Isolation and identification of a polygalacturonase inhibiting protein from Isatidis root

Isatis indigotica Fort. (Chinese woad) has been widely used as a dye- and medicinal-plant in traditional Chinese medicine. Although I. indigotica has been cultivated in various regions across China in recent years, its innate immunity is little known. In this study, a protein with MW 37.5 kDa was purified from the extract of fresh Isatidis roots by SP-5PW cationic exchange and POROS HP2 hydrophobic interaction chromatography. The N-terminal amino acid sequence of the purified protein was subsequently determined as T-D-L-C-H-K-D-P-K-N-T-L-L by Edman degradation. The N-terminal sequence and PG inhibitory activity identified the purified substance as a polygalacturonase inhibiting protein. This purified Isatidis PGIP with a specific activity of 7.64x10(4) U/mg showed strong inhibitory activity against 160 U of Aspergillus niger polygalacturonase. Compared with PGIPs from guava and bean pods, Isatidis PGIP showed very poor pH and heat stabilities, which may represent the different need of plant innate immunity between plant underground and aboveground organs.