利用蒙脫土由甘薯液中分離胰蛋白脢抑制劑sporamin之研究

Separation of Trypsin Inhibitor sporamin from Sweet Potato Juice by Using Bentonite

王秋淳(Chiou-Chuen Wang)

指導教授 ： 陳志成

大同大學/工程學院/生物工程學系所/碩士(2005年)

摘要

本實驗乃利用蒙脫土bentonite初步純化甘藷之胰蛋白酶抑制劑sporamin之研究，並將其與硫銨沉澱之結果做探討與比較。實驗結果發現：在45%的硫銨沉澱純化下可獲得活性回收率 10.4 %的sporamin，純化倍數為1.11；由蒙脫土純化，約可吸附75-80 %蛋白質，活性回收率為62 %，純化倍數為1.77。硫銨沉澱需反應8小時或過夜，而利用蒙脫土只需要於pH=6.5，25℃下反應一小時即可完成吸附，並可在pH=10，4℃下，以0.1M AlK(SO4)2反應3小時即可完成脫附，可脫附35 %的蛋白質，活性回收率為62 %。實驗結果並顯示出在甘薯液為5 mg/ml的濃度下時，每克的蒙脫土，約可吸附66毫克的蛋白質；利用蘭格謬爾 (Langmuir adsorption isotherm model)公式計算出來的最大吸附量 qmax= 236 mg/g，以及Langmuir常數KL= 10 mg/ml，並可利用這些計算出的數值，應用於計算工業生產時之DBC (單位體積的固定床之動能鍵結能力)所需的條件。1997年，Dr.Yeh等人將sporamin轉殖於菸草中發現對斜紋夜盜蛾等幼蟲的啃食具有抗食性，因此sporamin經由初步純化後，可應用於生物農藥上。蒙脫土為一價格便宜之自然界礦土，在使用上不具有類似硫銨排放後，造成河川污染之環保問題；並在操作及反應時間上較硫銨沉澱來的方便而快速，並可降低生物農藥之成本。

關鍵字

蒙脫土，胰蛋白酶抑制劑

並列摘要

Bentonite was used to purify sporamim which is a trypsin inhibitor from sweet potato in this study and contrasted with the result of ammonium sulfate. The data showed at 45% saturation ammonium sulfate, 10.4% activity yield of spoarmin was recovered and the purification fold was 1.11. The result of separating sporamin by bentonite showed that 75-80% protein (sporamin) were adsorbed at pH = 6.5, 25℃ for 1 h, the purification fold was 1.77, and the activity yield was 62 %. Desorption was proceeded at pH=10, 4℃, 0.1M AlK(SO4)2 for 3h, 35 % protein could be desorption and activity yield was 62 %. Data also showed that maximum amount of adsorption was 66mg protein by 1g bentonite as the concentration of sweet potato juice is 5 mg/ml. The Langmuir fitting appeared the maximum amount of adsorption qmax = 236 mg/g，and the Langmuir constant KL = 10 mg/ml. These values can substitute DBC (Dynamic Binding Capacity) equation and calculate the conditions of scale-up. In 1997, Dr. Yeh et al. expressed the sporamin in transgenic tobacco plants, it was showed a high degree of resistance to Spodoptera litura. Thus, using sporamin can produce biopesticides after partial purification. Bentonite is a cheap material to separate soramin, it can reduce the cost of biopesticides. The bentonite which is discharged into river after using did not contaminate the river.

並列關鍵字

bentonite ； purification ； sporamin

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