The Effect Of Dynamic High-pressure Microfluidzation On Enzymatic Properties And Conformation Change Of Trypsin And Bromelain

Dynamic high-pressure microfluidization (DHPM) is an emerging non-thermal technology, which uses the combined forces of high-velocity impact, high-frequency vibration, instantaneous pressure drop, intense shear, cavitation, and ultra-high pressures up to 200 MPa. This subject was to study the effect of DHPM on activity, reaction stability, and conformational change of trypsin and stem bromelain, and discuss the relationship between properties change and conformational change then.Trypsin was one of alkali protease, the optimum temperature and pH of trypsin was 35℃and pH7.6, respectively. It had been provided with good thermal stability and pH stability, after 100 min of incubation at 45℃, the relative activity of trypsin was 86%. The relative molecular mass of Trypsin was about 24 kDa by SDS-PAGE. The exposed SH contents and total SH contents were 6.21±0.4 and 12.7±0.22μmol/g, respectively. DSC analysis indicated that the denaturation temperature of trypsin was 93℃. The maximum UV and fluorescence absorption peak was at 280nm and 350nm, respectively.The effect of DHPM on the activity, reaction stability and conformation was showed as follows, DHPM treatment on the activity of trypsin showed no significance, with the relative activity of 98.5%(80 MPa),98.3%(100 MPa), and 97.8%(120 MPa). At 160 MPa which was considered as a reasonably high pressure, its relative activity was still as high as 97%. However, DHPM treatment enhanced the pH and thermal stability of trypsin. After 100 min of incubation at 45℃, the residual activity of trypsin treated at 80 MPa rised to 96%. The optimum pH of trypsin maintained surprising consistency (pH=7.6), nevertheless, its relative activity was about 102% and 103% at 100 and 120 MPa, respectively. In addition, DHPM-induced conformational changes of trypsin were observed. The increase in exposed SH contents and decrease in total SH contents suggested the trypsin was unfolded after DHPM treatment. This phenomenon was also reflected by FTIR spectra analysis, which showed a decrease in a-helix intensity. Besides, UV absorption spectra and fluorescence emission spectra change indicated the change of the microenvironments of the tryptophan and tyrosine residues and gradual exposure of hydrophobic groups initially buried in the interior of the molecules. Pymol was then used to model the conformation of native trypsin and unfolding trypsin, the loose structure of unfolding trypsin induced by DHPM was visually viewed.The effect of DHPM on the activity and stability of stem bromelain was studied. The results indicated that bromelain was inactivated by DHPM although the relative activity did not decrease with increasing pressure. The activity of bromelain was 94%, 93%,98%, and 92% under pressure of 60,80,100, and 120 MPa, respectively. The optimum temperature of bromelain treated under different pressure showed significant difference, whereas, the optimum pH value was not affected. However, the pH value stability of bromelain was greatly enhanced when bromelain was treated under 60 and 120 MPa. After incubation at pH 8.0 for 40 min, the relative activity of bromelain treated under 60 and 120 MPa was 85% and 82%, respectively, increasing by 14% and 11% in comparison with untreated bromelain.The effect of DHPM on the conformation change of stem bromelain was also analyzed:DSC analysis indicated that the absorb peak of bromelain disappeared after DHPM treatment. In addition, conformational change of bromelain induced by DHPM was reflected in the change of spectra. UV and fluorescence spectra indicated that DHPM induced the unfolding of bromelain and the change of microenvironments of the tryptoph and tyrosine residues. The secondary or tertiary structure of bromelain disturbed by DHPM was shown in FTIR spectra.