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。

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.

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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