Study On Purification And Passivation Mechanism Of Trypsin Inhibitor Of Soybean "Huaxia 3"

"Huaxia 3"soybean?HX3?is a new high-quality soybean,and anti-nutritional factors such as trypsin inhibitor?TI?will weaken the nutritional value of soybean.At present,there is no report on the study of HX3 trypsin inhibitor?HX3 TI?.Therefore,this study used HX3 TI as a research object to study it.Firstly,the coarse separation of HX3 TI was performed by sulfuric acid extraction.The recovery rate of one-step salting out was?26.98±0.00?%,and the purification multiple was?3.31±0.08?times;the recovery rate of two steps of salting out was?26.05±0.12?%,and the purification factor was?3.25±0.04?times.From the consideration of comprehensive resource conservation,the preliminary purification of HX3 TI by salting out would be performed using 0⁴5%ammonium sulfate concentration.The HX3 TI salt solution was further purified using four column methods:?1?Purification by DEAE-52 column chromatography,the HX3 TI purification factor is 3.60times.?2?Purification by Sephadex G-75 column chromatography,the HX3 TI purification factor was 3.14 fold.?3?Sephadex G-75 and DEAE-52 column chromatography were combined to separate and purify.The purification factor was 5.26 times.?4?DEAE-52 was combined with Sephadex G-75 column chromatography to separate and purify.The purification factor was 7.30 times.The experimental data showed that the two-step purification was more advantageous,and the best purification effect was the combination of DEAE-52 and Sephadex G-75 column chromatography.The electrophoretic detection of the HX3 TI with the highest purification factor was performed by three electrophoresis methods:PAGE,Gelatin-PAGE and SDS-PAGE.?1?PAGE electrophoresis showed that there were more miscellaneous proteins in the HX3 TI salting solution lane;there was only one electrophoresis band in the purified sample lane,indicating that the effect of the purified HX3 TI was significant and there was no impurity protein band.?2?The specificity of Gelatin-PAGE electrophoresis was used to detect the activity of trypsin inhibitor.There were 5 bands in the salting solution lanes,indicating that there were 5 kinds of HX3 TI species,in which the electrophoresis bandwidth and dark staining were high,indicating that the activity of HX3 TI was high.Only one band was detected in the column chromatographically purified sample lanes,indicating that there was only one type of purified HX3 TI.?3?The molecular weight of HX3 TI was determined by SDS-PAGE electrophoresis to be about 20 kDa.Then the influencing factors of HX3 TI's passivation were discussed.At a metal ion concentration of 0.8 mol/L and heating at 100°C for 15 min:Compared with NaCl and AlCl₃,the activity of HX3 TI in CaCl₂ decreased by 13.28%and 11.49%,respectively,and the residual rate also decreased by 13.28%and 11.47%,respectively,indicating that metal ions had a greater influence on the passivation of HX3 TI.And Ca²⁺had more obvious effect on HX3 TI passivation.Under the conditions of 0⁰.5 mol/L CaCl₂,the HX3 TI residual rate showed a U-shaped change,of which 0.2 mol/L CaCl₂ had the best effect on HX3 TI,and the residual rate was only 27.04%without CaCl₂.Further analysis of the inactivation of HX3 TI by Arrhenius equation shown that CaCl₂ makes HX3 TI passivated in accordance with y=b1 e^-b2x+?1-b1?e^-b3x equation and the inactivation energy was104.15 kJ In addition,the inactivation energy without CaCl₂ was 150.61 kJ/mol,and the energy required for adding CaCl₂ was 46.46 kJ/mol lower than that without CaCl₂.Therefore,Ca²⁺significantly reduces the energy required for the inactivation of HX3 TI to achieve rapid inactivation of HX3 TI.The influence factors of alkaline protease passivation HX3 TI were further discussed.?1?The optimal conditions for single factor experiments were:pH 10,65°C,50 U/mL enzyme dosage and 5 h action time.?2?The order of primary and secondary influences of each factor in the orthogonal test L₉?4³?is:enzyme dosage>temperature>pH>action time.The optimal inactivation conditions were:75 U/mL enzyme dosage,65°C,pH 9,5 h action time,HX3 TI's residual rate was 0.5%.?3?The best passivation conditions for the response surface analysis were:69.01 U/mL enzyme dosage,65.59°C,pH 9.56,6.21 h action time,HX3 TI residual rate was the lowest,0.33%.?4?In the response surface analysis,the HX3 TI residual rate was reduced by 10.58%over the commercial soybean TI,indicating that the HX3 TI is more easily passivated than the commercially available soybean TI.