Abstract 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.

中文翻译：

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在不同 α-淀粉酶抑制活性的小麦品种上饲养的多米尼克根霉 (F.) 后代的 α-淀粉酶的差异生化和动力学特性

摘要 本研究旨在深入了解在不同 α-淀粉酶抑制活性的小麦品种中饲养的后代中表达的多米尼克根霉的 α-淀粉酶同种型（命名为 RdA70、RdA79 和 RdA90）的生化和动力学特性。在后代的淀粉酶活性和小麦抑制活性之间观察到反比关系。选择具有高和低侵染率的小麦样品（分别命名为 HI-Borlaug 和 LI-Villa Juarez）以简化研究。通过疏水相互作用色谱法分离子代淀粉酶，而通过尺寸排阻色谱法分析小麦样品的α-淀粉酶抑制活性。LI-Villa Juarez 后代的同工型 RdA70 和 RdA90 显示出比 HI-Borlaug 更高的酶活性（分别为 73.8% 和 43.4%）。当测试淀粉酶同种型对抑制性白蛋白抑制的敏感性时，LI-Villa Juarez 的那些比 HI-Borlaug 的更敏感。动力学参数的测定表明，来自 HI-Borlaug 后代的 RdA70 的淀粉亲和力比来自 LI-Villa Juarez 的低 3.0 倍（Km 为 12.3 ± 1.8 对 4.0 ± 0.3）。其余的 α-淀粉酶在 HI-Bourlag 中没有显示出与 RdA​​70 相同的模式，因为它们的淀粉亲和力进一步降低（RdA79 Km = 23.1 ± 4.3，RdA90 Km = 17.1 ± 2.9）。IC50 值的估计证实了 LI-Villa Juarez 后代的三种 α-淀粉酶对小麦 α-淀粉酶抑制剂的高度敏感性。LI-Villa Juarez 后代中 RdA70 的抑制剂常数 Ki 最低，表明抑制剂与该同工酶紧密结合。