- 學位論文
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
The isolation of chitinase from Streptomyces thermocarboxydus and its application in the preparation of chitin oligomers
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。
並列摘要
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.