【摘要】 目的:为了研究胰蛋白酶抑制剂的活性位点,揭示Ft TI结构与功能的关系。将Ft TI和突变体aFtTI-R65L,aFtTI-D67V和aFtTI-R65L/D67V经IPTG诱导培养5h,收集菌液经过超声波破碎得到粗产物,经过纯化后的胰蛋白酶抑制剂对胰蛋白酶的摩尔抑制比分别为1∶1,1∶1.15,1∶1.3,1∶1.2;抑制常数Ki分别为1.62n M,1.69 n M,1.9 n M,1.8 n M(BAp NA作为底物)。结果:SDSPAGE分析表明突变前和突变后表达产物胰蛋白酶抑制剂的大小一致,均为9.5 k Da。对突变体aFtTI-R65L,aFtTI-D67V和aFtTI-R65L/D67V抑制反应温度研究表明,其最适反应温度均为40℃。在10~80℃保温30 min后,突变体对胰蛋白酶的抑制活性仍保留80%以上;在90℃保温30min,突变体的抑制活性开始显著下降,只保留其39%。具有较高的耐热性。将aFtTI在pH 3.0~10.0的不同缓冲溶液中放置30 min后,其抑制活性可保留90%左右,在pH 2.0条件下,aFtTI抑制活性丧失约31%;在pH 11.0条件下,aFtTI抑制活性丧失约43%。结论:对苦荞麦蛋白酶抑制剂Ft TI的定点突变并不会改变它是一种偏碱性的胰蛋白酶抑制剂的性质,突变前后均保持了耐碱性的特点。更多还原

【Abstract】 Objective: In order to study the active sites of trypsin inhibitor and reveal the relationship between structure and function. Methods: The Ft TI gene from tartary buckwheat was mutated by site-directed mutagenesis technology and final three mutant strains R65 L,D67V,R65 L / D67 V were obtained. The expression products were isolated,purified and the inhibition activity measured. The mutants R65 L,D67V,R65 L / D67 V were induced for 5 h by IPTG,inhibition molar ratios are respectively 1 ∶ 1,1 ∶ 1. 15,1 ∶ 1. 3 and 1 ∶ 1. 2. The inhibition constants( Ki) are respectively 1. 62 n M,1. 69 n M,1. 9 Nm and 1. 8 n M( BAp NA as substrate).Results: The SDS-PAGE analyses of expression products showed that premutation and after mutation of trypsin inhibitor have the same molecular weight as 9. 5 k Da. The optimum temperature of mutants’ aFtTI-R65 L,aFtTID67 V and aFtTI-R65 L / D67 V is still 40 ℃. The thermal stability results showed that all three mutants have high heat resistance. After 10 ~ 80 ℃ for 30 min,aFtTI trypsin inhibitory activity remains more than 80%. After 90℃ for 30 min,the aFtTI inhibitory activity begin to decreased significantly and only reserved it ’s inhibitory activity about 39%. Thus,a FTt I has high heat resistance. aFtTI inhibitory activity could retain about 90%,after placed different buffer solutions( pH 3. 0 ~ 10. 0) for 30 minutes. Under pH 2. 0 conditions,the inhibitory activity of aFtTI loses about 31%. Under pH 11. 0 conditions,the aFtTI inhibitory activity loses about 43%.Conclusions: The experiment showed that site-directed mutagenesis of Ft TI did not change the alkaline property and maintained the resistance alkali characteristics with or without mutations.更多还原