| 摘要: | 本研究係探討Bacillus cereus TKU006所生產幾丁質分解酵素及其他抗氧化質之研究，探討其較適培養條件，進行酵素純化與定性為研究目的。
TKU006以1％烏賊軟骨粉末為碳/氮源培養於100 mL含 0.1％ K2HPO4及0.05％ MgSO4．7H2O之液態培養基，於25℃搖瓶培養三天可得較高幾丁聚醣酶活性；以2％蝦頭粉末為碳/氮源於25 mL液態培養基培養二天得較高幾丁質酶活性。所得發酵上清液經硫酸銨沉澱、陰離子交換層析、膠體過濾層析步驟純化得幾丁質酶、幾丁聚醣酶。幾丁質酶分子量為39 kDa，Fe2+完全抑制酵素活性，最適反應溫度、pH分別為60℃、pH 5，熱安定性、pH安定性分別為≦50℃、pH 2-8。幾丁聚醣酶分子量為44 kDa，受到Fe2+、Cu2+完全抑制酵素活性，最適反應溫度與pH分別為60℃與pH 7，熱安定性、pH安定性分別為≦50℃與pH 3-10。
以25 mL含1％烏賊軟骨、0.1％ K2HPO4及 0.05％ MgSO4．7H2O之液態培養基，於25℃搖瓶培養二天，可得較佳DPPH自由基清除能力。發酵上清液之自由基清除能力、亞鐵離子螯合能力、還原力分別為75％、97％與441 μg/mL CE，而酚類化合物含量為815 μg/mL GAE。總酚類化合物與自由基清除能力之相關係數為r=0.92。
The purpose of this thesis is to study the production of chitinolytic enzymes and other antioxidants from Bacillus cereus TKU006.
The optimum culture conditions for chitosanase and other antioxidants production were composed of 1% squid pen powder, 0.1% K2HPO4, 0.05% MgSO4．7H2O shaken at 25℃ in 100 mL of liquid medium in an Erlenmeyer flask (250 mL) for 3 days and 25 mL of liquid medium for 2 days, respectively. The optimum culture carbon/nitrogen source for chitinase production was 2% shrimp head powder incubated for 2 days. A chitinase and a chitosanase were purified by chromatography procedures of ion exchange and gel filtration. The molecular mass of the chitinase and chitosanase determined by SDS-PAGE were approximately 39 kDa and 44 kDa, respectively. The chitinase was completely inactivated by Fe2+, and the chitosanase was completely inactivated by Fe2+ and Cu2+. The optimum temperature, optimum pH, thermal stability and pH stability of chitinase were 60℃, pH 5, ≦50℃, pH 2-8 . The optimum temperature, optimum pH, thermal stability and pH stability of chitosanase were 60℃, pH 7, ≦50℃, pH 3-10.
After squid pen powder fermented, the antioxidant properties by the assays of radical scavenging, metal chelating, reducing power and total phenolic content were reached at 75%, 97%, 441 μg/mL cysteine equivalent and 815 μg/mL gallic acid equivalent, respectively. Correlation between DPPH scavenging ability and total phenolic content were r=0.92. |
