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    題名: Bacillus cereus TKU022所生產幾丁聚醣酶及蛋白酶之特性分析與應用
    其他題名: Characterization of a chitosanase and a protease from Bacillus cereus TKU022 and their applications
    作者: 謝佳霖;Hsieh, Jia-lin
    貢獻者: 淡江大學生命科學研究所碩士班
    王三郎
    關鍵詞: 臘狀芽孢桿菌;幾丁聚醣酶;蛋白酶;蝦頭粉;Bacillus cereus;chitosanase;protease;shrimp head powder
    日期: 2010
    上傳時間: 2010-09-23 16:09:55 (UTC+8)
    摘要: Bacillus cereus TKU022係以蝦頭粉為主要碳/氮源,篩選自台灣北部土壤之幾丁聚醣酶生產菌,發酵蝦頭粉所得離心上清液具有幾丁聚醣酶及蛋白酶活性。幾丁聚醣酶較適生產條件為 1.5% 蝦頭粉、0.1% K2HPO4、0.05% MgSO4.7H2O 之50mL液態培養基(pH5)於 37℃振盪培養 2天。發酵所得離心上清液經硫酸銨沉澱、DEAE-Sepharose、Phenyl Sepharose 、Macro-Prep DEAE及Sephacryl S-100等層析步驟後,可純化出一種幾丁聚醣酶及一種蛋白酶,經 SDS-PAGE測定分子量分別為 44 kDa及45kDa。其幾丁聚醣酶及蛋白酶之最適反應pH、最適反應溫度、pH 安定性、熱安定性分別為 pH 7, 60 ℃, pH 7-10, <40 ℃及pH 10, 50-60℃, pH 6-10, <50 ℃;幾丁聚醣酶活性會受Cu2+及Mn2+所抑制,而非離子型界面活性劑 Triton X-100、Tween 20及離子型界面活性劑SDS 則不具抑制效果;蛋白酶則會受到Mn2+、EDTA及SDS所抑制,在2 mM之存在下Triton X-100、Tween 20、Tween 40還維持其原活性97 %、105 %及94 %。
    TKU022以較適生產幾丁聚醣酶培養基(1.5 % SHP, 37 ℃, pH 5)培養8天,所得發酵上清液於第5天有最高之還原醣(649 µg/mL)產量;並0.05 %之還原醣添加至MRS broth中可提升Lactobacillus paracasei TKU010之生長速率。
    The chitosanase -producing bacterium, Bacillus cereus TKU022, was isolated from soil in the north Taiwan by using shrimp head powder as the sole carbon/nitrogen source. The supernatant of the culture medium contains the chitosanase and the protease the activity. The optimized condition for chitosanase production was found when the culture was shaken at 37°C for two days in 50mL of medium containing 1.5% SHP, 0.1% K2HPO4, 0.05 % MgSO4.7H2O ( pH 5). The chitosanase and protease were purified from the culture supernatant by ammonium sulfate precipitation, DEAE-Sepharose, Phenyl Sepharose, Macro-Prep DEAE and Sephacryl S-100. The molecular masses of the chitosanase and protease determined by SDS-PAGE were approximately 44 and 45 kDa, respectively. The optimum pH, optimum temperature, pH stability and thermal stability of TKU022 chitosanase and protease were pH 7, 60 ℃, pH 7-10, <40 ℃and pH 10, 50-60 ℃, pH 6-10, <50 ℃, respectively. The chitosanase activity was inhibited by Cu2+ and Mn2+, but not by Tween 20, Triton X-100 (nonionic surfactant) and SDS (anionic surfactant).The protease activity was inhibited by Mn2+, EDTA and SDS, but retained 97 %, 105 % and 94% of its original activity in the presence of 2 % Triton X-100, 2 % Tween 20 and 2 % Tween 40, respectively.
    B. cereus TKU022 was incubated for 8 days under the optimized culture conditions(1.5 % SHP, 37 ℃, pH 5)and analyzed the reducing sugar. TKU022 culture supernatant incubated for 5 days had the highest reducing sugar(649 µg/mL)and enhanced the growth rate of Lactobacillu paracasei TKU010 in MRS broth.
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