Purification, Characterization And Biofunction Analysis Of TBTI-Ⅱ From Tartary Buckwheat

Proteinase inhibitor is widely distributed in nature including many kinds of animals, plants and microorganisms. It plays important roles in maintaining the interaction balance of enzyme proteolysis in vitro and in vivo. According to the different enzymes inhibited, the inhibitors are generally classified into four types: serine proteinase inhibitor, cysteine proteinase inhibitor, asparagine proteinase inhibitor and metalloproteinase inhibitor.Plant proteinase inhibitor is present in almost every kind of plant, and mainly concentrated in plant seeds and tubers. Researchers have identified it as a defense protein against insects and fungi attacks as well as a possible anti-nutritional factor.Buckwheat originates in the southwest of China and attracts popular attention in Asia, Europe and other areas in the world. It is special in it high contents of proteins and antioxidant substances. Due to the wider availability of common buckwheat, many researchers pay much more attention to it than tartary buckwheat.In this paper, we obtained a purified trypsin inhibitor from the seeds of tartary buckwheat (TBTI-II) and analyzed its physiochemical properties on its molecular weight, pI, thermostability, amino acid composition, and inhibition kinetics. The results showed that with a MW of about 9.39kDa, TBTI-II contained high contents of acid and basic amino acids and low content of Ala. Its pI was about 6.55-6.85 and it had high tolerance against heat treatment. The inhibition constant of TBTI-II Ki was measured as 1.93 10-4M. Comparing with other reports on buckwheat proteinase inhibitors, we concluded that TBTI-II had similar properties with the temporary proteinase inhibitor and may belong to Potato Proteinase inhibitor-I family.In order to explore the anti-insect activity of TBTI-II, we mixed different concentrations of 60U, 140, 200U of it into 500g artificial diet per (wet weight) for Helicoverpa armigera. By comparing the insect growth rate of the control and sample insects, it demonstrated that retarding effect of TBTI-II on the growth of larva was significant but not proportional to the dosage of TBTI-II. This is related with the complicated changes of the digestion system in the larva midgut.Above all, we isolated a small trypsin inhibitor from tartary buckwheat (TBTI-II) and investigated its biochemical and physiochemical properties. The research also found that it had significant inhibitory effects on the larva growth. This work is to enrich the scientific research of tartary buckwheat, and it will be useful for further studying buckwheat in the fields of nutrition, medicine and plant self-protection and so on.