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

Abstract

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.