Study On The Mechanism Of Trypsin Hydrolysis Reaction Enhanced By Ultrasound

The application of ultrasound to enzymatic reaction can increase enzyme activities and accelerate enzymatic reactions. In this thesis studied the effects of ultrasound treatment on the hydrolysis of casein by trypsin, the optimal parameters were determined: ultrasound power 225W, frequency 20KHz, ultrasound treatment time to pause time 1:1, total ultrasound time 10min. Under above-determined optimal conditions, the kinetics of enzymatic action were further studied. The temperature characteristics of trypsin-catalyzed reactions with ultrasound treatment was greatly changed. The hydrolysis rate were 54%and 73% at 5 and 20, increased about 4.4times and 3.3 times in treatment of ultrasound than the control (without ultrasound treatment). The optimal pH of trypsin was not affected by the ultrasound treatment. Km was decreased and Vm increased; the hydrolysis rate of different casein concentrate were all higher than the control. The hydrolysis rates with enzyme concentrations of 0.02% and 0.04% under ultrasound treatment were equal to those with enzyme concentrations of 0.08% and 0.2% without ultrasound treatment respectively.The molecular weight distribution of the hydrolysates were determined by high performance size exlusion chromatography( HPSEC). Three different samples were prepared under the reaction 10min with ultrasound, reaction 10min and 40min without ultrasound respectively. The results showed that ultrasound treatment didn't affect the specific property of trypsin, but the molecular mass distribution of casein tryptic hydrolysis were changed. Compared the samples obtained with ultrasound for 10min, the total peptides amounts were almost equal, but peptides of MW between 200 and 2000 increased by 15% in reaction without ultrasound for 40min. The amount of total peptides increased by 54.8% in reaction 40min without ultrasound than those of reaction 10min without ultrasound treatment.Finally the hydrolysis mechanism of casein under ultrasound by trypsin was investigated. UV absorption spectrum and fluorescence emission spectrum of trypsin and casein pretreated by ultrasound were measured, and the activation energy (Ea) were calculated by Arrhnius plot. The results suggested that the ultrasound treatment could decrease the activation energy of reaction of trypsin, enhance mass transformation, decrease the effect of inhibition of substrate and products, the ultrasound under optimum condition could increase the hydrolysis rate.