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| Study on Tartary Buckwheat Trypsin Inhibitor Activity Sites by Using Site-directed Mutagenesis |
| RUAN Jing-jun1,2, YANG Yi1, TANG Zi-zhong1, CHEN Hui1 |
1. College of Life, Sichuan Agricutural University, Ya'an 625014, China;
2. College of Biological Science and Technology, Wuhan Institute of Biological Engineering, Wuhan 430415, China |
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Abstract Objective:In order to study the active sites of trypsin inhibitor and reveal the relationship between structure and function. Methods:The FtTI gene from tartary 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 the inhibition activity measured. The mutants R65L, D67V, R65L/D67V 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 1.62nM,1.69 nM,1.9 Nm and 1.8 nM (BApNA 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 kDa. The optimum temperature of mutants' aFtTI-R65L, aFtTI-D67V and aFtTI-R65L/D67V 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, aFTtI 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 FtTI did not change the alkaline property and maintained the resistance alkali characteristics with or without mutations.
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Received: 20 July 2015
Published: 22 December 2015
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