| After the enzyme is stimulated by the outside,a transition state will be appeared.The intermediates are a relatively stable structure in the transition state.This structure is looser relative to the structure of the natural state,and relative to the completely degenerate structure is more orderly,which is a more stable and highly active state.Enzymatic hydrolysis is an important biochemical reaction in the food industry.Ultrasonic treatment is one of the important measures to improve the enzyme activity.In this paper,trypsin and pepsin were used as research objects,the urea-induced trypsin and pepsin degeneration were set as control.Using the transition state theory,the effects of different modes of ultrasound such as energy-gathered,slit divergent and plate divergent ultrasound,on the protease induced to activate and its intermediate formation were studied.The relationship between the changes of enzyme activity and the folding intermediates was studied.The thermal stability,secondary structure and thermodynamic parameters of the protease intermediates were analyzed to reveal the mechanism of changes in protease activity.The main research work and the results are as follows:(1)The effects of different modes of ultrasound on the structure and activity of trypsin were studied.After trypsin was treated by urea for 24 h with concentration of 0-8 M,by energy-gathered ultrasound for 5 min at power density of 0-7×103 W/L and by slit divergent ultrasound for 20 and 30 min at power density of 20-180 W/L,each treatment respectively found a folded intermediate of 1T,2T,5T and 6T during the induction process,and the enzyme activity was 30.3%,21.6%,31.8% and 31.2%higher than that of the control,respectively.After trypsin was treated by energy-gathered ultrasound for 7.5 min,two folding intermediates of 3T and 4T were respectively found during the induction.The activity of former was 14.1% lower than those of control,and later increased by 21.6%.Slit divergent,energy-gathered ultrasound respectively for 10 min and plate divergent ultrasound for 10,20,30 min cannot induce trypsin intermediates.So the activity of trypsin intermediate 5T was the highest when the slit divergent ultrasound treated for 20 min.(2)The effects of slit divergent ultrasound frequency and their combination on the structure and activity of trypsin were studied.During trypsin treated by slit divergent ultrasound for 20 min at mode of single frequency of 28,33 and 40 k Hz at power density of 20-180 W/L,a folded intermediate was respectively found during the induction,which was 5T,7T and 8T,the enzyme activity respectively increased by31.8%,42.4% and 32.0%.The best frequency of 33 k Hz was selected.Based on 33 k Hz,trypsin treated by slit divergent ultrasound for 20 min at frequency combination of 33/28 and 33/40 k Hz at power density of 20-180 W/L,a folded intermediate with high enzyme activity was respectively found,which was 9T and 10 T,the enzyme activity respectively increased by 44.5% and 28.1%.Slit divergent ultrasound at single-frequency of 20,23,25 k Hz and frequency combination of 33/20 and 33/25 k Hz cannot induce intermediates.So the activity of trypsin intermediate 9T induced at33/28 k Hz was the highest.(3)The thermostability,secondary structure and thermodynamic parameters of the five trypsin intermediates were analyzed.The enzyme activity,the residual enzyme activity and enzyme activity residual rate of the five intermediates were significantly improved compared with the control.The enzyme activity of the intermediate 9T was the highest,and the thermal stability was the lowest.The thermal stability of intermediate 8T is the best.Except the intermediate 7T,the secondary structure of the remaining trypsin intermediates was significantly different from the control.The thermodynamic parameters indicated that for all the intermediates,the reaction rate of was significantly increased,the heat absorbed by the reaction was significantly reduced,degree of confusion in structure was significantly reduced,and degree of difficulty in reaction was not significantly different from that of the control.The thermodynamic parameters changes of the intermediate 9T were the most significant.(4)The effects of different modes of ultrasound on the structure and activity of pepsin were studied.After pepsin was treated by urea for 24 h with concentration of 0-8 M,and by slit divergent ultrasound for 20 and 30 min at power density of20-180 W/L,two intermediates were respectively found during induction,whichV respectively were 1p?2p?5p?6p?7p and 8p.For two intermediates activity induced by urea,the former increased by 21.0% and the later decreased by 15.3%.For four intermediates induced by slit divergent ultrasound,the activities respectively increased by 41.7%,27.1%,22.3% and 17.2%.After pepsin was treated by energy-gathered ultrasound for 5 and 7.5 min at power density of 0-7×103 W/L,a folded intermediate respectively found,which were 3p and 4p,the activity was increased by 27.8% and 20.9%,respectively.Slit divergent,energy-gathered ultrasound respectively for 10 min and plate divergent ultrasound for 10,20,30 min cannot induce pepsin intermediates.The activity of pepsin intermediate 5p was the highest when the slit divergent ultrasound treated for 20 min.(5)The effects of slit divergent ultrasound frequency and their combination on the structure and activity of pepsin were studied.During pepsin treated by slit divergent ultrasound for 20 min at mode of single frequency of 25 and 33 k Hz at power density of 20-180 W/L,a folded intermediate was respectively found during the induction,which was 9p and 10 p,enzyme activity of the former increased by27.6%,the later decreased by 12.7%,so the best frequency of 28 k Hz was selected.Based on 28 k Hz,pepsin treated by frequency combination of 33/28 and 33/40 k Hz,two folded intermediates were respectively found,which were 11p?12p?13p and 14 p,the enzyme activity respectively increased by 43.1%?33.8%?62.7% and 32.3%.Slit divergent ultrasound at single-frequency of 20,23,40 k Hz and frequency combination of 28/20,28/23 and 28/40 k Hz cannot induce intermediates.So the activity of pepsin intermediate 13 p induced at 28/33 k Hz was the highest.(6)The thermostability,secondary structure and thermodynamic parameters of the eight pepsin intermediates were analyzed.The enzyme activity,the residual enzyme activity and enzyme activity residual rate of the rest of pepsin intermediates except the intermediate 10 p,were significantly higher than that of the control.The intermediate 13 p enzyme activity is the highest,the third highest thermal stability.The thermal stability of the intermediate 11 p is the best.The secondary structure of intermediates 10 p,12p and 14 p was significantly different from the control,but the other pepsin intermediates were not.The thermodynamic parameters indicatedthat for all the intermediates,the reaction rate of was significantly increased,the heat absorbed by the reaction was significantly reduced,degree of confusion in structure was significantly reduced,and degree of difficulty in reaction was not significantly different from that of the control.The thermodynamic parameters changes of the intermediate 13 p were the most significant. |
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