ISOLATION OF A TRYPSIN–CHYMOTRYPSIN INHIBITOR AND ITS FUNCTIONAL PROPERTIES

Abstract

A novel trypsin inhibitor with thermal and pH stability, designated Merrtine, was isolated from Glycine max L. merr. The procedure involved ammonium sulfate precipitation, ion-exchange chromatography on CM-Sephadex C-50, and affinity chromatography on Affi-gel blue gel. The 20 N-terminal amino acid sequences were determined to be DEYSKPCCDLCMCTRRCPPQ, demonstrating high homology with the sequence of Bowman–Birk type trypsin inhibitors. The molecular mass and isoelectric point of the inhibitor were estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing to be 20.0 kD and 5.8, respectively. Trypsin could be completely inhibited by Merrtine when the molar ratio was 8.1. The inhibitory activity of Merrtine was unaffected after exposure to temperatures up to 85°C, as well as within the pH range 2–12. Besides inhibiting trypsin–chymotrypsin, the inhibitor demonstrated additional antifungal activity against the species of Alternaria alternate, Fusarium oxysporum, Pythium aphanidermatum, Physalospora piricola, Botrytis cinerea, and Fusarium solani. We herein report not only the trypsin inhibitor's extraction and isolation for the first time, but also its physiochemical and antifungal properties.

Keywords:

• antifungal
• Glycine max L.merr
• thermostable
• trypsin inhibitor

ACKNOWLEDGMENTS

This work was supported by the Open Project Program of State Key Laboratory of Food Science and Technology, Jiangnan University, China (No. SKLF-KF-201301), the Fujian Natural Science Foundation, China (No. 2013J01132), the S&T projects of Fujian Provincial Science & Technology Hall (No. 2012N0015 & 2012S0053), and the Project of Science and Technology of Nantong City, China (No. HS2011001).

Notes

Note. The presented N-terminal sequences in this table are from the NCBI database (http://www.ncbi.nlm.nih.gov). Underlined characters, identical amino acids with the purified Merrtine..

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