| Native trypsin (NT) is likely subjected to various kinds factor in environment which cause a decrease of thermal stability and an increase of autolysis. Previous research indicated that the unfolded trypsin (DT) induced by dynamic high-pressure microfluidization (DHPM) treatment had enhanced thermal stability. However, the unfolded protein is in a transitional thermodynamically unfavorable state, it generally refolds or aggregates by interacting with other molecules. This subject was to study the effect of monomethoxy poly(ethylene glycol) activated by succinimidyl carbonate and succinimidyl succinate (mPEG-SC and mPEG-SS) on activity, thermal stability, autolysis, kinetic parameter and conformational change NT and DT.There was no significant difference in the relative activity of NT after modification by mPEG-SC with molecular masses1000,2000,5000,10000and20000Da. However, mPEG-SC modified NT showed an increase of thermal stability and effect to resist autolysis, and NT showed the best thermal stability and effect to resist autolysis after modification by mPEG-SC with molecular masses5000Da. After modification by mPEG-SC (5000Da), the relative activity of NT was increased from32.1%to79.4%. at50℃, and the relative activity of NT was also increased from42.3%to73.9%after incubation120min at40℃. In addition, the conformatinal change of NT was observed after modification. UV absorption and the fluorescence intensity was decreased accompanied by a slight blue shift, indicating the change of the microenvironments and gradual hide of hydrophobic group. CD spectra displayed an increase in the negative ellipticity, and there is an increase of β-structure content accompanied by a decrease in the random coil content. After modification by mPEG-SC (5000Da), the conformation of NT showed the most change. For example, the β-structure content of NT was increased from51.2%to56.7%after modication by mPEG-SC(5000Da).The effect of mPEG-SC (5000Da) on DT showed as follows, mPEG-SC (5000Da) modification on the activity of DT displayed no significance. However, mPEG-SC (5000Da) modification enhanced the thermal stability of DT and effect to resist autolysis. Meantime, mPEG-SC (5000Da) modified DT showed higher catalytic efficiency and affinity to substrate. After modification, the Km value had a decrease from0.35mM to0.15mM, and the Kcat/Km value had an increase from11.83s-1mW-1to20.13s-1mM-1. In addition, the conformatinal change of DT was observed after modification. UV absorption and the fluorescence intensity was decreased and the fluorescence emission peak had an obvious blue shift, indicating the change of the microenvironments and gradual hide of chromophore and hydrophobic group, and there is an increase of β-structure content accompanied by a decrease in the random coil content. The β-structure content of DT was increased from49.7%to58.4%after modication. Compared with NT, after modification, the characteristics of DT had more obvious improvement and the conformation had also more obvious change. Moreover, these observations indicated that there may be a correlation between enzyme properties and conformation change of modified trypsin.The conformational changes of mPEG-SC (5000Da) modified DT have been investigated during the denaturation by urea of different concentration. Results indicated that DT displayed a significant effect to resist the denaturation of urea after modification. As increase of urea concentration, the conformation of sample was gradually damaged. The structure of NT, DT and modified DT was extremely damaged at urea concentration6mol/L,4mol/L and8mol/L, respectively.The effect of mPEG-SS(5000Da) on the characteristics of NT and DT was studied. The results indicated that the relative activity of NT and DT was hardly changed after modification. However, compared with modified NT, the modified DT showed higher thermal stability and affinity to substrate and better effect to resist autolysis. Moreover, the effect of modification of mPEG-SC to NT or DT was better than mPEG-SS. |
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