Purification and Trypsin Inhibitor Activity of a Sporamin B from Sweet Potato (Ipomoea batatas Lam. 55-2)

Sporamin is a soluble protein in sweet potato, and falls into two distinct homology groups, subfamilies A and B. In this research, a sporamin B was purified and its amino acid sequences, trypsin inhibitor activity (Ti activity) were analyzed. This sporamin B was isolated from sweet potato tubers [Ipomoea batatas (L.) Lam cv. 55-2] through extraction of the water-soluble fraction, dialysis, ultrafiltration and ion-exchange chromatography. Homology determined by polyacrylamide gel electrophoresis showed that mainly one bond appeared in gel after being reduced by SDS (sodium dodecyl sulfate), or by SDS and 2-mercaptoethanol, or in native situation. By comparing the data of the polypeptide mass Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry with those of the mass of the theoretical amino acid sequences from NCBI protein database, it was revealed that it was Q40091|Q40091_IPOBA, sweet potato sporamin B – Ipomoea Batatas (sweet potato) (Batate). The sequence coverage was 70.6%. N-terminal sequence was SETPV (Ser-Glu-Thr-Pro-Val). There is a linear relationship between trypsin inhibitor activity (Ti activity) and amounts of this sporamin B (3-18 μg mL−1). The equation of linear regression was y = 2.5809x + 17.049 (r2 = 0.9966). There was a curvilinear relationship between Ti activity and amounts of this sporamin B (21–150 μg mL−1). The equation of curvilinear regression is y = 14.417ln(x) + 23.26 (r2 = 0.9924). The concentration of sporamin B with Ti activity after heating at 40°C may induce part denature of this sporamin B, and there was no statistic difference after heating at 40, 50, 60°C for 20 min. Heat treatment at more than 90°C leads to a dramatic decrease of trypsin inhibitor efficiency. The results suggested that Q40091|Q40091_IPOBA was the major sporamin B in sweet potato tubers [Ipomoea Batatas (L.) Lam cv. 55-2], which had strong Ti activity, and was stable to both thermal and DTT (DL-dithiothreitol) relatively.