Degradation Mechanisms Of Soybean Protein B₃ Subunit In Soy Sauce By Prolyl Endopeptidase And Ultrasonic-assisted The Enzyme

Soy sauce is apt to form a mud-like precipitate at the bottom of the bottle during storage and sale,which is the secondary precipitate of soy sauce.The secondary precipitate seriously affects the appearance quality and market competitiveness of soy sauce.Previous studies have proved that the B₃subunit of soy globulin G₄is the main ingredient of secondary precipitate protein and inducer of soy sauce secondary precipitate.Meanwhile,previous studies also proved that the prolyl endopeptidase from Aspergillus niger can degrade the B₃subunit in soy sauce.In this study,the protease capable of degrading B₃subunit in soy sauce was isolated and identified from Aspergillus niger,and the degradation mechanisms of B₃subunit in soy sauce catalyzed by the enzyme and ultrasound assisted the enzyme were clarified.Firstly,a prolyl endopeptidase（APE）was isolated and identified from Aspergillus niger using buffer extraction,ammonium sulfate precipitation,cation and anion column chromatography,test tube centrifugation,SDS-PAGE and MALDI-TOF/TOF-MS.APE was a salt-tolerant serine protease with a molecular weight of about 62 k Da.The optimal p H value and temperature were 4.0 and 40℃,respectively.The activity of APE did not change significantly in 3 M Na Cl solution in the first 3 days,and then decreased,but maintained more than 69%relative activity within 10 d.Result of bioinformatics tools analysis showed that B₃subunit contained10 proline residues and was a potential substrate of APE.Secondly,2 U APE/g koji was added to the soy sauce moromi at 20 d during fermentation.The relative content of B₃subunit in soy sauce was determined using SDS-PAGE and MALDI-TOF/TOF-MS,and the composition of free amino acids in soy sauce was determined by using HPLC.The results showed that APE could degrade approximately 50%of B₃subunit,reduce 61%of soy sauce secondary precipitate and increase proline content by 24%in soy sauce simultaneously.Sensory evaluation showed that the addition of APE had no significant effect on soy sauce taste.Thirdly,the mechanism of APE catalyzing the degradation of B₃subunit in soy sauce fermentation was studied by combining experiments and computer simulation.The results of circular dichroism and intrinsic fluorescence spectra showed that the secondary and tertiary structures of APE were not affected in high salinity environment for 3 days,thereafter the structure of APE changed significantly,which was consistent with the changes of APE activity.The variation of RMSD,Rg,RMSF,active centers,intermolecular and intramolecular hydrogen bonds and salt bridges of APE in 0 M and 3 M Na Cl solutions were studied by molecular homology modeling,molecular dynamics simulation and molecular docking.The results showed that 3 M NaCl had no significant effect on the active center,hydrogen bond,salt bridge and internal hydrophobicity of APE,which was the reason for the strong stability of APE spatial structure and activity in 3 M Na Cl solution.However,3 M Na Cl solution enhanced the rigidity of B₃subunit and reduced the hydrogen bond between APE and B₃subunit,indicating that the steric hindrance caused by 3 M Na Cl weakened the affinity of between APE and B₃subunit,leading to the lowering of catalytic activity of APE on B₃subunit,which was the reason why APE could not degrade B₃subunit completely during soy sauce fermentation.Finally,based on the preliminary experiments,the optimal ultrasonic conditions（ultrasonic frequency 68 k Hz,ultrasonic intensity 60 W/L,ultrasonic time 10 min/time,a total of 8 ultrasonic times,10 s stop 5 s each ultrasonic time）were selected to study the ultrasonic assisted APE catalytic degradation effect on B₃subunit during soy sauce fermentation.The results showed that ultrasonic assisted APE could degrade85.6%of B₃subunit and reduce 91%of secondary precipitation in soy sauce,indicating that ultrasonic assisted APE could further improve the degradation rate of B₃subunit and reduce the secondary precipitate in soy sauce.The results of circular dichroism analyses showed that ultrasound decreased the ordered structures of APE and increased the disordered structures,indicating that ultrasound increased the molecular flexibility of the enzyme and substrate.Intrinsic fluorescence analyses showed that ultrasound enhanced the fluorescence intensities of APE and B₃subunits,suggesting that some hydrophobic amino acid residues buried in interiors of the enzyme and substrate might be exposed to the molecular surfaces.It was reasonable to speculate that ultrasound destroyed the‘dense’ordered structure of APE and exposed the restriction sites.Meanwhile,ultrasound might enlarge the substrate channel of enzyme and expose the enzyme active center,reduce the steric hindrance between substrate and enzyme activity,and enhance the degradation activity of APE on B₃subunit.In this study,it was the first time to report that APE had strong salt tolerance and the ability to degrade B₃subunit in soy sauce and reduced the secondary precipitate of soy sauce.Meanwhile,the mechanism of the salt tolerance of APE and degradation of B₃subunit in soy sauce were clarified,which provided a new method and theoretical basis for solving the problem of secondary precipitate of soy sauce.