The present study was accomplished to gain insights into the biochemical and kinetic properties of Rhyzopertha dominica's α-amylase isoforms (named RdA70, RdA79, and RdA90) expressed in progenies reared in wheat varieties differing in α-amylase inhibitory activity. An inverse relationship was observed between the progenies’ amylase activity and the wheat inhibitory activity. Wheat samples with a high and low infestation (named HI-Borlaug and LI-Villa Juarez, respectively) were chosen to simplify the study. The progenies amylases were isolated by hydrophobic interaction chromatography, while the wheat samples were analyzed in α-amylase inhibitory activity by size exclusion chromatography. The isoforms RdA70 and RdA90 from LI-Villa Juarez progeny showed higher enzyme activities (73.8 and 43.4%, respectively) than those from HI-Borlaug. When the amylase isoforms were tested in susceptibility to inhibition by the inhibitory albumins, those from LI-Villa Juarez were more susceptible than those of the HI-Borlaug. Determination of the kinetic parameters revealed that RdA70 from the HI-Borlaug progeny showed 3.0-fold less starch affinity than that from the LI-Villa Juarez (Km of 12.3 ± 1.8 versus 4.0 ± 0.3). The rest of the α-amylases did not show the same pattern as RdA70 in the HI-Bourlag as that their starch affinity was further reduced (RdA79 Km = 23.1 ± 4.3, RdA90 Km = 17.1 ± 2.9). Estimation of IC50 values confirmed the high sensitivity of the three α-amylases of the LI-Villa Juarez progeny to wheat α-amylase inhibitors. The inhibitor constant Ki was the lowest for RdA70 in the LI-Villa Juarez progeny indicating the inhibitor’ tight binding to that isoenzyme. These results suggest that R. dominica uses RdA70 to bind large amounts of wheat α-amylase inhibitors than RdA79 and RdA90 as a physiological defense mechanism.
Bibliographical noteFunding Information:
This research was funded by the Consejo Nacional de Ciencia y Tecnología ( CONACYT Mexico) grant CB-2011-169476 .
This research was funded by the Consejo Nacional de Ciencia y Tecnolog?a (CONACYT Mexico) grant CB-2011-169476.
- Biochemical properties
- Kinetic properties
- Rhyzopertha dominica
- Wheat varieties
- α-Amylase inhibitors
- α-Amylase isoforms