淡江大學覺生紀念圖書館 (TKU Library)

系統識別號 U0002-2005201916570500
中文論文名稱 Streptomyces thermocarboxydus 所生產 幾丁質酶之分離及其於幾丁質寡糖製備之應用
英文論文名稱 The isolation of chitinase from Streptomyces thermocarboxydus and its application in the preparation of chitin oligomers
校院名稱 淡江大學
系所名稱(中) 化學學系碩士班
系所名稱(英) Department of Chemistry
學年度 107
學期 2
出版年 108
研究生中文姓名 陳氏娥
研究生英文姓名 Thi-Ngoc, Tran
學號 606185014
學位類別 碩士
語文別 英文
口試日期 2019-05-07
論文頁數 27頁
口試委員 指導教授-王三郎
中文關鍵字 甲殼素  幾丁質酶  烏賊軟骨  Streptomyces thermocarboxydus  抗氧化  Prebiotic 
英文關鍵字 Chitin  Chitinase  Squid pens  Streptomyces thermocarboxydus  Anti-oxidant  Prebiotic 
學科別分類 學科別自然科學化學
中文摘要 微生物幾丁質酶於醫藥、生物以及農業之應用,近年來備受矚目。本研究利用含有烏賊軟骨粉做為唯一碳氮源之培養基,從台灣土壤篩選到超過五十株幾丁質酶生產菌。依據幾丁質分解活性分析結果,選擇具有較強幾丁質酶活性之Streptomyces thermocarboxydus TKU045進行後續研究。較適培養條件探討結果顯示,於含有1%烏賊軟骨粉液態培養基,菌株TKU045於45 °C經過36小時培養結果, 能生產最高之幾丁質酶活性(52.985 U/mL)。分離所得幾丁質酶之定性結果顯示,具有較其他Streptomyces屬幾丁質酶更低之分子量(12.8 kDa,SDS-PAGE定量)以及較酸之最適反應酸鹼值(pH4)。經由HPLC以及MALDI-TOF分析結果顯示,TKU045幾丁質酶水解懸浮態幾丁質所得幾丁質寡糖之聚合度分布在1-7。此低聚合度幾丁質寡糖具有抗氧化以及促進乳酸菌生長之活性。綜合此結果,TKU045幾丁質酶水解懸浮態幾丁質所得幾丁質寡糖之抗氧化以及益生素活性,於醫藥以及保健食品方面具有應用潛力。
英文摘要 Microbial chitinase has received great attention due to its medical, biological, and agricultural applications. In this study, over 50 bacterial strains were isolated from Taiwanese soils using medium containing squid pen powder (SPP) as the sole source of carbon/nitrogen. Based on chitinolytic activity, Streptomyces thermocarboxydus TKU045 was selected for further study. Optimized culture conditions revealed S. thermocarboxydus TKU045 could produce the highest chitinase activity (52.985 U/mL) when cultured in a medium containing 1% (w/v) SPP at 45 °C for 36 h. Characterized TKU045 chitinase showed novel properties with a smallest molecular weight (12.8 kDa by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis) and more acidic optimal pH (pH 4) than those of other Streptomyces chitinases. A combination of high-performance liquid chromatography and matrix-assisted laser desorption ionization time of flight mass spectrometer data revealed that chitin oligomers (COS) obtained from the hydrolysis of colloidal chitin by TKU045 chitinase comprise oligomers with multiple degrees of polymerization (DP) varying from 1 to 7. The COS with low DP exhibited enhanced 2,2-diphenyl-1-picrylhydrazyl radical scavenging capability and promoted the growth of Lactobacillus lactis. Taken together, the COS obtained by hydrolyzing colloidal chitin with TKU045 chitinase could have the potential to be used in medicine or nutraceuticals due to its active anti-oxidant and prebiotic contents.
論文目次 Catalog Page

Acknowledgment I
Abstract II
Abbreviation used IV
Catalog V
List of tables VII
List of figures VIII
1. Introduction 1
2. Experimental section 3
2.1. Materials 3
2.2. Screening and identification of microorganisms 3
2.3. Chitinase activity assay 3
2.4. Optimization of culture conditions for the chitinase-producing strain 4
2.5. Purification and characterization of Streptomyces thermocarboxydus TKU045 chitinase 4
2.6. Effects of temperature and pH on the activity and stability of TKU045 chitinase 4
2.7. Preparation of chitin oligomers 5
2.8. MALDI-TOF mass spectrometry analysis 5
2.9. HPLC analysis 5
3. Results and discussion 6
3.1. Isolation, screening and identification of a chitinase-producing strain 6
3.2. Effects of the C/N source on chitinase production 6
3.3. Optimization of culture conditions 7
3.4. Purification of TKU045 chitinase 10
3.5. Effects of temperature and pH on the activity and stability of TKU045 chitinase 12
3.6. Chitin hydrolysis 13
3.7. Anti-oxidant activity of COS 15
3.8. Effects of COS on the growth of lactic acid bacteria 16
4. Conclusions 18
Reference 19
Appendix 24
Appendix 1: Identification of strain TKU045 24
Appendix 2: List of publication 27

List of tables Page
Table 3.1. Comparison of culture conditions before and after optimization 9
Table 3.2. Purification of the chitinase from S. thermocarboxydus TKU045 10
Table 3.3. Comparison of chitinases produced by TKU045 and other Streptomyces strains 12

List of figures Page
Figure 3.1. Production of chitinase by S. thermocarboxydus TKU045 in 1% of different chitin-containing medium. 7
Figure 3.2. The effects of some parameters on chitinase production by S. thermocarboxydus TKU045 8
Figure 3.3. Cultivation curves for chitinase production before and after optimization 9
Figure 3.4. A typical elution profile of S. thermocarboxydus TKU045 chitinase on Macro-Prep High Q 11
Figure 3.5. SDS-PAGE analysis of the chitinase produced by S. thermocarboxydus TKU045 11
Figure 3.6. Effects of temperature and pH on TKU045 chitinase activity 13
Figure 3.7. MALDI-TOF-MS spectra of the COS mixtures obtained during the colloidal chitin hydrolysis for 5 h with the TKU045 chitinase. 14
Figure 3.8. HPLC spectrum (c) and content analysis (d) of the COS obtained during colloidal chitin (1%) hydrolysis with TKU045 chitinase at pH 5 (50 mM sodium acetate buffer) for 5 h. 15
Figure 3.9. DPPH radical scavenging activities of colloidal chitin (C.C.) hydrolysates, high DP COS, and low DP COS 16
Figure 3.10. Effects of supplement MRS broth with colloidal chitin (C.C) hydrolysate, high DP COS, and low DP COS on the growth of Lactobacillus lactis BCRC 10791 17
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