醯化程度達到96.79%。添加10 ppm鐵離子可獲得幾丁聚醣最大產量每公升43.6毫克,以及最大乾燥菌體的幾丁聚醣含量0.79%。改以蔗糖當作碳源時,添加10 ppm的鐵離子可獲得最高細胞乾重,每公升6.4公克。培養基添加5 ppm錳離子,可獲得幾丁聚醣最大產量每公升22.4毫克,以及最大幾丁聚醣含量占乾燥菌體的0.46%。添加5 ppm鋅離子或10 ppm鐵離子可增加幾丁聚醣的分子量。鐵離子可以提高幾丁聚醣去乙醯化程度。培養基中添加5 ppm鋅離子或錳離子可以促進β-呋喃果糖苷酶的活性,其中,添加5 ppm錳離子可獲得最大的酵素活性。
The production of chitosan was conducted by a submerged culture of Asperjillus japonicus. During the fermentation using glucose as carbon source, adding 5 ppm of zinc ion resulted in cell growth up to 8.2 g/L, the chitosan with a molecular weight of 65 kDa and the degree of deacetylation of 96.79%. The maximum yield of chitosan at 43.6 mg/L and the chitosan content of 0.79% were achieved upon ferrous ion at 10 ppm. When sucrose was used as carbon source, the highest production of biomass at 6.4 g/L was obtained upon ferrous ion at 10 ppm. Adding 5 ppm of manganese ion resulted in chitosan up to 22.4 mg/L and a chitosan content of 0.46%. Adding 5 ppm of zinc ion or 10 ppm of ferrous ion gave the molecular weight of chitosan up to 49 kDa. The degree of deacetylation of chitosan was increased by adding ferrous ion. The activity of β-fructofuranosidase was enhanced by adding 5 ppm of zinc or manganese ion in the culture medium. The maximum activity was obtained upon manganese ion at 5 ppm.
TABLE OF CONTENTS
ACKNOWLEDGEMENTSi
ABSTRACTii
CHINESE ABSTRACTiii
TABLE OF CONTENTSiv
LIST OF TABLESviii
LIST OF FIGURESx
CHAPTER 1 INTRODUCTION1
CHAPTER 2 LITERATURE REVIEWS3
2.1. Chitin and chitosan3
2.1.1. Structure of chitin and chitosan6
2.1.2. Properties of chitin and chitosan8
2.1.2.1. Characteristics of chitosan8
2.1.2.2. Deacetylation of chitosan9
2.1.2.3. Molecular weight11
2.1.3. Commercial uses and potential applications13
2.1.3.1. In the Wastewater Treatment13
2.1.3.2. In the Food Industry14
2.1.3.3. In Medical14
v
2.1.3.4. In Cosmetics14
2.2. Fungal chitosan16
2.2.1. Fungal cell wall17
2.2.2. Isolation from the chitosan-glucan complex of fungal cell wall18
2.3. Chitin and chitosan biosynthesis 21
2.3.1. Chitin synthases23
2.3.2. Chitin deacetylase25
2.4. Effect of metal ion on microorganism27
2.5. Fructooligosaccharides30
2.5.1. Effect of metal ion on the activity of β-fructo- furanosidase33
2.6. Aim of the study34
CHAPTER 3 MATERIALS AND METHODS35
3.1. Microoganisms35
3.2. Instruments35
3.3. Chemicals37
3.4. Cultivation of Aspergillus japonicus38
3.4.1. Culture media38
vi
3.4.2. Culture of Aspergillus japonicus38
3.4.3. Batch fermentation39
3.4.4. Control of fermentation41
3.5. Determination of biomass44
3.6. Analysis of carbohydrates by HPLC44
3.7. Assay of β-fructofuranosidase activity45
3.8. Analysis of chitosan46
3.8.1. Isolation of chitosan from fungal cell wall46
3.8.2. Determination of chitosan relative molecular weight by HPLC-GPC47
3.8.3. The degree of deacetylation of chitosan48
CHAPTER 4 RESULTS AND DISCUSSION49
4.1. Screening of microorganisms49
4.2. Cleavage of chitosan-glucan complex by crude α-amylase..49
4.3. Production of chitosan from Aspergillus japonicus BCRC 9300752
4.4. Effect of metal ions on the production of fungal biomass and chitosan54
4.4.1. Effect of metal ions on the yield of biomass and chitosan
vii
using glucose as carbon source55
4.4.2. Effect of metal ions on the yield of biomass and chitosan using sucrose as carbon source56
4.5. Effect of metal ion on the molecular weight of chitosan59
4.6. Effect of metal ion on the degree of deacetylation of chitosan62
4.7. Effect of metal ion on the activity of β-fructofuranosidase65
CHAPTER 5 CONCLUSION67
REFERENCES69
APPENDIXES74
APPENDIXE 1: Standard Curve of Chitosan Molecular Weight74
APPENDIXE 2: The HPLC-GPC Diagram of Chitosan75
APPENDIXE 3:Polymer Average Molecular Weight76
APPENDIXE 4: The First Derivative UV-Spectra for Various Standard Solutions of N-acetylglucosamin Solution77
APPENDIXE 5: Standard Curve for the Determination of N-acetylglucosamin78

LIST OF TABLES
Table 2.1 Chitin content of selected crustacea, insects, molluscan organs and fungi5
Table 2.2 Applications of Chitosan15
Table 2.3 Characteristics of chitin deacetylases from different sources26
Table 2.4 Compounds and ions influencing the activity of chitin synthase and chitin deacetylase29
Table 4.1 Yields and molecular weights of chitosan after the treatment of three kinds of α-amylases51
Table 4.2 Effect of metal ions on the yield of biomass and chitosan using glucose as carbon source57
Table 4.3 Effect of metal ions on the yield of biomass and chitosan using sucrose as carbon source58
Table 4.4 Effect of metal ions on the molecular weight of chitosan using glucose as carbon source60
Table 4.5 Effect of metal ions on the molecular weight of chitosan using sucrose as carbon source61
Table 4.6 Effect of metal ion on the degree of deacetylation of chitosan using glucose as carbon source63

Table 4.7 Effect of metal ion on degree of deacetylation of chitosan using sucrose as carbon source64
Table 4.8 Effect of metal ion on the activity of β-fructofuranosidase produced by submerged culture of A. japonicus66

LIST OF FIGURES
Figure 2.1 The structures of chitin (a), chitosan (b) and cellulose (c)7
Figure 2.2 Proposed molecular structure of β(1→4) glucosaminoglycan (chitin/chitosan), β(1 → 3) and (1 → 6) glucan chain, and chitosan bound to glucan connected by an α(1→4) glucosidic bond20
Figure 2.3 Model depicting the spatial regulation of chitin and chitosan biosynthesis22
Figure 2.4 The reaction of FOS production31
Figure 2.5 The reaction of FOS production32
Figure 3.1 Scheme of procedures in the fermentative experiments40
Figure 3.2 A display of the original ADVENTECH GENIE strategy of fermentation (main board)42
Figure 3.3 A display of original ADVENTENCH GENIE strategy for fermentation (connecting system)43
Figure 4.1 Yields of chitosan after the treatment of three kinds of α-amylases50
Figure 4.2 Time courses of biomass and chitosan during submerged fermentation of Aspergillus japonicus BCRC 93007 at 25°C using sucrose as carbon source53
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