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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/14983

Title: Bacillus subtilis YJ1納豆激酶之純化與特性
Purification and Characterization of from Bacillus subtilis YJ1
Authors: Kar-Tat Sham
Contributors: NTOU:Department of Food Science
Keywords: 納豆激酶;純化
Date: 2007
Issue Date: 2011-06-30T07:54:06Z
Abstract: 中文摘要 本研究之目的在純化發酵食品篩選分離的 Bacillus subtilis YJ1 之納豆激酶與生化特性探討。Bacillus subtilis YJ1 菌株在複合培養基 (含1% skim milk、1% husk、0.5% NaCl、0.2% glucose) 中,於 37oC、150 rpm 下振盪培養96小時,收集胞外粗酵素液,其蛋白酶活性約為 23.50 FU/mL。將粗酵素液經硫酸銨分劃、透析後,進行 CM-Sepharose Fast Flow 管柱層析可得到具有活性之納豆激酶,其比活性為1791.86 FU/mg,回收率為 9.50%,純化倍率為9.15倍。經SDS-PAGE 電泳分析呈現單一色帶之純化納豆激酶,其分子量為35.5 kDa。納豆激酶最適酸鹼度在 pH 8.5,其在 pH 6.0-10.0之間有較佳之安定性,而其最適溫度為50oC,在10oC-40oC之間安定性較佳。純化之納豆激酶會受到Fe3+、Hg2+、Cu2+ 及Zn2+ 金屬離子的抑制。純化之納豆激酶明顯被 PMSF 抑制,而 Leupeptin 及 EDTA 則會輕微抑制酵素活性,故推測此納豆激酶為絲胺酸蛋白酶且其活化能為49.30 kcal/mole。
Abstract Bacilus subtilis YJ1, isolated from fermented product, could produce nattokinase when it was cultivated in a medium containing 1 % skim milk, 1 % husk, 0.5% NaCl, 0.25% glucose. The nattokinase activity reached 23.50 FU/mL after 96 hrs incubation at 37oC. After being filtered through a 0.45 µm membrane to remove the bacterial cells, the nattokinase was purified to electrophoretical homogeneity after ammonium sulfate fractionation and CM-Sepharose Fast Flow chromatography. About 1791.86 FU/mg of specific activity, 9.50% of yield and 9.15 of purification fold were achieved at this stage. The purified nattokinase, with a MW of 35.5 kDa, had optimal temperature and pH at 50oC and 8.5, respectively, and was stable at pH 6-10 and 10~40oC. Fe3+, Hg2+, Cu2+, Zn2+ and PMSF could inhibit, while leupeptin and EDTA slightly inhibited its activity. According to the substrate and inhibitor specificities, it was considered to be a serine protease. The activation energy was 49.30 kcal/mole.
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0M94320001
Appears in Collections:[食品科學系] 博碩士論文

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