he biochemical analysis of sporamin for trypsin inhibitor!activity and its homology modeling

• Main Authors: Lin, Ya-Huei, 林雅慧
• Other Authors: Yeh Kai-Wen
• Format: Others
• Language: zh-TW
• Published: 1998
• Online Access: http://ndltd.ncl.edu.tw/handle/85870958859314541415

碩士 === 國立臺灣大學 === 植物學系研究所 === 86 === Sporamin, the major storage protein in the tuberous root ofsweet potato, had been found to have tryspin inhibitor activity.The expression of sporamin gene in transgenic tobacco can confer resistance against Spodopetra litura. This t hesis is focused on the biochemical analysis of sporamin for its trypsininhibi tor activity and on searching for its possible reactivesites. Sporamin is exp ressed in E. coli and then purified for the biochemical analysis. In the kinet ics analysis, sporamin belongs to mix-type inhibition.The thermo-stability of sporaminsis up to 65℃ for 10 minutes; its optimal reaction temperatureis in t he range of 35-45℃; its optimal reaction pH ranges from pH7-10. Sporamin can tolerence alcohol up to 30%. Low concentration (<7.5mM) of Cd++ and Mg++ can e nhance its abilityto inhibit trypsin activity; low concentration (<7.5mM) of E DTAcan decrease its ability to inhibit trypsin activity and thehigher concentr ation of Ca++ can lower the trypsin inhibitoryactivity Sporamin have 30% iden tity and 50% homology with soybean Kunitz type trypsin inhibitor. Based on seq uence alignment,secondary structure prediction, and protein data bank (PDB)thr ee-dimensional structure analysis, it is predicted that sporamin has a similar structure to Erythrina caffra trypsininhibitor DE-3 (ETI). Using the ETI stru cture as a template,the conformation of sporamin was predicted by homology mod eling.The result indicates that sporamin also has 12 beta-sheet like other Kun itz type trypsin inhibitors and the putativereactive site is located in a loop . However, the reactiveloop is composed of negative charge amino acid residue s whichis quite different with other trypsin inhibitors