Study On Tartary Buckwheat Trypsin Inhibitor Activity Sites By Using Site-directed Mutagenesis

The buckwheat belongs to serine protease inhibitors, which contain rich trypsin inhibitor. Buckwheat protease inhibitors can inhibit trypsin activity, having a high thermal stability and a certain degree of pH stability. Also it can lower blood pressure, blood fat, lowering blood sugar, resistant to bollworm and anti-fungal, anti-aging and anti-AIDS action. One kind of FtTI was separated in early stage of laboratory studies. Studies have that it can inhibit pumpkin vine blight strongly, chayote leaf blight, tomato early blight and anthracnose and other pathogenic fungal hyphae pepper growth.The important theoretical significance and practical value that depth study of the structure and function has been confirmed, because of it’s a functional protein. We replaced the arginine (65 site) and aspartic acid (67 site) by Leu. and Val. The amino acid sequence and active center region of active groups are researched. Next, FtTI activity center was analyzed by bioinformatics.The FtTI gene from buckwheat was mutated by site-directed mutagenesis technology and final three mutant strains R65L, D67V, R65L/D67V were obtained. The expression products were isolated, purified and inhibition activity.(1) After induced culture by IPTG from mutant R65L, D67V, R65L/D67V, the moore rejection ratio to trypsin is 1:1,1:1.15,1:1.3 and 1:1.2. The inhibition constants (Ki) is 1.62nM,1.69 nM,1.9 nMand 1.8 nM (BApNA as substrate). Theinhibitors vitality of mutation aFtTI-R65L, aFtTI-D67V and aFtTI-R65L/D67V have decreased by 13.04%, 23.85% and 16.67%.(2) The SDS-PAGE analysis of expression products showed that mutation premutation and after of trypsin inhibitor have the same size both 9.5kDa.(3) The mutant aFtTI-R65L, aFtTI-D67V, aFtTI-R65L/D67V has more high heat resistance and the optimum temperature (40℃) does not change significantly. The thermal stability results showed that all three mutants have high heat resistance. After 10-80℃ for 30 min, aFtTI inhibitor activity to trypsin 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, aFTtl has high heat resistance.(4) FtTI inhibitory activity could retain about 90%, after placed different buffer solutions (pH 3.0-10.0) for 30 min. Under pH 2.0 conditions, the FtTI inhibitory activity loss of about 31% and pH 11.0 conditions, the FtTI inhibitory activity loss of about 43%. It’s indicated that FtTI has a same feature with original trypsin inhibitor consistent, which are relatively stable under the strong acids and bases conditions. The FtTI has the maximum inhibitory activity in the pH 8.0. This study showed that FtTI mutagenesis of buckwheat inhibitors did not changed the alkaline nature and maintained the alkali resistance characteristics after mutations before and after.(5) Under high temperature and pressure (greater than 0.101MPaand 100℃) conditions, FtTI soon be passivated. FtTI lost its inhibitory activity of 96% and 95%, at 198MPa,120℃, at 0.143MPa,110℃ conditions for 20 min. However, ultrasonic inactivation FtTI shoed proportional to its amplitutude and duration action.In this study, lay the foundation for future study the relationship between structure and function of protease inhibitors, research of anti-pest plant gene transfer and development of anticancer drugs.