mPEG-SS修饰对DHPM去折叠胰蛋白酶相对活性、热稳定性和动力学参数的影响

Effect of mPEG-SS modification on relative activity,thermal stability and kinetic parameters of DHPM unfolding trypsin

采用ω-琥珀酰酯酸琥珀酰亚胺酯活化的α-甲氧基-聚乙二醇(mPEG-SS),修饰经动态高压微射流(Dynamic high-pressure microfluidization,DHPM)诱导的去折叠态(unfolding)胰蛋白酶,研究经mPEG-SS修饰后其相对活性、热稳定性、动力学(米氏常数Km、最大反应速率Vmax)变化情况。结果表明:经mPEG-SS修饰后,天然态和去折叠态胰蛋白酶的相对活性都没有明显改变;修饰后去折叠胰蛋白酶(DTP)较天然胰蛋白酶(NTP)的耐热性有明显提高,当温度为55、45℃条件下保温180min,NTP和DTP的相对活性分别提高了2%、7%和19%、30%;经mPEG-SS修饰后天然胰蛋白酶和去折叠胰蛋白酶的Km分别为4.67、3.24mg/mL,Vmax分别为0.042、0.034U/min,均比未经修饰的要小。修饰后去折叠胰蛋白酶(DTP)的Km比天然胰蛋白酶(NTP)小,表明修饰后去折叠胰蛋白酶比修饰后天然胰蛋白酶具有更强的与底物(酪蛋白)的亲和能力。

DHPM unfolding trypsin was modified by mPEG-SS. The relative activity,thermal stability and kinetic parameters（Km, Vmax）of trypsin were measured. Results showed that mPEG - SS modification did not change the relative activities of native and DHPM unfolding trypsin. mPEG-SS modification enhanced the thermal stability of unfolded trypsin more obviously than native trypsin. When incubated at 55℃ or incubated at 45℃ 180min,the values of mPEG-SS modification enhancing the relative activity of native and unfolded trypsin were 2%,7% and 19%,30% respectively. The Km and Vmax of native and unfolded trypsin modified by mPEG-SS were 4. 67,3. 24mg/mL and 0. 042,0. 034U/min respectively,which were lower than native and unfolded trypsin. The Km of unfolded trypsin modified by mPEG-SS was lower than native trypsin modified by mPEG-SS,which meant that the former would affine casein easier than later.