Purification And Characterization Of A Trypsin Inhibitor From The Seeds Of Artocarpus Heterophyllus Lam

Artocarpus heterophyllus Lam is not only edible fruit, but also widely used as the medicinal plant in disease prevention and treatment. Because of its excellent therapeutic value, increasingly more attention has been paid to A. heterophyllus Lam. The focuses of A. heterophyllus Lam study are active small inorganic molecules, while there is little attention on the active protein of A. heterophyllus Lam. In this study, the activities of gelatinase, trypsin, trypsin inhibition, antioxidant, catalase and casein hydrolysis in seed, pericarp and pulp were measured, It was found that the seed had gelatinase activity and strongly inhibitory effect of trypsin; pericarp had gelatinase activity; pulp had gelatinase, antioxidant activity and inhibitory effect of trypsin, however the activity was weaker than that of the other parts.The productivity of A. heterophyllus Lam is higher than other tropical fruits. The quantity of seed in A. heterophyllus Lam is approximately100-500while weight of seeds can make up8-15%of the whole fruit. In this paper, a proteinaceous inhibitor against trypsin named as AHLTI (A. heterophyllus Lam trypsin inhibitor) was isolated from the seeds of A. heterophyllus Lam by successive ammonium sulfate precipitation, ion-exchange and gel-filtration chromatography.AHLTI was consisted of a single polypeptide chain with a molecular weight of28.5kDa, which was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel-filtration chromatography. The N-terminal sequence of AHLTI was DEPPSELDAS, which showed no similarity to other known trypsin inhibitor sequences. AHLTI completely inhibited bovine trypsin at a molar ratio of1:2(AHLTI:trypsin) analyzed by native polyacrylamide gel electrophoresis, inhibition activity assay and gel-filtration chromatography. AHLTI owned two trypsin reactive sites. Moreover, kinetic enzymatic studies were showed that AHLTI was a competitive inhibitor with an equilibrium dissociation constant(Ki) of3.7×10-8M and exhibited potent inhibitory activity towards trypsin. However, AHLTI was able to inhibit chymotrypsin (15%inhibition) and elastase (6.2%inhibition) weakly compared with complete inhibition towards trypsin (100%). AHLTI was stable over a broad range of temperatures20-80℃. However, there was approximately75%decrease at100℃. Pre-incubation of AHLTI at the pH range of2.0-10.0had a slight effect on its inhibitory activity. Even at extreme conditions (pH2-3or9-10), the inhibitory activity of AHLTI retained60%. The reduction agent, dithiothreitol, had no obvious effect on AHLTI and only lost about15%activity when treated with the100mM DTT for2h. According to the stability of AHLTI, we deduced that the active sites of AHLTI are far away from the disulfide bridges, or it has low cysteine content.The result of bacteriostasis experiment suggested that AHLTI had no inhibition to Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Pseudomonas fluorescens and Xanthomonas Campestris. The trypsin inhibition assays of AHLTI towards digestive enzymes from insect pest guts in vitro demonstrated that AHLTI was effective against enzymes from Locusta migratoria manilensis (Meyen).These results suggest that AHLTI might be a novel trypsin inhibitor from A. Heterophyllus Lam belonging to Kunitz family. The purification and study of AHLTI were conducive to the basic use of protease inhibitors and A. heterophyllus Lam seeds.