Bacillus subtilis TKU007 所生產納豆激酶之研究

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Title:	Bacillus subtilis TKU007 所生產納豆激酶之研究
Other Titles:	Studies on nattokinases from bacillus subtilis TKU007
Authors:	吳盈瑩;Wu, Ying-ying
Contributors:	淡江大學生命科學研究所碩士班王三郎;Wang, San-lang
Keywords:	Bacillus subtilis;納豆激酶;casein;Lactobacillus;促進生長;Bacillus subtilis;nattokinase;casein;Lactobacillus
Date:	2009
Issue Date:	2010-01-11 02:27:49 (UTC+8)
Abstract:	Baciilus subtilis TKU007 係一株以蝦頭粉為唯一碳/氮源，篩選自台灣北部土壤之納豆激酶生產菌。TKU007生產納豆激酶之較適培養條件為-含有1%蝦頭粉、0.1% K2HPO4及0.05% MgSO4‧7H2O之100 mL 液態培養基，於 pH 7、30℃，搖瓶培養 3天。所得發酵上清液經硫酸銨沉澱、DEAE-Sepharose、Phenyl Sepharose 及Sephacryl S-100 層析分離步驟，純化出二種納豆激酶，經SDS-PAGE測得分子量分別為 30 kDa (N1)、15 kDa (N2)。於最適反應pH、最適反應溫度、pH安定性、熱安定性方面，N1分別為 pH 8、40℃、pH 6-10、<50℃。納豆激酶 N1 活性會受到Mg2+、Fe2+、Cu2+所促進，然而會受到 Zn2+、PMSF所抑制，為絲胺酸型蛋白酶。將B. subtilis TKU007發酵所得上清液與酪蛋白於37℃反應2小時，添加所得水解液 (最終濃度2.5 %) 至乳酸菌培養基MRS進行乳酸菌Lactobacillus paracasei TKU010之培養，於25℃、搖瓶培養3天，L. paracasei TKU010菌落數可達到5.9×1012 CFU/mL；較對照組(未添加水解物) 增加2. 3倍，添加未水解的酪蛋白反而會抑制L. paracasei TKU010 生長。 Bacillus subtilis TKU007, cultured by shrimp head powder as the only carbon/nitrogen source, was isolated from the soil in Taiwan. For the production of nattokinase, B. subtilis TKU007 was grown in 100 mL of liquid medium in an Erlenmeyer flask (250 mL) containing 1% SHP, 0.1% K2HPO4 , 0.05% MgSO4‧7H2O and grown in an orbital shaking incubator for 3 days at 30℃ and pH 7. After incubation, the culture broth was centrifuged (4℃ and 10,870 ×g for 15 min) , and the supernatant was used for further purification by DEAE-Sepharose, Phenyl Sepharose and Sephacryl S-100. The molecular mass of TKU007 nattokinases determined by SDS-PAGE was approximately 28 kDa (N1) and 15 kDa (N2), respectively. The optimum pH, optimum temperature, pH stability, thermal stability of nattokinase N1 was pH 8, 40℃, pH 6-10、<50℃, respectively. N1 nattokinase activity was activated by Mg2+, Fe2+, Cu2+, inactivated by Zn2+, PMSF, indicating N1 nattokinase was serine protease. A culture supernatant of B. subtilis TKU007 was mixed with casein and incubated for 2 hour at 37℃. The hydrolysates were added to MRS medium (final concentration 2.5 v/v ) for Lactobacillus paracasei TKU010 culture and incubated for 3 days at 25℃. The growth of L. paracasei TKU010 was 5.9×1012 CFU/mL. Which than the control group (non-add hydrolysates) increase 2.3 fold . However, the addition of unhydrolyzed casein could inhibit the growth of L. paracasei TKU010.
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