Preparation Of Anticoagulant Peptides From Tenebrio Molitor By Enzymatic Hydrolysis And Exploration Of Its Mechanism

Tenebrio molitor is referred to as the"treasure of protein"due to its abundant protein resources.In this study,the Tenebrio molitor as the raw material was carried out with controllable enzymatic hydrolysis.The bioactive peptide with anticoagulant properties was isolated and purified from the hydrolyzate.The identified anticoagulant peptide was synthesized and its thrombin inhibition mechanism was investigated.The anticoagulant peptide was modified to enhance its thrombin inhibitory activity,and the inhibition mechanism was explored.The details are as follows:Tenebrio molitor larvae were hydrolyzed by pepsin and trypsin for preparing high anticoagulant activity hydrolysate.The significant variables were selected according to the Plackett-Burman design and further optimized by the Response Surface Methodology（RSM）.Four factors,including substrate concentration,pepsin digestion time,pepsin amount and trypsin amount,respectively,influenced significantly on the hydrolysis effectiveness.The best hydrolysis conditions obtained using RSM experiments are as following:substrate concentration of 19.8 mg/m L,pepsin digestion time of 1.8 h,pepsin amount of 1634 U/m L and trypsin amount of126 U/m L,respectively.Under the optimal hydrolysis conditions,the anticoagulant activity was 82.8%predicted by the RSM model,which was basically consistent with the 80.6%obtained in the three verification experiments.Moreover,the anticoagulant activity of the hydrolyzate had no linear relationship with the degree of hydrolysis.The hydrolysate was subjected to ion exchange chromatography gel filtration chromatography;the obtained anticoagulant activity values of the fractions were40.87%and 65.61%at 8.0 mg/m L,respectively.After further preparation by reverse-phase liquid chromatography,the peptides with anticoagulant activity of28.66%at 0.2 mg/m L were identified by liquid chromatography tandem mass spectrometry as SLVDAIGMGP and AGFAGDDAPR.The identified peptide AGFAGDDAPR（P2）was proved that had thrombin inhibitory activitywith IC₅₀ value of 163μM.In this study,the interactions of P2 with thrombin were investigated by fluorescence spectroscopy,UV-vis spectroscopy,circular dichroism,Fourier-transform infrared spectroscopy and Raman spectroscopy,respectively.The results showed that P2 bonded to thrombin via hydrogen bonding or van der Waals forces,resulting in fluorescence quenching.And,the secondary structure of thrombin changed,theβ-sheet decreased,and the random coil increased.P2 bonded to proline and lysine,and changed the space structure of thrombin,resulting in inhibition of thrombin activity.The molecular docking of with thrombin by Discovery studio had shown the best pose with“-CDOCKER ENERGY”of135.97 kcal/mol.The optimal docking mode of synthesized peptide P3（N-RGDAGFAGDDAPR）and thrombin was revealed by molecular docking with“-CDOCKER ENERGY”of178.68 kcal/mol.It was expected to be an inhibitor with higher antithrombotic activity.The results showed that the IC₅₀ of the P3 was determined by colorimetric method to be 115μM.And enzyme kinetic experiments showed that P3 was a competitive inhibitor of thrombin with K_i=106μM.Fluorescence spectra and three-dimensional fluorescence showed that peptide could alter the secondary structure of thrombin and the microenvironment of certain chromogenic amino acids.P3 can spontaneously bind with thrombin exosite 1 in the form of 1:1 mainly through hydrogen bonding or van der Waals force.Molecular dynamics simulations showed that thrombin-peptide complex in a stable state under the solvent-added environment,which meaned that P3might be used as a potent inhibitor of thrombin.The controllable enzymatic hydrolysis was used to obtain Tenebrio molitor anticoagulant peptide in this study,which provided a new idea for the source of anticoagulant drugs,and also enhanced the comprehensive utilization of Tenebrio molitor resources.