利用芽孢桿菌醱酵生產納豆激酶與環狀糊精葡萄糖苷轉移酶之研究

Produce of nattokinase and cyclodextrin glucanotransferase by Bacillus sp.

郭洲獎(Chou-Chiang Kuo)

指導教授 ： 段國仁

大同大學/工程學院/生物工程學系所/博士(2011年)

摘要

從市售日本納豆食品中篩選出納豆激酶 (nattokinase)生產量最高的納豆菌，再以API CHB 50 Bacillus sp.專用生化鑑定套組分析，確定此納豆菌 (おかね)為Bacillus subtilis。最適醱酵生產納豆激酶之液態培養基成分為：30 g/L黃豆粉、30 g/L glucose、30 g/L CaCO3、5 g/L KH2PO4及1 g/L MgSO4‧7H2O，於37 ℃、180 rpm條件下，經醱酵培養 24小時後得到酵素活性達 18.5 unit/mL，48小時後得到酵素活性達 23.9 unit/mL。
5 公升醱酵槽批次醱酵結果顯示，接種 10% (v/v)的種菌液，通氣量為1 vvm，攪拌速度為 300 rpm，於 37℃下進行醱酵培養，培養至第 30小時產生最高的酵素活性為 58.2 unit/mL。利用饋料醱酵策略，將葡萄糖濃度控制在 10~20 g/L之間，於第 29小時納豆激酶活性高達約 107.0 unit/mL。
納豆激酶經過管柱層析純化後，分析其分子量約為 27,470，酵素活性表現之最穩定的 pH值在 5.0～8.0之間，而且 Ca2+、Mg2+的存在能促進納豆激酶的活性表現，但是在 50 mM的 Cu2+及 Hg2+處理24小時，活性表現剩下原來的 23.3%及 3.4%。
本研究另外以 Bacillus circulans No. 38-2 (BCRC 10094)生產 刍-環狀糊精葡萄糖苷轉移酶 (CGTase)，以 20 g/L的可溶性澱粉為碳源並配合基礎培養基的成分：20 g/L 酵母萃取物、10 g/L Na2CO3、0.2 g/L CaCl2、1 g/L KH2PO4、1 g/L K2HPO4、0.2 g/L MgSO4．7H2O、0.2 g/L Mn(NO3)2，在5公升醱酵槽、33 ℃進行批次培養，第 10小時達最大菌體濃度 15.19 g/L，CGTase 酵素活性在培養第 14小時達最高的 15.48 unit/mL。
在5公升醱酵槽連續式培養中，稀釋率在 0.15 h-1、0.11 h-1與0.075 h-1的條件下，分別可維持酵素活性穩定範圍在 1.2~1.5 unit/mL、2.0~2.2 unit/mL以及 9.8~10.0 unit/mL。計算不同的醱酵策略之CGTase的單位體積產率（volumetric productivity）分別為：批次(batch) 約 1.11 unit/mL-h，連續式醱酵中，稀釋率 0.15 h-1、0.11 h-1及 0.075 h-1的單位體積產率分別為0.18~0.23 unit/mL-h、0.22~ 0.24 unit/mL-h以及0.74~0.75 unit/mL-h。

關鍵字

納豆激酶 ； 環狀糊精葡萄糖苷轉移酶 ； 芽孢桿菌

並列摘要

A strain was isolated from a traditional natto in Japan, and identified the bacteria as Bacillus subtilis by using API CHB50 kits. It was employed for production of nattokinase by shaking culture at 180 rpm and 37 牵C. The optimum medium composition was : 30 g/L soybean flour, 30 g/L glucose, 5 g/L CaCO3 , 5 g/L KH2PO4 and 1 g/L MgSO4．7H2O, with which the nattokinase activity of 23.9 unit/mL was obtained after 48 h.
The batch fermentation using the same medium as in the shaking culture at 300 rpm, 1 vvm of aeration, and 37 牵C, 58.2 unit/mL of nattokinase was achieved after 30 h cultivation.A fed-batch fermentation was performed to control the glucose concentration in the range of 10~20 g/L, in which the nattokinase activity of 107.0 unit/mL was achieved after 29 h.
The nattokinase mocular weight is 27,470 by gel filtration. It was stable at pH 5~8. Addition of and Ca2+ and Mg2+ could enhance the enzyme activity, but 23.3% and 3.4% activity at 24 h retained when adding 50 mM Cu2+ or Hg2+ in the medium.
The other study of this dissertation was focused on the production of cyclodextrin glucanotransferase (CGTase) by Bacillus circulans No. 38-2 (BCRC 10094) in alkaline medium under various conditions. The batch fermentation was performed to achieve CGTase activity of 15.48 unit/mL at 33 牵C using the following medium, 20 g/L soluble starch, 20 g/L yeast extract, 10 g/L Na2CO3, 1 g/L KH2PO4, 1 g/L K2HPO4, 0.2 g/L CaCl2, 0.2 g/L MgSO4．7H2O and 0.2 g/L Mn(NO3)2.
In a continuous fermentation, 1.2~1.5 unit/mL,2.0~2.2 unit/mL and 9.8~10.0 unit/mL were achieved at respective diluting rate of 0.15 h-1 , 0.11 h-1 and 0.075 h-1. The results indicated that the volumetric activity (0.74 ~ 0.75 unit/mL-h) was achieved for the continuous fermentation at a dilution rate of 0.075 h-1, (0.18 ~ 0.23 unit/mL-h) at 0.15 h-1, and (0.22 ~ 0.24 unit/mL-h) at 0.11 h-1, in comparison whit the volumetric activity of 1.11 unit/mL-h respectively for a batch fermentation.

並列關鍵字

nattokinase ； cyclodextrin glucanotransferase ； Bacillus sp.

參考文獻

Nattokinase之探討。民國九十一年度國科會專題研究計畫成果報告。

吳世佑。2005。從市售納豆健康食品中篩選納豆菌並探討最適化生產納豆激

林清安。2005。利用嗜鹼性芽胞桿菌Bacillus circulans No. 38 (BCRC 10094)

范瓊藝。2006。用饋料發酵生產納豆激酶之研究。大同大學生物工程所碩士

Esaki, H., H. Onozaki, and T. Osawa. 1994. Antioxidative activity of fermented