Extraction, Isolation And Purification Of Trypsin Inhibitors From Leguminosae Beans

Trypsin inhibitors are a series of proteins that can inhibit the activity of trypsin.They can cause not only the death of phytophagous insects, but also possess manyimportant bioactivities like anti-cancer and anti-radiation, which make them ahot-spot of research. China has abundant resources of leguminous plants, which areespecially rich with protease inhibitors. Therefore, the extraction and isolation oftrypsin inhibitors from leguminous plant seeds are of great significance consideringthe development of plant anti-nutritional factors. In the present study, the trypsininhibitory activities of15different species and origins of Leguminosae sp. beanswere measured. The distribution of trypsin inhibitors in different parts of beans wasalso studied. Four Phaseolus beans were selected and their trypsin inhibitors wereisolated and purified. Afterwards, the thermo stability of these trypsin inhibitorswas studied.Results showed that the activities of trypsin inhibitors from glycine seeds weresignificantly higher than those from vigna seeds. While Phaseolus seeds weregenerally higher than vigna seeds and lower than glycine seeds in the activities oftrypsin inhibitors. Geographic origins also had an effect on trypsin inhibitors.What’s more, cotyledon was found to have the highest protein content, specialactivity and total activity of trypsin inhibitor while seed coat was lowest in all thethree indexes, indicating that trypsin inhibitors mainly exist in the cotyledon ofseeds.In addition, black kidney bean, light speckled kidney bean, snail flower kidneybean and large white kidney bean were select for further isolation of their trypsininhibitors. Ammonium sulfate precipitation, DEAE-52ion-exchange columnchromatography and Sephadex G-100gel filtration chromatography weresuccessively performed. A protein fraction with a relative molecular weight around24.3kDa was isolated from black kidney bean. It was15.6kDa for light speckledkidney bean,11.4kDa and16.7kDa for snail flower kidney bean, and10.6kDa,17.8kDa and52.0kDa for large white kidney bean. In addition, trypsin inhibitorsfrom the four kinds of kidney beans all demonstrated very good thermo stability. After treatment at100oC for60min, trypsin inhibitors from black kidney bean, lightspeckled kidney bean, snail flower kidney bean and large white kidney beanretained respectively66.40%，47.53%，52.63%and51.76%of their originalinhibitory activities.Trypsin inhibitors are widespread in many leguminous plants. However, theirstudies are subjected to soybean, mung bean, cowpea and several other limitedspecies. Recent researches have changed their subjects to Phaseolus beans whileonly preliminary isolations are involved. It is reasonable to predict that deeperinvestigations into trypsin inhibitors from various leguminous species will be apotential research hot-spot.