利用毛細管電泳作蘇力菌素伴胞結晶蛋白之定量分析   全文下載

Quantitative Analysis of Parasporal Crystal Protein from Bacillus thuringiensis by Capillary Electrophoresis

蘇力菌素產生的伴胞結晶蛋白（δ-endotoxin）為目前最具潛力的生物農藥之一。在菌種的選擇及發酵方法的研發過程中，測試發酵液中伴胞結晶蛋白之量為一決定性的重要依據。傳統的方法是以生物檢測法或HPLC為主，這些方法一般較為耗時且準確度不高。本研究利用毛細管電泳分析經還原劑溶解後的伴胞結晶蛋白作定量之分析，以達到快而準確的分析。本實驗以較高濃度的硼酸（300 mM Boric acid）用1N的NaOH將pH調至10.0 ，用Lysozyme 作為內在標準品，在注入已知量的結晶蛋白溶解液及定量Lysozyme 之混合液至毛細管電泳儀，經過10 kV，二十分鐘之電泳後，從電泳圖譜上結晶蛋白溶解液及Lysozyme形成的波峰面積之比率與結晶蛋白之濃度作線性迴歸相關性分析。其結果顯示相關係數為0.9994 ，斜率為0.4095 ，交叉點為+0.0025 ，利用此一公式，可用定量之Lysozyme 加入所要測試的結晶蛋白溶解液中作快速、經確之定量分析。本實驗用三種品系的蘇力菌（HD-1，A3-4，及YIM303）分析其發酵液中結晶蛋白之含量作為例證。

The amount of parasporal crystal protein (δ-endotoxin) from the fermentation broth of the Bacillus thuringiensis is the best indication to assess the efficiency of the fermentation process or strain selection. Traditional methods for the assessment of insecticidal effect, such as bioassay or HPLC, were either time-consuming, inaccurate or inefficient. In this study, capillary electrophoresis (CE) was used for analyzing the amount of parasporal crystal protein after it was dissolved by adding a reducing agent, such as β-mercaptoethanol, to break the disulfide bonds. This soluble protein, δ-endotoxin, was then subjected to quantitative analysis by CE. The running buffer contained 300 mM boric acid, and pH was adjusted to 10.0 with 1 N NaOH. The dimensions of the capillary were 47 cm × 50 μm I.D. without coating. Lysozyme was used as internal standard for the quantitative assay of the δ-endotoxin. The migration time of the lysozyme peak was approximately 2 minutes earlier than that of the δ-endotoxin peak. The correlation between the concentration of δ-endotoxin and the ratio of the peak area of δ-endotoxin and the peak of lysozyme was calculated. The linear regression analysis showed that the correlation coefficient is equal to 0.9994, the slope is 0.4095 and the intercept is +0.0025. From this standard regression equation, the concentration of δ-endotoxin in fermentation broth or solution can be estimated easily by CE analysis. 蘇力菌素產生的伴胞結晶蛋白（δ-endotoxin）為目前最具潛力的生物農藥之一。在菌種的選擇及發酵方法的研發過程中，測試發酵液中伴胞結晶蛋白之量為一決定性的重要依據。傳統的方法是以生物檢測法或HPLC為主，這些方法一般較為耗時且準確度不高。本研究利用毛細管電泳分析經還原劑溶解後的伴胞結晶蛋白作定量之分析，以達到快而準確的分析。本實驗以較高濃度的硼酸（300 mM Boric acid）用1N的NaOH將pH調至10.0 ，用Lysozyme 作為內在標準品，在注入已知量的結晶蛋白溶解液及定量Lysozyme 之混合液至毛細管電泳儀，經過10 kV，二十分鐘之電泳後，從電泳圖譜上結晶蛋白溶解液及Lysozyme形成的波峰面積之比率與結晶蛋白之濃度作線性迴歸相關性分析。其結果顯示相關係數為0.9994 ，斜率為0.4095 ，交叉點為+0.0025 ，利用此一公式，可用定量之Lysozyme 加入所要測試的結晶蛋白溶解液中作快速、經確之定量分析。本實驗用三種品系的蘇力菌（HD-1，A3-4，及YIM303）分析其發酵液中結晶蛋白之含量作為例證。