The Influence Of Dynamic High Pressure Microfluidization On Activation And Conformational Changes Of Enzyme

Dynamic High-pressure microfluidization (DHPM) is an emerging nonthermal 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 new technology is based on a very high pressure capacity as well as on a new different reaction chamber geometry design, which can attain pressures 10-15 times higher than classical valve homogenizers. It operations where high pressures are experienced over very short times are different from static high pressures systems.The objective of this investigation was to determine the effect of DHPM on the activity and conformational changes of enzyme (polyphenoloxidase, trypsin and papain), of specific interest were activity, enzyme kinetics, storage stability and molecular conformation in the unfolding state.Polyphenoloxidase (PPO) from Chinese pear (Pyrus pyrifolia Nakai) was characterized using catechol as a substrate. PPO had a Vmax of 289.2 units/min and a Km of 3.8 mmol/L, which indicates that P. pyrifolia Nakai PPO has a great affinity for catechol. The catalyzing reaction velocity was proportional to the PPO concentration. The optimum pH and temperature for PPO activity were 4.5 and 45°C, respectively. In addition, an investigation was made on the effect of DHPM of treatment pressure, treatment pass and enzyme solution temperature on P. pyrifolia Nakai PPO. As the treatment pressure increased, the PPO relative activity was elevated from 100% untreated to 182.57% treated at 180 MPa. PPO relative activity was enhanced as the treatment pass increased. PPO solution temperature (25, 35, and 45°C) had a significant effect on PPO relative activity when treated at 120 and 140 MPa.DHPM could also lead mushroom PPO to activation. Treatment pressures had significant effect on the relative activity of mushroom PPO. The highest relative activity of 110.74% was exhibited after one pass treatment at the pressure of 110 MPa.When the enzyme solution was subjected to the pressure of 150 MPa 1, 2 and 3 passes, mushroom PPO was activated by 10.4%, 10.9% and 11.57%, respectively. The relative activity was 111.57% after treated three passes. The relasionship between activity and conformational change had also been investigated.The circular dichroism (CD) analysis demonstrated that some of secondary structures such asα-helix were destroyed. There were some indices that the increase of relative activity was accompanied by a decrease inα-helix content. The fluorescence emission spectra analysis indicated that Trp and Tyr residues in mushroom PPO were more or less exposed to solvent, and the result was in good agreement with that of UV absorption spectra analysis. The sulphydryl groups detection showed that the sulphydryl groups content on the surface of mushroom PPO was increased. We have found that the secondary conformation of mushroom PPO is changed by DHPM treatment and it is in an unfolding state.DHPM treatment on the activity of trypsin showed no significance; with the relative activity of 98.5% (80 MPa), 98.3% (100 MPa), 97.8% (120 MPa) and 97% (160MPa). 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 was still as high as 96% while the untreated trypsin retained only 86% of its original activity. The optimum pH of trypsin maintained surprising consistency (pH=7.6), nevertheless, its relative activity was about 97%, 102% and 103% at 80, 100 and 120 MPa, respectively. In addition, DHPM-induced conformational changes of trypsin were observed. The unfolding of trypsin induced by DHPM treatment was reflected in the increase in maximum emission fluorescence intensity and exposed SH contents as well as the decrease in total SH contents, UV absorbance andα-helix intensity.The unfolding trypsin induced by DHPM treatment had enhanced stability. However, according to thermodynamic hypothesis, the unfolding trypsin was in a transitional state and will refold or aggregate to be in the lowest energy state. In our opinion, patching the surface, doweling the exposed amino acid, or wedging in gap between amino acids all can stabilize the unfolding state. In this study, mPEG-SC was chosen to react with unfolding trypsin to patch the exposed surface. The result indicated that mPEG-SC showed significant effect on storage stability and thermal stability of DHPM-induced unfolding trypsin in comparison with that of native trypsin although the activity of both was not notably influenced. After storage at 4℃for 8 days, the activity of NT and NT_P remained 61% and 59.9%, respectively, the activity of D₈₀T_P and D₁₀₀T_P remained still 78% and 74% while the activity of D₈₀T and D₁₀₀T decreased sharply to 56.5% and 50%. As to thermal stability, D₈₀T_P and D₁₀₀T_P both remained still approximately 87% of the original activity at 55℃for 10 min in contrast the activity of D₈₀T and D₁₀₀T only remained 70%.Different explanations for thermal stability mechanism included change of surface hydrophobic/hydrophilic character and surface electric charge distribution, decrease in thermal denaturation and autolysis rate, formation of hydrogen bond, and lowering of the melting temperature of unfolding. The unfolding trypsin modified by mPEG-SC showed higher thermal stability might be due to the conformational change induced by the change of interactions between residues as well as residues and medium.Activity and conformational changes of papain treated by DHPM with M-chambers and L-chambers were observed. Both chambers resulted in the decrease in activity and the formation of SH group. Under the same condition of 160 MPa for three passes, the activity and total SH content of papain treated by M-chambers was 76.02% and 110.89%, while that of papain treated by L-chambers was 86.28% and 105.36%, respectively. Meanwhile, a violet-shifting of UV absorption peaks and red shift of maximum emission wavelength of Tyr and Trp residues were observed in papain treated by M-chamber, while the UV absorption and fluorescence spectra of papain treated by L-chambers remained unchanged. The phenomenon indicated that in spite of the same treatment pressures and passes, DHPM with different reaction chamber brought about different effect on the characteristics and conformational change of papain. The result proved what we call pseudo-pressure phenomenon.The three-dimensional structure of native papain was modeled by software of PyMOLWin based on the computational method of de novo structure prediction, and that of unfolding papain induced by DHPM with M-chambers was also predicted and simulated on the basis of conformational change we observed. Differences focused on more "loose" structure,rupture of S-S bonding,exposure of Tyr and Trp residues, and reduction ofα-helix content.