- 學位論文
Chryseobacterium taeanense TKU001發酵綠豆所生產澱粉酶及其生物活性物質之研究
Studies on Purification of Amylases and Bioactive Materials from Mung Bean Fermented by Chryseobacterium taeanense TKU001
摘要
Chryseobacterium 又稱Flavobacterium，為非發酵、無運動性之菌株，係屬革蘭氏陰性桿菌。C. taeanense TKU001 以綠豆為主要碳氮源，經發酵後可生產澱粉酶，並進一步探討其酵素純化及定性。澱粉酶之較適培養條件為1.5％綠豆粉（mung bean powder；MBP）、0.1％ K2HPO4 及0.05％ MgSO4．7H2O 之100 mL 液態培養基(pH 9)，於30℃振盪(150 rpm)培養5 天後可得較佳澱粉酶活性。 將發酵上清液經由冷凍乾燥（或減壓濃縮）、DEAE Sepharose CL-6B、Phenyl Sepharose 6 Fast Flow 等一系列管柱層析後進行生化性質分析。最適反應溫度與熱安定性分別為50℃與<60℃，最適反應pH 與pH 安定性分別為pH 9 與pH 7-11。在化學試劑穩定性測試中，Ca2+與Tween 40 可明顯提升酵素活性，但Fe2+、Cu2+及Mn2+則會完全抑制其活性。 抗氧化分析方面，以DPPH 自由基清除力、亞鐵離子螯合力、及還原力三種方式檢測，並測定其總酚含量。自由基清除試驗可達79%以上之有效清除率(1st 天)，亞鐵離子螯合力則為0.34 mg/mL EDTA equivalents (6th 天)，其總酚含量達0.38 mg/mL (7th 天)，其還原力相當於0.36 mg/mL cysteine equivalents (7th 天)。
並列摘要
Chryseobacterium, formerly known as Flavobacterium, is a nonfermenting, nonmotile, gram-negative aerobic rod. An amylase was produced from the culture supernatant of C. taeanense TKU001 with mung bean powder as the main nutirion source. The optimized conditions for amylase production was found when the culture was shaken at 30℃ for five day (150 rpm) in 100mL of medium containing 1.5% mung bean powder, 0.1% K2HPO4, 0.05% MgSO4．7H2O (pH 9). The amylase was purified from culture by lyophilization (or evaporation), DEAE sepharose CL-6B, and Phenyl Sepharose 6 Fast Flow column chromatography. The optimal temperature and thermal stability of this enzyme were 50℃ and <60℃. The optimal pH and pH stability of this enzyme were pH 9 and pH 7-11. This amylase was activated by Ca2+ ions and Tween 40, but completely inactivated by Fe2+, Cu2+, and Mn2+ ions. The antioxidative evaluations were analyzed by free radical-scavenging activity, metal-chelating ability, reducing power, and total phenolic contents. The DPPH scavenging rate, Fe2+- chelating activity, reducing power, and total phenolics of TKU001 culture supernatant were revealed 56% (1st day), 0.34 mg/mL EDTA equivalents (6th day), 0.38 mg/mL (7th day), and 0.36 mg/mL cysteine equivalents (7th day), respectively.