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Purification and characterization of nattokinase in fermented Porphyra dentate by Bacillus subtilis natto
|Contributors: ||NTOU:Department of Food Science|
|Issue Date: ||2011-06-30T08:17:51Z
|Abstract: ||傳統以納豆菌發酵黃豆所得之納豆激酶具血栓分解能力。紫菜含豐富醣類和蛋白質，以紫菜為基質，以搖瓶方式已建立其生產納豆激酶之生產培養液及生產條件。本研究首先利用十公升發酵槽探討通氣量、控制 pH 值等操作條件對納豆菌生產納豆激酶之影響，接著分離純化發酵液中納豆激酶，並探討其酵素特性。通入過濾空氣 0.8~3.0 vvm，隨著通氣量增加所得酵素活性隨之增加，於 2.0 vvm 達到最高酵素活性，其酵素活性為 986.83 U/mL。酵素活性主要在菌體生長之靜止期產生，以 48 小時活性最高。將大量生產所得之納豆激酶粗酵素液經 DEAE-Sepharose FF 離子交換層析及Sephadex G-50 膠體過濾層析純化所得酵素比活性為 269.44 U/mg，純化倍率為 5.73 倍。純化納豆激酶分子量為 46.54 kDa，等電點為 pI 8.35，Km 值為 0.044 mg/mL，Vmax 為 9.5 mM/min。納豆激酶最適作用溫度與 pH 值，分別為 55 oC 及 pH 8.0，其於 55 oC 保溫 1小時仍保有 80.33 % 之活性，而於 pH 5-9 37oC 保溫 1 小時仍保有 89.22 % 以上之酵素活性。10 mM 之 Cu2+、Fe3+ 及 2 % 之 Triton X-100可提昇酵素活性，相對活性分別為 137、 123 及 122 %。納豆激酶除了可水解血纖維蛋白外，亦可水解血紅蛋白及明膠。PMSF 及 leupeptin顯著抑制納豆激酶活性，故判定其為絲胺酸蛋白酶。|
Nattokinase (NK), from a fermented soybean product by Bacillus subtilis natto, is a strong fibrinolytic enzyme. Porphyra dentate is abundant in carbohydrates and protein. Optimal conditions for the production of nattokinase from Porphyra dentate by shaking flask method has been evaluated previously. In this study, factors of aeration and pH control in a ten-liter fermentor for nattokinase production was further evaluated. Then nattokinase from fermented Porphyra dentate substrate by B. subtilis natto was purified and characterized. The NK activity was increased with increasing of aeration, with maximal of 986.83 U/mL at 2 vvm. A homogeneous nattokinase was obtained after DEAE-Sepharose FF anion exchange and Sephadex G-50 gel filtration chromatography. The NK specific activity was 269.44 U/mg with purification of 5.73-fold. The molecular mass and pI of the enzyme were 46.54 kDa and pH 8.35. It’s kinetic parameters of Km and Vmax were 0.044 mg/mL and 9.5 mM/min, respectively. The optimal temperature and pH of nattokinase were 55 oC and pI 8.0, respectively. The residual activity of nattokinase at 55 oC for 1 hr was 80.33 %. The enzyme activities remained over 89.22% at pH 5-9 for 1 hour. The enzyme activity was increased in the presence of 10 mM Cu2+, Zn2+ (136 and 123 %) and 2 % Triton X-100 (122 %). Besides of fibrinogen, NK also hydrolyzed fibrin, hemoglobin and gelatin. PMSF, leupeptin and EDTA greatly inhibited the activity of nattokinase. These results indicated that the purified enzyme is a serine-like proteas.
|Appears in Collections:||[食品科學系] 博碩士論文|
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