An Investigation into Bioactive Proteins and Their Changes During Imbibition, Germination and Development of Red Kidney Bean Seeds (Phaseolus vulgaris L.)
Thesis DisciplinePlant Biotechnology
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
Red kidney bean seeds (Phaseolus vulgaris) contain a variety of bioactive proteins including lectins, enzyme inhibitors, hydrolytic enzymes and antifungal proteins. The aim of this research was to investigate activities of selected low pH and heat-stable bioactive proteins extracted from different parts of red kidney bean seed, seedling and pod as well as seed and root exudates.
Crude red kidney bean seed extracts inhibited growth of Alternaria alternata as well as its protease activity, but not its amylase activity. A protein with inhibitory activity against growth of A. alternata was purified from extracts of the red kidney bean cotyledons and embryonic axis. This purified bean protein was devoid of chitinase and β-1, 3- glucanase activities. Also, it did not inhibit porcine pancreatic α-amylase, bovine trypsin, amylase and protease of A. alternata suggesting that the antifungal activity of the protein is not related to these activities.
Proteinaceous extracts of red kidney bean cotyledons induced melanin and conidia formation in mycelium of A. alternata. A protein responsible for this conidiation inducing effect was shown for the first time to be a mannose-binding lectin which is also known as PvFRIL (Phaseolus vulgaris fetal liver tyrosine kinase 3-receptor interacting lectin).
An unexpected finding was that extracts of the embryonic axis stimulated rather than inhibited porcine α-amylase activity. Phytohemagglutinin (PHA-L in particular), co-extracted with α-amylase inhibitor from red kidney bean seeds, was implicated as an α-amylase stimulator with the potential of greatly assisting digestion of starch. In cotyledonary extracts, amylase stimulatory activity was masked by amylase inhibitory activity that was inactivated when the extracts were boiled for 10 min. An in-gel non-denaturing electrophoretic method was used to show presence of porcine α-amylase isoinhibitors in extracts of the cotyledons and embryonic axis. All other seedling parts as well as seed and root exudates had amylase stimulatory activity.
Another improved non-denaturing electrophoretic method with immobilized azoalbumin was developed for in-gel detection of isoinhibitors of bovine trypsin in seed parts. It eliminates the need for both time-consuming and labourious staining, destaining or renaturation steps used in other methods.
Accumulation of most of the selected bioactive proteins during seed development in different seed parts appeared to start at 20 days after flower abscission. The activities of these proteins decreased to lower levels after 11 days of germination. Besides these observed developmental changes, under abiotic (UV-C irradiation) and biotic (seedlings co-cultured with A. alternata) stress, increased activity of some of the selected bioactive proteins were detected. In conclusion, this study has contributed to a better understanding of antifungal activity and the selected bioactive proteins in extracts of red kidney bean.