Extraction Of Taro Protein And Its Trypsin Inhibitor

This thesis studies the effect of different solvents on composition and activity of taro extracts. Trypsin inhibitor was purified from taro, and its structure and stability were also analysised. This study provided theoretical foundation for the development and utilization of taro trypsin inhibitor and would expand trypsin inhibitor resources at the same time.Firstly, Zhangxi taro was taked as raw materials, water, salt, ethanol, tris and alkali solution were used as extraction agents to extract the protein from the taro, and protein, amino acid, total sugar, total phenol of the five extractives are measured. In order to get a better extraction method with which more active substance can be obtained, its antioxidant capacity, including hydroxyl radical scavenging capacity and ABTS· scavenging capacity, coagulating activity and trypsin inhibitory activities from the five extractives were compared. The results showed that protein, total sugar, phenolics exist in the five extractives; all these extractives had some ABTS· scavenging capacity and hydroxyl radical scavenging ability, and tris extractive had better effect than others; taro extractives showed agglutinating activity against rabbit erythrocytes but not against sheep erythrocytes. The comparation of specificity hemagglutination activity from the five extractives is salt extractive（3.24×10³ HU/mg）>tris extractive（2.88×10³ HU/mg）>alkali extractive（1.86×10³ HU/mg）>water extractive（1.41×10³HU /mg）>ethanol extract（0.98×10³ HU/mg）. Among the 5 extractives, ethanol extractive has no trypsin inhibitory activity, salt extractive, water extractive, tris extractive had a higher trypsin inhibitory activity, were 134 TIU/mg 130.51 TIU/mg, 113.30 mg/TIU, respectively. In conclusion, tris extractive contain more protein with higher activity among the five extractives, and has more protein bands than others by SDS-PAGE.Taro trypsin inhibitor with Electrophoretic purity was obtained by ammonium sulfate precipitation, DEAE Sepharose FF ion exchange column chromatography, Trypsin-Sepharose-4B affinity column chromatography, Sephadex G-100 gel column chromatography. SDS-PAGE showed that the molecular weight was 24 ku, HPGPC and MALDI-TOF-MS showed that the molecular weight is 36 ku with a single peak. the UV, FTIR, Raman and CD were used to identify of its structure, secondary structure, the results showed that the purified protein is mainly composed of β-sheet, random coil, with a small amount of α-helix, β-turn.Based on dynamic test, TTI was known as a non-competitive inhibitors, Ki=2.28×10^-8 mol/L << Km. Temperature had a significant impact on the activity and secondary structure of TTI, inhibitory activity of TTI remained about 10% when processed with 120 ℃ for 10 min, and the secondary structure of TTI changed significantly. TTI had strong acid and alkali resistance, inhibitory activities of TTI were over 80% when pH extended from 2 to 11, TTI reached the highest inhibitory activity at pH8. With ultrasonic treatment for 15 min, TTI still had about 70% inhibitory activity, and the secondary structure without obvious changes. DTT and BME reduced the inhibitory activities of TTI significantly and made its secondary structure change obviously. Denaturants processed TTI individually had not big influence on the activity of TTI, but the inhibitory activity of TTI would decreased significantly with the change of secondary structure when combined with heat treatment. Above all, TTI was stable under some factors, but its stability be significantly reduced under the condition of high temperature, strong reducing agents and denaturants, especially when combined with heat treatment.