Isolation of a thermostable trypsin inhibitor with exploitable potential

A novel trypsin inhibitor with considerable thermal and pH stability, designated Glytine, was isolated from seeds of the Chinese black soybean Glycine max (L.) Merr. The purification procedure involved ammonium sulfate precipitation, ion-exchange chromatography on CM-Sephadex C-50, gel filtration chromatography on Sephacryl S-200HR, and gel filtration chromatography on POROS HS-20. The 20 N-terminal amino acid sequences were determined to be DEYSKPCCDLCMCTRRCPPQ, demonstrating close homology with the sequences of leguminous trypsin inhibitors. The molecular mass and isoelectric point of the inhibitor were estimated by SDS-PAGE and isoelectric focusing to be 19.9 kDa and 6.2, respectively. Trypsin could be completely inhibited by Glytine when the weight ratio was 1.5. The inhibitory activity of Glytine was unaffected by exposure to temperatures up to 100 °C, or within the pH range 2–12. Besides trypsin–chymotrypsin inhibition activity, Glytine demonstrated other biological activities including antiproliferative activity against tumor cells including human liver hepatoma cells Bel-7402 and neuroblastoma cells SHSY5Y. In addition, the inhibitor showed antifungal activity against Pythium aphanidermatum, Fusarium oxysporum, Alternaria alternata (Fr.) Keiss, Fusarium solani, and Botrytis cinerea. This study extended research on leguminous trypsin–chymotrypsin inhibitor and suggested exploitable potential.