Purification And Bioactivities Of Trypsin Inhibitor From Black Soybean

Trypsin inhibitor,TI is a widespread active material in nature,which has excellent effects on anti-fungal and plant resistance to diseases.People pay more and more important attention to their health functions and applications in the field of anti-cancer,especially the recent discoveries indicate the contact between cancer and TI.Beans are an important source of Trypsin inhibitor,while black soybeans not only are an important part of the beans,but also a well-known healthy food.But the current researches about Trypsin inhibitor mainly focus on soybean and mung bean,and there are not much research reports about Trypsin inhibitor in black bean,so it is necessary to deep into Trypsin inhibitor in black soybean.This thesis aims at the extraction,purification and identification of its activity of Trypsin inhibitor in black soybean.Methods:crush and defat black beans by petroleum ether,conduct 40%-60%ammonium sulfate fractionation,then pass through desalting column,and further purify by Sephacry1TMS-100HR gel column and DEAE-FF anion column,test by enzyme assaya and collect the target protein.Their molecular weight is determined by SDS-PAGE gel electrophoresis and mass spectrometry and the protein concentration by Lowry method.The study of physicochemical properties of inhibitors includes:temperature,pH,metal ions' influence on the inhibitor activity of Trypsin inhibitor.By Lineweaver-Burk plot method and Dixon mapping method can we realize the kinetic characterization of Trypsin inhibitor and determine the type and the potency of it.Characterization of the biological activity of Trypsin inhibitors can be divided into two aspects:experiments of inhibiting proliferation of cancer cells and anti-fungal experiments.Results obtained are as follows:1.Black soybean Trypsin inhibitor?BSTI?having chitinase activity,is obtained by the purafication of black beans,then by SDS-PAGE and mass spectrometry can we find out a molecular weight of 20.3 kD and pI value of 4.6.The homology analysis of database shows that homology between soybean Trypsin inhibitor and it reaches 49%and the protein concentration measured by Lowry is 0.409 mg/ml.2.BSTI has good thermal stability,whose its inhibitory activity is still more than 80%while processing 1 h at 100?;BSTI are relatively stable under acidic and neutral conditions,but its inhibitory activity gradually decreases with the increase of pH under alkaline conditions;Fe³⁺,Mn²⁺,Ca²⁺would inhibit BSTI activity,and Mn²⁺ has the most obvious inhibitory effect;Fe²⁺,Zn²⁺,K+,Mg²⁺ would activate the inhibitor,and Mg²⁺ the strongest;according to Lineweaver-Burk plot method,the linear equations derived from the reaction between trypsin and BApNA are y = 13867x + 101.1,Km =137.127 mmol/L.The same Km' is obtained by the reaction between the trypsin and substrate as the existence and reaction between Km and BSTI,from which BSTI can be determined as a noncompetitive inhibitor type;by Dixon mapping method can we learn the kinetic linear equation of BSTI y =19725 x+173.57 and the inhibition constant Ki = 8.8×10-3 mmol/L.Ki is much less than Km,which indicates that BSTI has a strong Trypsin inhibitor activity.3.By the initial screening of MTT can we find that the inhibitory effect is not obvious to mammary cancer MCF-7 cells when the concentration of BSTI is at 0-150?g/mL;the inhibitory effect on MCF-7 appears and shows a concentration-dependence when the concentration above 200?g/mL;the inhibitory ability to HepG2 cells appears when the concentration at 250?g/ml.Above preliminary results show that BSTI has a certain inhibitory effect on the proliferation of MCF-7 and HepG2 cells when its concentration reaches above 250?g/mL.By anti-fungal assay can we find that BSTI is significantly inhibited due to the growth of Trichoderma and Fusarium oxysporum.This paper aims at the extraction and purification of a kind of black bean Trypsin inhibitor and the measurement of its physical properties and the text of its biological activity.The research result accumulates the scientific research and production experience about the later study of black bean Trypsin inhibitor and its industrial applications.