本研究分為兩部分實驗，第一部份在探討紫田薯塊莖中β-澱粉?基本性質，而第二部分是含耐熱β-澱粉?轉殖馬鈴薯其酵素活性之初步探討。 在紫田薯塊莖研究中，塊莖間差異性大且所萃取之粗酵素液黏度太高，以致於無法成功分離純化β-澱粉?，而有效降低黏度之方法以添加2% 陽離子介面活性劑後再加入甲醇的效果最佳。經由初步處理之粗酵素液其基本性質為：最適溫度為60℃，最適pH值為6.0，Km值為1.786 %，最大反應速率為357.1 μmole / μL / min、在金屬離子方面只有鋅離子會促進β-澱粉?之活性、在化學修飾劑方面尿素會明顯抑制β-澱粉?，而PMSF和EDTA則會促進酵素活性，但EDTA中含有鈣離子的緩衝溶液對β-澱粉?則會抑制。而不同的受質種類對紫田薯中β-澱粉?活性影響很大。 另外，利用馬鈴薯基因轉殖的部分，因耐熱β-澱粉?基因的特性可在E. coli BL21表現。因此首先研究E. coli BL21中耐熱β-澱粉?之表現來建立分析轉殖株方法。原始耐熱β-澱粉?在E. coli BL21中表現的性質為：最適溫度為60 ℃，粗酵素液可於60 ℃維持一個半小時之活性、Km 值為3.2%、最大反應速率為137.0μmole / μL / min。目前已有數個轉殖成功之含耐熱β-澱粉?基因的轉殖株。為了要成功分析是否有轉殖成功，先要建立一個分析耐熱β-澱粉?的方法。此方法為去除內生性β-澱粉?，條件為粗酵素液先透析4小時，之後再於60 ℃加熱20分鐘。利用此前處理方法所分析轉殖株中耐熱β-澱粉?，只有ptAmy-sp 1E、ptAmy 2A、ptAmy 5B及ptAmy 7F有明顯活性表現。 There were two parts in this research. The part one was to study the β-amylase activity from Dioscorea alata L. var. purpurea M. Pouch and part two was to study the thermostable β-amylase activity in trans- genic potatos. In part one, it was difficult to purify β-amylase because the crude extract contained sticky substances. After sequentially adding 2% cetylpyridinium and methanol, we were successful to remove the sticky substances. The pH and temperature optima for theβ-amylase activities were pH 6.0 and 60 ℃; the Km and Vmax values were 1.786 % and 357.1 μmole / μL / min separately. The β-amylase was specifically activated by Zn2+ , PMSF and EDTA, but was strongly inhibited by urea. The catalytic efficiency of β-amylase was siguificautly affected by using different substrates. In part two, characteristics of the original thermostable β-amylase expressed in E. coli BL21 was investigated. The temperature optium for this thermostable β-amylase activity was 60 ℃, the Km and Vmax values were 3.2% and 137.0 μmole / μL / min separately. Several transgenic potato lines conferred with thermostable β-amylase gene were obtained. In order to confirm the successful transformation and expression of β-amylase in potatos, a method to determine thermostable β-amylase activies was established. This included dialysis of crudeβ-amylase extract from transgenic potatos for 4 hrs followed by heating under 60 ℃ for 20 mins to denature the endogenousβ-amylase. Using this method to assay all available transgenic potato lines, only ptAmy-sp 1E、ptAmy 2A、ptAmy 5B and ptAmy 7F showed β-amylase activities .
