Heat stable trypsin inhibitors in dry beans: Screening cultivars of dry beans and purification and characterization of trypsin inhibitors from Great Northern beans (Phaseolus vulgaris)

Donald Bergeron, Purdue University

Abstract

The objectives of this study were: (1) to investigate the heat stability of trypsin inhibitors (TIs) in nine cultivars of dry beans, specifically in the intact whole bean matrix, ground bean flour, and bean albumin (water soluble protein) extract, all under the same experimental conditions, (2) to purify TIs from Great Northern beans using a variety of chromatographic techniques including anhydrotrypsin-Sepharose affinity chromatography, and (3) to separate, identify, and partially characterize the trypsin isoinhibitors present in Great Northern beans. Nine dry bean cultivars exhibited evidence of heat stable trypsin inhibitory activity (TIA). TIA exhibited minimum, medium, and maximum heat stability in the whole intact bean matrix, ground bean flour and bean albumin extract, respectively. Boiling (30 min) either soaked intact beans or bean albumin extract resulted in 2.5 to 5% and 97 to 100% retention of TIA, respectively, when compared to unheated controls. Purification of heat stable TIs from Great Northern beans was performed using extraction with 2% NaCl, anion exchange, hydrophobic interaction, and affinity (anhydrotrypsin-Sepharose) chromatographic techniques. Eleven isolated TIs with isoelectric points of approximately pH 4 to 5 were observed by TI staining of an isoelectric focusing gel. Some of the eleven isoinhibitors that exhibited TIA also exhibited chymotrypsin inhibitory activity. An isoelectric focusing gel of albumin extracts from nine dry bean cultivars revealed differences between cultivars in the actual isoelectric points and relative amounts and/or activities of individual trypsin and chymotrypsin isoinhibitors present. The apparent molecular weight of the pool of TIs was approximately 18,000 by SDS-PAGE, 16,000 by size exclusion chromatography, 8,000 to 17,000 by SDS-PAGE of S-carboxymethylated TIs, and 8,406 and 8,957 by mass spectrometry. Amino acid compositional analysis of one Great Northern bean isoinhibitor revealed high levels of Cys, Asp, Ser, and Glu, low levels of Tyr, Leu, Phe, and Arg, and no Val or Met. N-terminal sequence analysis of five Great Northern bean TIs revealed that four contained Ser at the N-terminus, and one contained Ser at the second position. Three of the five isoinhibitors were homologous with each other and with other plant source TIs.

Degree

Ph.D.

Advisors

Nielsen, Purdue University.

Subject Area

Food science|Agricultural chemicals

COinS