Study On Extraction,Separation And Purification Of Soybean Bowman-Birk Inhibitor

Trypsin inhibitor is widely consist in animals,plants and microorganism, generally, they are Polypeptide and Protein which can inhibite activities of trypsin. It can also great a dynamic balance with corresponding proteolytic enzymes to adjust many important life activities in the lives.It has been researched that there are five kinds of trypsin inhibitors in soybean, but only two kinds of them, Kunitz soybean inhibitor and Bowman-Birk soybean inhibitor were extracted.Study on extraction of rough BBI. At first we extracted BBI with ethanol from defatted soybean powder. Through the single factor experiment, we can get suitable condition scope of ethanol extraction. The conditions include concentrate of ethanol 40%~80%,fluid and material ratio(quality of Alcohol g/quality of defatted drying soybean powder g)4~6,time of extraction 30~60min,the temperature of extraction 30~60℃. through orthogonal experiments of four factors and three levels, the best conditions of ethanol extraction were determined. After ethanol extraction. Adjust pH to the isoelectric point of BBI, pH4.2. the trypsin inhibitor has the maximum precipitation at the isoelectric point. The acetone was added into it for precipitating the trypsin inhibitors in the liquor. Through the single factor experiment the best suitable quality of acetone can be determined. Then ultrafiltration was given. Through experiment the parameters of three kinds of ultrafilteration can be determined. They include pressure,time and temperature of ultrafiltration. Finally, vacuum drying was used to get rough BBI crysta。Study on the purification of the rough BBI. In this step, DEAE-25 ion-exchange chromatography and Sephadex G-50 gel filtration column were used. The pure BBI which we get through exmperiment and the standard BBI of Sigma were in comparison in trypsin inhibitory activity. The BAPNA method was used to determine the inhibitory activities of rough BBI, purified BBI and BBI of Sigma. By contrast, identify relationship between BBI in the quality and value of absorbance changes. And compare the purified BBI which got by experiment and BBI of Sigma.The experiments also were done on the stability of BBI. Separately changes affect BBI inhibitory activity conditions. To get BBI stability data. The factors that affect BBI hihibitory activity include temperature, pH, reductant. Do with BBI at different temperature. Meanwhile, taking BAPNA determination BBI inhibite trypsin changes in absorbance vaules. Through this , we can get the thermal stability of BBI. Preparation of different pH buffer handling BBI. pH3.0 by 1.0mol/L glycine-HCl buffer. pH4.0~6.0 by 0.1 mol/L citrate-sodium citrate buffer. pH7.0 by 0.1 mol/L sodium dihydrogen phosphate-two sodium phosphate buffer. pH8.0~9.0 by 0.1 mol/L Tris-HCl buffer. pH10.0~12.0 by 0.1 mol/L glycine- NaOH buffer. With 12 hours to observe the changes in the inihibitory activity. And then do with it again with the buffer pH8.0 for 12 hours. The pH change the inhibitory activity of BBI is reversible inhibition. At last adoption of the reductant DTT which influence BBI inhibitory activity to draw a conclusion that BBI has strong stability against DTT.Although the trypsin inhibitor of plant is essential to plant, and also has very important physiological funcitons. The trypsin inhibitor in feed which has the negative effects on the growth and development of livestock. Therefore, the study also exmines the BBI deactivation. Using ultrasonic method. Ultrasound frequency of 100kHz, in different time ultrasound treatment, determined the inhibitory activities of BBI.